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Overview of Protein Purification and Characterization

AIMS AND OBJECTIVES


Protein purification has an over 200-year history: the first attempts at isolating substances from plants having similar properties to egg albumen, or egg white, were reported in 1789 by Fourcroy. Many proteins from plants were purified in the nineteenth century, though most would not be considered pure by modern standards. A century later, ovalbumin was the first crystalline protein obtained (by Hofmeister in 1889). The year 1989 may not go down in history as a milestone in protein chemistry, but since then there has been a resurgence of interest in proteins after more than a decade of gene excitement. The aims of protein purification, up until the 1940s, were simply academic. To then, even the basic facts of protein structure were not fully appreciated, and pure proteins were needed just to study structure and test the rival theories of the pre-DNA days. During the Second World War, an acute need for blood proteins led to development of the Cohn fractionation procedure for purification of albumin and other proteins from serum (Cohn et al., 1946). This was the inception of large-scale protein purifications for commercial purposes; Cohn fractionation continues to be used to this day. As more proteins, and particularly enzymes, were purified and crystallized, they started to be used increasingly in diagnostic assays and enzymatic analyses, as well as in the largescale food, tanning, and detergent industries. Many enzymes used in industry are not in fact very pure, but as long as they do the job, that is sufficient. Process enzymes such as -amylase, proteases, and lipases are pro-duced in ton quantities, mainly as secretion products in bacterial cultures, and may undergo only limited purification processes to mini-mize costs. At the other extreme, enzyme products for research and analysis require a high degree of purification to ensure that contaminating activities do not interfere with the intended use. Anyone familiar with molecular biology enzymes will appreciate how minute levels of contamination of DNase or RNase can completely destroy carefully planned experiments. The 1960s and 1970s could be described as the peak years for protein and enzyme research, and most of the methods used in protein purification were established by then, at least in their principles. More recent developments have been mainly in instrumentation designed to optimize the application of each methodology. Developments in instrumentation have been stimulated by the rapid progress in molecular biology, because gene isolation has often been preceded by isolation of the gene product. Because such products can now be characterized sufficiently (i.e., partially sequenced) using minute amounts of protein, the need for large-scale or even moderate-scale procedures has decreased. Hence there has been an explosive development of modern equipment designed specifically for dealing with amounts of protein in the milligram to microgram range. On the other hand, structural studies using X-ray crystallography and nuclear magnetic resonance (NMR) require hundreds of milligrams of pure protein, so larger-scale equipment and procedures are still needed in the research laboratory. The nature of the proteins studied has also changed substantially. Whereas enzymes were once the most favored subjects, they have now been superceded by nonenzymatic proteins such as growth factors, hormone receptors, viral antigens, and membrane transporters. Many of these occur in minute amounts in the natural source, and their purification can be a major task. Heroic efforts in the past have used kilogram quantities of rather unpleasant starting materials, such as human organs, and ended up with a few micrograms of pure product. It is now more usual, however, to take the genetic approach: clone the gene before the protein has been isolated or even properly identified, then express it in a suitable host cell culture or organism. The expression level may be orders of magnitude higher than in the original source, which will make purification a relatively simple task. It can be useful to know beforehand some physical properties of the protein, to facilitate the development of a suitable purification protocol from the recombinant source. On the other hand, there are now several ways of preparing fusion proteins, which can be purified by affinity techniques without any knowledge of the properties of the target protein. Moreover, there are ways of modifying the expressed product to simplify purification further.

UNIT 1.1

Strategies of Protein Purification and Characterization

Contributed by R.K. Scopes


Current Protocols in Protein Science (1995) 1.1.1-1.1.6 Copyright 2000 by John Wiley & Sons, Inc.

1.1.1
CPPS

Overview of Protein Purification and Characterization

Thus the approach to protein purification must first take into account the reason it is being done, as the methods will vary greatly with different requirements. At one extreme is the one-of-a-kind purification, in a well-financed and equipped laboratory, that is carried out to obtain a small amount of product for sequencing so that gene isolation can proceed. In this case, expense of equipment and reagents may be no problem, and a very low overall recovery of product can be acceptable, provided it is pure enough. At the other extreme are the requirements of commercial production of a protein in large amounts on a continuing basis, where high recovery and economy of processing are the chief parameters to be considered. There are many intermediate situations as well. Many publications in the area of protein research are entitled Purification and characterization of, and describe a purification procedure in sufficient detail that it can be reproduced in another laboratory. The characterization section may include structural, functional, and genetic information, and carrying out such studies is likely to require at least milligram quantities of pure protein. Ideally the purification should involve a small number of steps, with good recovery at each step. If the recovery is poor (<50% at any step), however, there should be some indication of what happened to the missing activity. Has it been discarded in the other fractions for the sake of purity, or does it represent a true loss of activity? If the latter, then the end-product may be less than fully active despite apparent homogeneity indicated by standard analysis. The choice between recovery and purification at each step can be problematical; taking a narrow cut of a chromatographic peak may provide a very pure fraction, at the expense of losing a good deal of less pure active component on either side. In making such decisions, the objective of the exercise must be kept in mind: if yield is not important, then the choice of poor yield for the sake of purity may be logical. By far the most important requirement of a publication is reproducibility of the method reported. It is not sufficient to have carried out the process only once if it is expected that other investigators will want to repeat it. There are always factors that influence the process that may be overlooked at first, and which if varied slightly can have a major effect on the purification procedure. The reported process should always be repeated exactly as described before submitting the manuscript for publication. There is one exception, namely, the case where

purification was conducted simply to obtain enough protein for sequencing and gene isolation; if that was achieved, there should be no need to provide instructions for repetition.

SOURCES OF MATERIAL FOR PROTEIN PURIFICATION


For many people embarking on a protein purification project, there is no choice of material. They are studying a particular biological tissue or organism, and the objective is to purify a protein from that source. However, there may be approaches that can make the project simpler. If, for instance, the source is difficult to obtain in large amounts, it may be best to carry out at least preliminary trials on a source species more readily obtained. The most obvious and relevant example is when the species being studied is Homo sapiens, and tissue samples are not readily available for practical or ethical reasons, or both. In this case, it is usual to go to where mammalian tissue is readily available (i.e., an abattoir) and work with bovine, ovine, or porcine sources. Alternatively, if quantity of tissue is not a problem, the humble laboratory rat may suffice. Once a protocol for purifying the protein from substitute sources has been worked out, it will be much easier to develop one using human materialthe identical procedure may work satisfactorily. Proteins differ to a fairly small extent between species that have diverged within about 100 million years, a time frame that groups together most higher mammals. Thus the behavior of proteins derived from different animals with respect to the various fractionation procedures is likely to be similar, and a protocol worked out for pig tissues is likely to need only minor adjustments for application to human tissues. A second example is where the interest is mainly on the function of a protein, especially an enzyme, for which functions and actions have generally been strongly conserved through evolution. In that case, a preliminary screening of potential sources, or, better still, the literature, should come up with a raw material that is best suited to the investigators purposes. Considerations should include the following: (1) What functions are required of the end product? For instance, an enzyme having a low Km may be needed, so selecting the source with the highest activity may not suffice. (2) How convenient is it to grow or obtain the raw material, and are there problems concerning pathogenicity or extractability? (3) Does the quantity of the protein vary with growth conditions or age, and does it deteriorate in situ

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Current Protocols in Protein Science

if left too long? Obviously one requires a source that reliably produces the highest amount of the desired protein per unit volume to maximize the chances of developing a good purification procedure. (4) What storage conditions are required for the raw material? It is important to consider that fresh raw material may not be immediately available whenever a purification is attempted. The above considerations are relevant to the traditional situation for commencing a protein purification project. It is becoming increasingly common, however, for proteins to be purified as recombinant products using techniques in which the gene is expressed in a host organism or in cultured cells. This of course requires the gene encoding the protein of interest to be available. Until the mid-1980s, such material was usually obtained by hybridization of an oligonucleotide synthesized according to amino acid sequence information. This required the protein to have been purified first, so the initial task of protein purification still needed to be done at least once. More recently, genetic techniques have permitted the isolation of many genes encoding known proteins, even though the proteins may never have been studied directly. Moreover, with the expansion of the Human Genome Project and related DNA sequencing efforts, many genes for both known and unknown proteins will become available and will be able to be expressed in recombinant form without ever being purified from the host species. As a result some completely new considerations for protein purification come into play, including the possibility of modifying the gene structure not only to increase expression level and alter the protein product itself to enhance a desired function, but equally importantly to aid in purification. Recombinant proteins may be expressed in bacteria, yeasts, insect cells, and animal tissue cultures. Further details may be found in UNIT 1.2.

semiquantitative, indicating which fraction contains the most of the desired protein is essential. Assays may range from the quick-and-easy type (e.g., instantaneous spectrophotometric measurement of enzyme activity) to long and tedious bioassays that may take days to produce an answer. The latter situation is very difficult, because by the time one knows where the protein is, it may be was, owing to degradation or inactivation. Moreover, this may not become clear until the next step has been completed and its products assayed. Any assay that is quick is therefore advantageous, even if it means a sacrifice of accuracy for speed. Measurement of total protein is useful, as it indicates the degree of purification at each step. However, unless the next step critically depends on how much protein is present, total protein measurement is not extremely important: a small sample can be put aside and measured later, when the purification is complete. It is, however, very important to know how much protein is present in the final, presumed pure sample, as this will indicate the specific activity (if the protein has an activity), which can be compared with other preparations. The general object is to obtain as high a specific activity as possible (taking into account recovery considerations), which means retaining as much of the desired protein as possible while ending up with as little total protein as possible.

METHODS FOR SEPARATION AND PURIFICATION OF PROTEINS


The methods available for protein purification range from simple precipitation procedures used since the nineteenth century to sophisticated chromatographic and affinity techniques that are constantly undergoing development and improvement. Methods can be classified in several alternative waysperhaps one of the best is based on the properties of the proteins that are being exploited. Thus the methods can be divided into four distinct but interrelated groups depending on protein characteristics: surface features, size and shape, net charge, and bioproperties.

DETECTION AND ASSAY OF PROTEINS


During a protein purification procedure there are two measurements that need to be made, preferably for each fraction. Measurements both of the total protein and of the amount (usually bioactivity) of the desired protein must be made. Details of the most commonly used assay methods are given in Chapter 3. It is not possible to isolate a protein without a method of determining whether it is present; an assay, either quantitative or at least

Methods Based on Surface Features of Proteins


Surface features include charge distribution and accessibility, surface distribution of hydrophobic amino acid side chains, and, to a lesser extent, net charge at a given pH (see discussion of net charge). Methods exploiting surface fea-

Strategies of Protein Purification and Characterization

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Current Protocols in Protein Science

tures mainly depend on solubility properties. Differences in solubility result in precipitation by various manipulations of the solvent in which the proteins are solubilized. Methods for obtaining an extract containing the desired protein in soluble form are given in Chapter 4. The solvent, nearly always water containing a low concentration of buffer salts, can be treated to alter properties such as ionic strength, dielectric constant, pH, temperature, and detergent content, any of which may selectively precipitate some of the proteins present. Conversely, proteins may be selectively solubilized from an insoluble state by manipulation of the solvent composition. The surface distribution of hydrophobic residues is an important determinant of solubility properties; it is also exploited in hydrophobic chromatography, both in the reversed-phase mode (UNIT 11.6) and in aqueousphase hydrophobic-interaction chromatography (UNIT 8.4). Also included in this group is the highly specific technique of immunoaffinity chromatography, in which an antibody directed against an epitope on the protein surface is used to pull out the desired protein from a mixture.

cation. The net charge of a protein depends on the pHpositive at very low pH, negative at high pH, and zero at some specific point in between, termed the isoelectric point (pI). It should be stressed that at the pI a protein has a great many charges; it just happens that at this pH the total negatives exactly equal the total positives. The most charged state (disregarding the charge sign) is in the pH range 6.0 to 9.0. This is the most stable pH range for most proteins, as it encompasses common physiological pH values. Ion exchangers consist of immobilized charged groups and attract oppositely charged proteins. They provide the mode of separation that has the highest resolution for native proteins. High-performance reversedphase chromatography has equivalent or even better resolution, but it generally involves at least partial denaturation during adsorption and so is not recommended for sensitive proteins such as enzymes. Protein purification using ion-exchange chromatography has mainly employed positively charged anion exchangers, for the simple reason that the majority of proteins at neutral pH are negatively charged (i.e., have a low isoelectric point). Details of methodology are found in UNIT 8.2.

Methods Based on Whole Structure: Size and Shape


Although the size and shape of proteins can have some influence on solubility properties, the chief method of exploiting these properties is gel-filtration chromatography (UNIT 8.3). In addition, preparative gel electrophoresis makes use of differences in molecular size. Proteins range in size from the smallest classified as proteins rather than polypeptides, around 5000 Da, up to macromolecular complexes of many million daltons. Many proteins in the bioactive state are oligomers of more than one polypeptide (see UNIT 1.2), and these can be dissociated, though normally with loss of overall structure. Thus many proteins have two sizes: that of the native state, and that (or those) of the polypeptides in the denatured and dissociated state. Gel-filtration procedures normally deal only with native proteins, whereas electrophoretic procedures commonly involve separation of dissociated and denatured polypeptides.

Methods Based on Bioproperties (Affinity)


A powerful method for separating the desired protein from others is to use a biospecific method in which the particular biological property of the protein is exploited. The affinity approach is limited to proteins that have a specific binding property, except that proteins are theoretically able to be purified by immunoaffinity chromatography (UNIT 9.1), which is the most specific of all affinity techniques. Most proteins of interest do have a specific ligand: enzymes have substrates and cofactors, and hormone-binding proteins and receptor molecules are designed to bind specifically and tightly to particular hormones and other factors. Immobilization of the ligand to which the protein binds (or of antibody to the protein) enables selective adsorption of the desired protein in the technique known as affinity chromatography (Chapter 9). There are also nonchromatographic modes of exploiting biospecific interactions.

Methods Based on Net Charge


The two techniques that exploit the overall charge of proteins are ion-exchange chromatography (by far the most important) and electrophoresis (Chapter 10). Ion exchangers bind charged molecules, and there are essentially only two types of ion exchangers, anion and

Overview of Protein Purification and Characterization

CHARACTERIZATION OF THE PROTEIN PRODUCT


Once a pure protein is obtained, it may be employed for a specific purpose, such as enzymatic analysis (e.g., glucose oxidase and lac-

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Current Protocols in Protein Science

tate dehydrogenase), or as a therapeutic agent (e.g., insulin and growth hormone). However, it is normal, when a protein has been isolated for the first time, to characterize it in terms of structure and function. Several features are generally expected in characterization of a new protein. These include molecular weight, or at least the size of the subunit(s), determined by SDS-polyacrylamide gel electrophoresis (Chapter 10) and/or gel filtration (UNIT 8.3). Spectral properties such as the UV spectrum (Trp and Tyr content), circular dichroism (CD) spectrum (secondary structure), and special characteristics of proteins with prosthetic groups (e.g., quantitation and spectra) may be presented. The quantity and nature of carbohydrates on glycoproteins should be determined (Chapter 12). Also, if the gene has not already been reported, some amino-terminal sequence analysis should be given, if at all possible, along with the results of a database search for similar sequences (UNIT 2.1). Functional proteins should be demonstrated to have the appropriate function, and for enzymes detailed kinetic characterization is appropriate. Ultimately the full three-dimensional structure of the protein may be determined, which will require crystals: any successful crystallization attempts should be reported.

THE PROTEIN PURIFICATION LABORATORY


The requirements for a protein purification laboratory cannot be exactly formulated because they depend greatly on the types and amounts of proteins being isolated. To cover all eventualities, it would be necessary to have one set of equipment to deal with submicrogram quantities and another set to deal with multigram quantitiesa range of around 108! One laboratory dedicated to protein purification may not need small-scale equipment if, for example, it works with plasma proteins that are always available in large quantities. Another may have all the latest in high-performance equipment but not be able (nor need) to handle quantities of protein in excess of a few milligrams. If it is assumed that neither extreme in quantity is to be attempted, and that the laboratory is handling a variety of protein types and sources, then certain basic pieces of equipment are needed. Obtaining the starting material and making an extract of it require homogenization equipment and centrifuges to remove insoluble residues. Preliminary fractionation, when starting with a crude extract of tissue or cells,

requires equipment and materials that will not become clogged by particulates. Adsorbents and similar materials used at the first step should be relatively inexpensive so that when performance falls off after a few uses, owing to intransigent impurity buildup, they can be discarded. It is also relevant that a larger amount is handled at the initial step than later steps; therefore, reagent expense can be an important consideration. After the first one or two steps, the sample should be sufficiently clean and clear to enable use of high-performance equipment. High-performance liquid chromatography, or HPLC, is a term with a variety of meanings. To some it refers exclusively to reversed-phase chromatography; to others it includes all sorts of chromatography provided that the equipment is fully automated and high-performance adsorbents are used. A high-performance system designed specifically for proteinsintroduced by Pharmacia Biotech (see SUPPLIERS APPENDIX) called Fast Protein Liquid Chromatography, or FPLCuses standard protein chromatographies such as ion exchange, hydrophobic interaction, and gel filtration. Scaleup is possible with larger equipment based on the FPLC design, so that laboratory development can be quickly translated to large-scale production. FPLC is designed to separate proteins in their native active configuration, whereas reversed-phase HPLC often causes at least transient denaturation during adsorption and elution. Reversed-phase HPLC has a high resolving power, but it is best suited to peptides and proteins smaller than 30 kDa. Chromatography run with older-style low-pressure adsorbents is sometimes referred to as low-performance or open-column chromatography; neither of those descriptions is necessarily accurate. Simple fraction collector and monitoring equipment is needed. This equipment will be used for larger-scale operations (tens of milligrams of protein and upward), probably at an earlier stage in the protocol than with HPLC. Various columns, both prepacked with proprietary adsorbents and empty for self-packing, will be needed, with the sizes and types depending on the scale of operations. Several anionexchange columns (different sizes), one or two cation-exchange columns, and gel-filtration media are essential, along with a range of alternative adsorbents such as hydrophobic interaction materials, dyes, hydroxyapatite, and chromatofocusing and specialist affinity media. Fully equipped protein purification laboratories should also have preparative electropho-

Strategies of Protein Purification and Characterization

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Current Protocols in Protein Science

resis and isoelectric focusing apparatuses for rare occasions when other techniques fail to give sufficient separation. In addition to equipment used in the actual fractionation processes, a variety of other items are needed. In particular it should be possible to change buffers quickly and to concentrate protein solutions with ease. These operations require such things as dialysis membranes (APPENDIX 3B), ultrafiltration cells, and gel-exclusion columns of various sizes (UNIT 8.3). Finally, equipment for assaying and analyzing the preparations is needed. Most such equipment is fairly standard in biochemical laboratories and includes spectrophotometers, scintillation counters, analytical gel and capillary electrophoresis apparatuses, immunoblotting materials, and immunochemical reagents. A listing of standard equipment is found in APPENDIX 2D.

Key References
Deutscher, M.P. (ed.) 1990. Guide to protein purification. Methods Enzymol. 182:1-894. Extensive collection of purification methods with some general protocols and examples. Janson, J.-C. and Ryden, L.G. 1989. Protein Purification: Principles, High Resolution Methods, and Applications. VCH Publishers, New York. A useful collection of methods and examples. Kennedy, J.F. and Cabral, J.M. (eds.) 1993. Recovery Processes for Biological Materials. John Wiley & Sons, New York. A useful introduction to the problems of large-scale methods. Kenny, A. and Fowell, S. (eds.) 1992. Practical protein chromatography. Methods Mol. Biol. 11:1-327. Extensive descriptions of affinity chromatographic techniques with protocols and recipes. Scopes, R.K. 1993. Protein Purification, Principles and Practice, 3rd ed. Springer-Verlag, New York and Heidelberg. General principles of all the main techniques used in purifying proteins. A useful laboratory handbook; does not include recipes or procedures for specific proteins.

Literature Cited
Cohn, E.J., Strong, L.E., Hughes, W.L., Mulford, D.J., Ashworth, J.N., Melin, M., and Taylor, H.L. 1946. Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the proteins and lipoprotein components of biological tissues and fluids. J. Am. Chem. Soc. 68:459-475.

Contributed by R.K. Scopes La Trobe University Bundoora, Australia

Overview of Protein Purification and Characterization

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Strategies for Protein Purification


CLASSIFICATION OF PROTEINS
As with most heterogeneous collections of things, proteins can be classified in several different ways, such as by function, by structure, or by physicochemical characteristics. Each protein species consists of identical molecules with exactly the same size, amino acid sequence, and three-dimensional shape. In this way a solution of a mixture of proteins differs from a solution of synthetic polymers or sheared DNA, both of which contain a complete spectrum of possible sizes centered around the average. The protein mixture has only discrete sizes of molecules corresponding to each type of protein present. Although we could classify proteins by size, it would be of limited use, as there is usually no obvious relationship between size and function. A more useful structural classification takes into consideration shape and oligomeric structure (Table 1.2.1). In part, structure reflects biological location and origin. Simple, fairly rigid protein molecules occur in the extracellular environment, more complex and readily deactivated molecules are found intracellularly, and hydrophobic proteins are associated with membranes. Classification by function is more relevant (Table 1.2.2). Proteins can be simply stores of amino acids, can be structural, or can have specific binding functions. The most funcTable 1.2.1

UNIT 1.2

tional proteins are enzymes, which have both binding and catalytic roles. In part, this reflects the degree to which the detailed structure is a requirement for the proteins function, which in turn relates to conservation of structure through evolution. But as with every attempt at classification, there are going to be examples that do not fit the pattern well. Most proteins of interest to the pharmaceutical industry belong to the general class of binding proteins, for instance, hormones [e.g., insulin and bovine somatostatin (BST)], viral antigens (e.g., hepatitis B antigen), growth factors [e.g., interferons, interleukins and colony-stimulating factors (CSFs)], and antibodies.

STRATEGIES FOR PROTEIN PURIFICATION Soluble Extracellular Proteins


The source of soluble extracellular proteins is the extracellular medium, whether it be an animal source such as blood or spinal fluid, or a culture medium in which bacterial, fungal, animal, or plant cultures have been grown. Generally these do not contain a large number of different proteins (blood is an exception), and the desired protein may be a major component, especially if produced as the result of recombinant expression. Nonetheless, the protein in the starting material may be quite dilute,

Classification of Proteins by Structural Characteristics

Structural characteristic Monomeric Oligomeric Identical subunits

Examples Lysozyme, growth hormone

Comments Usually extracellular; often have disulfide bonds Mostly intracellular enzymes; rarely have disulfide bonds Allosteric enzymes; different subunits have separate functions Readily solubilized by detergents Require lipid for stability Many extracellular proteins contain carbohydrate
Strategies of Protein Purification and Characterization

Mixed subunits Membrane-bound Peripheral Integral Conjugated

Glyceraldehyde-3-phosphate dehydrogenase, catalase, alcohol dehydrogenase, hexokinase Aspartate carbamoyltransferase, pertussis toxin Mitochondrial ATPase, alkaline phosphatase Porins, cytochromes P450, insulin receptor Glycoproteins, lipoproteins, nucleoproteins

Contributed by R.K. Scopes


Current Protocols in Protein Science (1995) 1.2.1-1.2.4 Copyright 2000 by John Wiley & Sons, Inc.

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CPPS

Table 1.2.2

Classification of Proteins by Function

Function Amino acid storage Structural Inert With activity Binding Soluble Insoluble With activity

Examples Seed proteins (e.g., gluten), milk proteins (e.g., casein) Collagen, keratin Actin, myosin, tubulin Albumin, hemoglobin, hormones Surface receptors (e.g., insulin receptor), antigens (e.g., viral coat proteins) Enzymes, membrane transporters (e.g., amino acid uptake systems, ion pumps)

and a large volume may therefore need to be processed. The starting fluid may also contain many compounds other than proteins, whose behavior must be taken into account. The first stage should aim mainly to reduce the volume and get rid of as much nonprotein material as possible; some protein-protein separation is also useful, but not essential. No general rules can be given, but a batch adsorption method using an inexpensive material such as hydroxyapatite, ion-exchange resin, immobilized metal affinity chromatography (IMAC) medium, or affinity adsorbent is best, if feasible. Following the first step, the sample should be in a form that is amenable to standard purification processes such as precipitation and column chromatography (Chapter 8).

Membrane-Associated Proteins
There are two approaches to isolating a membrane-associated protein. In one method, the relevant membrane fraction can first be prepared and then used to isolate the protein. Alternatively, whole tissue can be subjected to an extraction that solubilizes the membranes and releases the cytoplasmic contents as well. The former is much better in that purification is accomplished by isolating the membranes: the specific activity of the solubilized membrane fraction will be much higher than in the second method. However, the process of purifying the membrane fraction may lead to substantial losses, and it may be difficult to scale up. If total recovery of the protein is more important than purity, a whole-tissue extract is likely to be more appropriate. Although this means that a greater degree of purification is needed, the fact that membrane proteins have, by definition, properties somewhat different from those of cytoplasmic proteins permits some very effective purification steps (e.g., hydrophobic chromatography or fractional solubility separation). Peripheral membrane proteins are only loosely attached and may be released by gentle conditions such as high pH, EDTA, or low (nonionic) detergent concentrations. Once in solution, some peripheral proteins no longer require the presence of detergent to maintain their solubility. Integral membrane proteins are much more difficultthey require high concentrations of detergent for solubilization (i.e., complete solubilization of the membrane is needed to release them) and generally are neither soluble nor stable in the absence of detergent. It is sometimes necessary to maintain

Intracellular (Cytoplasmic) Proteins


To obtain soluble intracellular proteins (which are mainly enzymes), cells must be broken open or lysed to release their soluble contents. The ease with which cell disruption can be accomplished varies considerably; animal cells are readily broken, as are many bacteria, but plants and fungi have tough cell walls. Methods for obtaining cell extracts are given in Chapter 4. The macromolecular soluble contents of cells are mainly proteins, with nucleic acids as a minor but significant component. Bacterial extracts may be viscous unless DNase is added to break down the long DNA molecules. Although chromatographic procedures can be applied to crude extracts, valuable highperformance materials should not be employed in the first step, as there are always compounds, including unstable proteins, that may bind to them and be difficult to remove.

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natural phospholipids in association with the proteins in order to maintain activity. Even though the final objective may not require activity (e.g., amino acid sequencing), there is a need for some sort of assay during the purification process to determine where the protein is. If a particular band on a gel is known to be the desired protein, then no other assay is needed and loss of bioactivity can be allowed. Purification processes may be affected by the presence of detergents. The problem of association with detergent micelles makes purifying integral membrane proteins difficult; the close association of the different proteins originating from membranes often results in very poor separation in conventional fractionation procedures.

Soluble Recombinant Proteins


Recombinant proteins that are not expressed in inclusion bodies either will be soluble inside the cell or, if using an excretion vector, will be extracellular (or, if E. coli is the host, possibly periplasmic). They can be purified by conventional means. In some systems, expression is so good that the desired product is the major protein present and its purification is relatively simple. In systems where the expression level is low, the purification process can be tedious, though easier, it is hoped, than isolation from the natural source. It should be remembered that a procedure developed for isolating a protein from natural sources may not work successfully with the recombinant product, because the nature of the other proteins present influences many fractionation procedures. Because of the difficulties often experienced in purifying recombinant proteins, a variety of vector systems (see UNIT 6.1 and Sassenfeld, 1990, for some examples) have been developed in which the expressed prod-uct is a fusion protein containing an N-terminal polypeptide that simplifies purification. Such tags can be subsequently removed using a specific protease. A further advantage is that the expression level is dictated mainly by the transcription and translation signals for the fusion portion of the protein, which are optimized. Tags used include proteins and polypeptides for which there is a specific anti- body, binding proteins that will interact with columns containing a specific ligand, polyhistidine tags with affinity to immobilized metal columns, sequences that result in biotinylation by the host and enable purification on an avidin column, and sequences that confer insolubility under specified conditions (UNIT 6.1 & UNIT 6.5). Unstable proteins may be modified by the molecular biological technique of site-directed mutagenesis to remove the site of instability for instance, an oxidizable cysteine. Such techniques are appropriate for commercial production of proteins, but may of course alter natural functioning parameters. Increased thermostability can be one modification, although it is not easy to predict mutations that will improve that parameter. Thermostable proteins originating from thermophilic bacteria do not need structural modification and, if expressed in large amounts, can be purified satisfactorily in one step by simply heat-treating the extract at 70C for 30 min, which denatures virtually all the host proteins (e.g., see Oka et al., 1989). The host bacteria used for production of recombinant proteins are usually Escherichia

Insoluble Proteins
Natural proteins that are insoluble in normal solvents are generally structural proteins, which are sometimes cross-linked by posttranslational modification. The first stage of purification is obviousit involves extracting and washing away all proteins that are soluble, leaving the residue containing the desired material. Further purification in a native state, however, may be impossible; extracting away other proteins using more vigorous solvents or attempting to solubilize the target protein may destroy the natural structure. Cross-linked proteins such as elastin or aged collagen cannot be dissolved without breaking the cross-links, and the individual proteins may even be crosslinked together.

Insoluble Recombinant Proteins (Inclusion Bodies)


A major new class of insoluble proteins are recombinant proteins expressed (usually in Escherichia coli) as inclusion bodies. These are dense aggregates found inside cells that consist mainly of a desired recombinant product, but in a nonnative state. Inclusion bodies may form for a variety of reasons, such as insolubility of the product at the concentrations being produced, inability to fold correctly in the bacterial environment, or inability to form correct, or any, disulfide bonds in the reducing intracellular environment. Their purification is simple, since the inclusion bodies can be separated by differential centrifugation from other cellular constituents, giving almost pure product; the problem is that the protein is not in a native state, and is insoluble. Some methods for obtaining an active product from inclusion bodies are described in UNIT 6.3.

Strategies of Protein Purification and Characterization

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Current Protocols in Protein Science

coli, or Bacillus subtilis; they may express proteins at 1% to over 50% of the cellular protein, depending on such variables as the source, promoter structure, and vector type. Generally the proteins are expressed intracellularly, but leader sequences for excretion may be included. In the latter case, the protein is generally excreted into the periplasmic space, which limits the amount that can be produced. Excretion from gram-positive species such as B. subtilis sends the product into the culture medium, with little feedback limitation on total expression level.

Janson, J.-C. and Ryden, L.G. 1989. Protein Purification: Principles, High Resolution Methods, and Applications. VCH Publishers, New York. A useful collection of methods and examples. Kennedy, J.F. and Cabral, J.M. (eds.) 1993. Recovery Processes for Biological Materials. John Wiley & Sons, New York. A useful introduction to the problems of large-scale methods. Kenny, A. and Fowell, S. (eds.) 1992. Practical protein chromatography. Methods Mol. Biol. 11:1-327. Extensive descriptions of affinity chromatographic techniques with protocols and recipes. Scopes, R.K. 1993. Protein Purification, Principles and Practice, 3rd ed. Springer-Verlag, New York and Heidelberg. General principles of all the main techniques used in purifying proteins. A useful laboratory handbook; does not include recipes or procedures for specific proteins.

Literature Cited
Oka, M., Yang, Y.S., Nagata, S., Esaki, N., Tanaka, M., and Soda, K. 1989. Overproduction of thermostable leucine dehydrogenase of Bacillus stearothermophilus and its one-step purification from recombinant cells of Escherichia coli. Biotechnol. Appl. Biochem. 11:307-316. Sassenfeld, H.M. 1990. Engineering proteins for purification. Trends Biotechnol. 8:88-93.

Key References
Deutscher, M.P. (ed.) 1990. Guide to protein purification. Methods Enzymol. 182:1-894. Extensive collection of purification methods with some general protocols and examples.

Contributed by R.K. Scopes La Trobe University Bundoora, Australia

Strategies for Protein Purification

1.2.4
Current Protocols in Protein Science

Protein Purification Flow Charts


Protein purification flow charts are presented to give a broad outline of the methods used for different types of proteins. They cannot give any detail, as the process appropriate for each protein will have its own variations at each stage. In most cases, the first stage is to obtain a solution containing the desired protein, after which it can be dealt with by the many separation techniques described in the following chapters. In some cases the insolubility of the desired protein can be exploited by removing soluble fractions. Purification procedures are commonly divided into three stages: (a) the primary steps, which deal with crude mixtures of proteins and other molecules present in the raw material; (b) the secondary processing, which generates a product near to homogeneity; and (c) the polishing steps, which remove minor contaminants, a process that is especially important for therapeutic proteins. tration step before proceeding, especially if the protein has been excreted into the culture medium. Normally ultrafiltration is used, although other techniques are possible, especially if the extract contains particulates that block ultrafiltration membranes. Recombinant expression in the cytoplasm of bacteria, followed by extraction via total cell disruption, results in large amounts of nucleic acids being solubilized with the protein. A number of treatments to remove nucleic acids are possible. Streptomycin is used to precipitate ribosomal material, and cationic polymers such as protamine (a basic protein) and polyethylenimine will form insoluble complexes (at low ionic strength) with nucleic acids. In addition, viscosity caused by DNA can be reduced by adding small amounts of DNase.

UNIT 1.3

INSOLUBLE RECOMBINANT PROTEINS


It has been found that many proteins expressed in bacteria (mainly in Escherichia coli) do not fold correctly, and as a result aggregation occurs, leading to large insoluble inclusion bodies within the cytoplasm of the cells (see Chapter 6). Although this creates major difficulties in obtaining satisfactory amounts of active native product, it greatly simplifies the initial stage of purification. The purification scheme for recombinant insoluble proteins is outlined in Figure 1.3.2. After cell disruption, inclusion bodies can be obtained in a fairly pure state by differential centrifugation. They must now be solubilized, however, and the active protein generated by encouraging correct folding. Solubilization is usually accomplished with guanidine hydrochloride and/or urea, and thiols such as 2-mercaptoethanol or glutathione are included to disrupt any disulfides that have formed and prevent more from forming. Folding the protein correctly may require a variety of additions to the solution, as well as slow removal of the denaturant. The latter can be carried out by simple dilution or by dialysis. Folding occurs best at low protein concentrations, so dilution may be adequate. If the native protein does contain disulfides, then it is important to create redox conditions such that some (but not excessive) oxidation of thiols can occur. A combination of oxidized and reduced glutathione is commonly used. In addition, the action of the enzyme protein disulfide isomerase, which can

SOLUBLE RECOMBINANT PROTEINS


Proteins expressed in a recombinant manner may be (1) soluble in the cytoplasm, (2) insoluble as inclusion bodies (see section on Insoluble Recombinant Proteins), (3) excreted from the cells into the culture medium, (4) excreted into the periplasmic space (e.g., in gram-negative bacteria), or (5) associated with organelles or membrane fractions. In addition they may be expressed (6) as the normal, mature, naturally occurring protein, (7) containing a natural leader peptide that would normally be processed, (8) as a fusion protein with a peptide that is not natural to the protein, or (9) lacking glycosylation or other post-translational modification, or incorrectly modified. Possibilities (1) to (5) affect the method of extraction used to obtain the starting material for purification. Cases (6) to (9) can affect the methods used for purification. The scheme for purifying soluble recombinant proteins is outlined in Figure 1.3.1. The first stage is to obtain a clarified solution containing the desired protein, with as little in the way of unwanted proteins as possible. For soluble cytoplasmic proteins, case (1), it is not normally possible to exclude any significant amount of unwanted soluble proteins, but in cases (2) to (5) the compartmentalization away from the cytoplasm allows such separation in the initial stage. It may be necessary to carry out a concenContributed by R.K. Scopes
Current Protocols in Protein Science (1995) 1.3.1-1.3.7 Copyright 2000 by John Wiley & Sons, Inc.

Strategies of Protein Purification and Characterization

1.3.1
CPPS

cells with protein soluble in cytoplasm

cells with protein excreted to medium

cells with protein in periplasmic space

cells with protein organelleassociated

break up cells; centrifuge or ultrafilter to remove insolubles

centrifuge or filter to remove cells

gently treat with lysozyme to minimize cell lysis

separate organelles; perform differential centrifugation

treat to remove nucleic acids

remove protoplasts and cell debris

extract to solubilize proteins, remove insolubles

perform concentration step

starting material: soluble fraction containing the protein

perform purification steps


use salt fractionation, ion-exchange chromatography, hydrophobic chromatography, affinity and pseudoaffinity chromatography, gel filtration, or other less conventional steps (i.e., preparative electrophoresis) for tagged fusion protein, use affinity column chromatography and polishing steps

purified protein

Figure 1.3.1 Purification scheme for soluble recombinant proteins, which may be excreted or located in the periplasm, in the membrane fraction, or most commonly the cytoplasm. The first step is to obtain an extract containing the desired protein in soluble form. After this, conventional purification steps may be carried out, or affinity purification of tagged fused proteins can be performed.

Protein Purification Flow Charts

make and unmake disulfides by exchange reactions, has been found to be beneficial in many cases. If the native protein is of intracellular origin, it probably will not contain disulfides; it will, however, contain cysteines, so a full reducing potential should be maintained. Specific methodology is discussed in UNIT 6.5. Not all proteins can fold unassisted by other cellular components. Chaperonins are proteins whose role is to refold denatured proteins such as those that form during heat shock (Zeilstra-

Ryalls et al., 1991). The most studied, and just becoming commercially available as of 1995, are the E. coli chaperonins GroEL and GroES, both of which are needed, together with ATP, to renature many proteins. Proline residues can adopt two isomeric conformations in proteins, and the wrong conformation is switched to the correct one by the enzyme prolyl isomerase, aiding the process of protein folding. At present these are not large-scale prospects, both because of the cost of the chap-

1.3.2
Current Protocols in Protein Science

cells containing protein

disrupt cells
perform differential centrifugation

wash inclusion bodies


dissolve in denaturing agents

dilute with buffer or dialyze to dilute denaturing agents add appropriate reducing agents and/or folding factors concentrate
perform purification procedure: remove unwanted proteins and incorrectly folded species (e.g., by ion-exchange chromatography, immunoaffinity methods, gel filtration)

purified protein

Figure 1.3.2 Purification scheme for insoluble recombinant proteins that are produced as inclusion bodies in the cytoplasm of host cells. The cells must be broken open, and then the insoluble inclusion bodies are separated by differential centrifugation. Solubilization is achieved by the use of denaturing solvents, and renaturation of the dissolved protein occurs on removal of the denaturant. Further polishing steps will be needed to remove small amounts of contaminating proteins as well as incorrectly folded species. Additional information can be found in UNIT 6.3-6.5.

eronins and because the agents operate best in vitro at very low protein concentrations. Once the proteins are folded, the purification process consists of removing small amounts of still incorrectly folded protein plus any other host proteins that were trapped with the original inclusion bodies. The former may be difficult, since incorrectly folded species have a size and charge similar to those of the correct product. However, subtle differences arising from the folded conformation can be exploited by chromatographic techniques. In ideal cases immunoaffinity techniques using antibodies specific for either the incorrectly folded form or the correct one can be used to resolve the mixture.

SOLUBLE NONRECOMBINANT PROTEINS


There are so many sources of soluble proteins that it is not possible to give a complete overview of methods used to obtain starting extracts from which a desired protein can be isolated. The sources can be classified as either microorganisms, plants, or animals, as shown in Figure 1.3.3, but these in turn should be subdivided according to how the starting extract is obtained. In particular there is a distinction between extracellular and intracellular proteins. With the latter it is necessary to disrupt the cells and release the proteins, whereas with the former, if the extracellular fluid can be obtained directly, there need be no contamination with intracellular proteins. Extracellular

Strategies of Protein Purification and Characterization

1.3.3
Current Protocols in Protein Science

microorganisms

plants

animals

disrupt cells, e.g., using French press, bead mill, or sonication with 5-10 vol buffer

disrupt cells, e.g., using French press or by grinding with sand using 1-2 vol buffer

homogenize with 2-5 vol buffer or mince and stir with buffer

treat to remove nucleic acids

treat to remove phenols

centrifuge
extracellular proteins

starting extract (5-20 mg/ml protein)

perform initial fractionation: use salt fractionation, precipitation with organic solvents, affinity methods, and/or two-phase partitioning perform secondary fractionation: use ion-exchange chromatography, hydrophobic chromatography, affinity methods, other adsorbents, and/or gel filtration perform polishing step: use HPLC reversed phase, HPLC ion exchange, or isoelectric focusing

purified protein

Figure 1.3.3 Purification scheme for soluble proteins present in their natural host cells. Cells must be disrupted to release the proteins, usually in the presence of 2 to 10 ml of a suitable buffer per gram weight. After removal of insoluble material, the process will generally require several steps, using various standard fractionation procedures in a suitable order. For production of highly pure protein, a final polishing step may be required to remove final trace contaminants. Additional information can be found in UNIT 6.2.

Protein Purification Flow Charts

sources include microorganism culture medium, plant and animal tissue culture medium, venoms, milk, blood, and cerebrospinal fluids. Soluble proteins may also occur within organelles such as mitochondria; these may be best obtained by first isolating the organelle, then disrupting it to release the contents. The starting extract normally contains be-

tween 5 and 20 mg protein per milliliter, though lesser concentrations can be dealt with, especially if working on a small scale. It may be necessary to include a concentration step before starting the purification process in order to approach that level. There are exceptions to every rule, however, and very high protein concentrations can be handled, for example,

1.3.4
Current Protocols in Protein Science

whole tissue
homogenize without detergent; centrifuge disrupt cells; gently isolate organelles

supernatant

residue
homogenize with detergent; centrifuge

supernatant containing membrane-associated proteins


perform primary fractionation: use hydrophobic chromatography, precipitation with organic solvents, ion-exchange chromatography, and/or phase partitioning
perform secondary fractionation: use ion-exchange chromatography, affinity methods, and/or gel filtration

residue containing insoluble proteins

suspend and extract with other solvents (e.g., ethanol, urea, or SDS)

purified protein

insoluble residue

perform polishing step: use HPLC or isoelectric focusing

purified protein

Figure 1.3.4 Purification scheme for membrane-associated and poorly soluble proteins (nonrecombinant). An initial purification can be achieved by isolation of organelles containing the desired protein. Membrane proteins are normally solubilized with a nonionic detergent, although loosely associated proteins may be extracted without detergent at high pH, with EDTA, or with small amounts of an organic solvent such as N-butanol. Normal fractionation procedures may need some modification if the detergent is required throughout to maintain the integrity of the protein.

with two-phase partitioning (Walter and Johansson, 1994). When isolating proteins on a large scale, the volumes being manipulated become of increasing concern, so maximizing protein concentration can be an important aim. The starting extract should be clarified, usually by centrifugation; on a large scale, ultrafiltration methods are becoming more widely used. Pretreatment of certain extracts to remove ex-

cessive amounts of nucleic acids, phenolics, and lipids may be necessary in order to obtain an extract that is amenable to standard fractionation procedures. Fractionation procedures can somewhat arbitrarily be divided into three steps: initial fractionation, secondary fractionation, and polishing. Initial processing, which deals with a large amount of extract that is not all protein, may

Strategies of Protein Purification and Characterization

1.3.5
Current Protocols in Protein Science

involve materials that become soiled and unable to be used many times. Consequently, preferred methods do not require expensive reagents or adsorbents such as are used in highperformance liquid chromatography (HPLC). Classic salt fractionation and the less-used organic solvent fractionation can achieve, if not a high degree of purification, a useful level of concentration and removal of much unwanted nonproteinaceous material. Alternatively, a highly selective affinity procedure may be used as the first step, but only if the affinity material is inexpensive to make and/or the extract is a simple, clear solution, as opposed to a turbid whole-cell homogenate. Secondary processing achieves the main purification, and may involve two or more steps in difficult situations. Ion-exchange and hydrophobic-interaction chromatography, gel filtration, and affinity techniques (see Chapter 8 and Chapter 9) are the main procedures. Finally, it may be necessary to remove traces of contaminants by polishing, using high-resolution procedures such as reversed-phase HPLC (UNIT 11.6) and isoelectric focusing (IEF; UNIT 10.2 & UNIT 10.4). Because every protein has unique characteristics, it is impossible to make general statements about procedures to be followed.

MEMBRANE-ASSOCIATED AND INSOLUBLE NONRECOMBINANT PROTEINS


Proteins that are not physiologically soluble can be purified after extracting and removing soluble proteins, thereby achieving a substantial degree of purification at the extraction step (Fig. 1.3.4; also see UNITS 6.1 & 6.2). To carry out a purification it is nearly always necessary to obtain the desired protein in a soluble form, which will often require the addition of solubilizing agents such as detergents. Some proteins remain insoluble even with detergent treatment, and so can be substantially purified by removing the soluble fractions. Some membrane-associated proteins become partly solubilized during breaking up of the tissue, and recovery in the particulate fraction may be poor. In such cases it may be best to solubilize the whole tissue by including detergent in the homogenizing buffer. Extraction of insoluble residues using detergents can be done differentially; some proteins are released at low detergent concentration, whereas others require complete solubilization of the membrane fraction. Suitable detergents include nonionic (e.g., Triton) and weakly acidic types (e.g., cholic acid deriva-

tives). Strongly acidic detergents such as sulfate esters (e.g., sodium dodecyl sulfate) usually result in denaturation. Detergents can be removed either by adsorption of the protein on a column, and subsequent elution without detergent, by use of special detergent-adsorbing beads, or even by extraction with nonmiscible organic solvents in which the detergent partitions. On the other hand, many membrane proteins require the presence of detergent at all times in order to remain in solution and in a native conformation. These include most integral membrane proteins, for example, cytochrome: P450, transmembrane receptors, and transporters. The most sensitive proteins require a particular combination of natural lipids (in addition to the detergent) to maintain structural integrity. Purification methods include most of those used for soluble proteins, but some techniques are not recommended if detergent is needed at all times. For instance, ammonium sulfate precipitation will often cause a detergent-protein complex to come out of solution and float rather than sink on centrifugationthis can be useful, but the floatate, when redissolved, may have a high detergent content. Hydrophobic chromatography can be very useful, as membrane proteins are naturally hydrophobic. Integral membrane proteins that are completely insoluble in normal detergents may be solubilized by denaturation using compounds such as sodium dodecyl sulfate and guanidine hydrochloride. Some cross-linked proteins such as elastin are not soluble without disruption of the covalent linkages.

Literature Cited
Walter, H. and Johansson, G. (eds.) 1994. Aqueous two-phase systems. Methods Enzymol. 228:1725. Zeilstra-Ryalls, J., Fayet, O., and Georgopoulos, C. 1991. The universally-conserved GroE (Hsp60) chaperonins. Annu. Rev. Microbiol. 45:301-325.

Key References
Deutscher, M.P. (ed.) 1990. Guide to protein purification. Methods Enzymol. 182:1-894. Extensive collection of purification methods, with some general protocols and examples. Janson, J.-C. and Ryden, L.G. 1989. Protein Purification: Principles, High Resolution Methods, and Applications. VCH Publishers, New York. A useful collection of methods and examples.

Protein Purification Flow Charts

1.3.6
Current Protocols in Protein Science

Kennedy, J.F. and Cabral, J.M. (eds.) 1993. Recovery Processes for Biological Materials. John Wiley & Sons, New York. A useful introduction to the problems of large-scale methods. Kenny, A. and Fowell, S. (eds.) 1992. Practical protein chromatography. Methods Mol. Biol. 11:1-327. Extensive descriptions of affinity chromatographic techniques with protocols and recipes.

Scopes, R.K. 1993. Protein Purification, Principles and Practice, 3rd ed. Springer-Verlag, New York and Heidelberg. General principles of all the main techniques used in purifying proteins. A useful laboratory handbook; does not include recipes or procedures for specific proteins.

Contributed by R. K. Scopes La Trobe University Bundoora, Australia

Strategies of Protein Purification and Characterization

1.3.7
Current Protocols in Protein Science

Purification of Glutamate Dehydrogenase from Liver and Brain


Because of the wide variety of procedures that may be used to purify any given enzyme, it is not possible to provide a single protocol that includes them all. In this unit two protocols for the purification of glutamate dehydrogenase (GDH)also referred to as + L-glutamate-NAD(P) oxidoreductase (deaminating; EC 1.4.1.3:GDH)have been selected as examples because they present two very different approaches to the purification of the enzyme. In the first (see Basic Protocol 1), affinity chromatography using a column consisting of the allosteric inhibitor guanosine 5-triphosphate (GTP) bound to Sepharose is described. Support protocols detail the preparation of the GTP-Sepharose needed for this procedure (see Support Protocol 1), assessment of incorporation of GTP into the GTP-Sepharose gel, if necessary (see Support Protocol 2), the activation of Sepharose 4B with CNBr, if necessary (see Support Protocol 3), and reuse and equilibration of the chromatography media (see Support Protocols 4 and 5). An alternative purification procedure (see Alternate Protocol) uses a bifunctional ligand composed of two NAD+ molecules linked together by a spacer arm to precipitate the enzyme. Protocols for the synthesis of the bis-NAD compound (see Support Protocol 7), resolution of NAD+ derivatives (see Support Protocol 8), and instructions for a pilot affinity study (see Support Protocol 9) complement this alternative approach. Of value to both studies are methods for concentrating the protein product (see Support Protocol 6), determining the protein concentration (see Basic Protocol 2), and assaying for GDH activity (see Basic Protocol 3). Although some of these techniques can be found elsewhere in this manual, instructions are included in this unit that are specific to the systems mentioned here. As discussed below, some tissues, including the brain but not the liver, contain an additional isoenzyme of glutamate dehydrogenase (GLUD2) as a relatively minor component. The procedures described here are for purifying the major form (GLUD1). Since this unit is concerned with the purification of mammalian glutamate dehydrogenase, a detailed review of its properties and functions is not appropriate. However, some of the essential properties are briefly summarized below. Further details can be found in the references cited. References to earlier work on the enzyme can be found in Tipton and Coue (1988) and are not repeated here. GDH catalyzes the reversible oxidative deamination of glutamate to 2-oxoglutarate (-ketoglutarate). Either NAD+ or NADP+ can serve as cofactor, although the kinetic and regulatory behavior depend on which is used. The native enzyme is a hexamer of six identical subunits, each consisting of 505 amino acids, with approximate relative molecular mass of 56,700. However, at high protein concentrations, the hexamers may aggregate further to form high-molecular-weight polymers. The crystal structure of the enzyme has been recently determined (Peterson and Smith, 1999; Smith et al., 2001). The enzyme activity is allosterically modulated by a range of metabolites, including adenosine, guanosine, and inosine di- and trinucleotides, as well as L-leucine (see Coue and Tipton 1989a). The exact behavior of these compounds depends on the conditions under which activity is measured and whether NAD+ or NADP+ is used as cofactor. The kinetic behavior in the direction of glutamate oxidation is complex. Substrate inhibition by high concentrations of 2-oxoglutarate occurs in the reaction leading to NADPH oxidation, but this is not apparent in the reaction pathway leading to NADH oxidation. For that reason, activity determinations are usually performed in the direction of 2-oxoglutarate amination with NADH as the cofactor. Phospholipids, such as cardiolipin, also
Contributed by Martha Motherway, Keith F. Tipton, Alun D. McCarthy, Ivan Coue, and Jane Irwin
Current Protocols in Protein Science (2002) 1.4.1-1.4.34 Copyright 2002 by John Wiley & Sons, Inc.

UNIT 1.4

Strategies of Protein Purification and Characterization

1.4.1
Supplement 29

affect enzyme activity (Coue and Tipton, 1989b) and the enzyme is also inhibited by antipsychotic drugs such as perphenazine (Coue and Tipton, 1990a,b; Yoon et al., 2001). GDH is localized in the mitochondrial matrix, although small amounts of the activity have been detected associated with microtubules (Rajas and Rousset, 1993) and endoplasmic reticulum (Lee et al., 1999). A nuclear-associated activity has also been reported (McDaniel, 1995) and the enzyme has been found to be capable of functioning as an RNA-binding protein (Preiss et al., 1993). In addition to the ubiquitously distributed GDH isoenzyme (GLUD1), which is encoded by a gene on chromosome 10, brain, retina, and testis tissue also contain a second form of the enzyme (GLUD2), encoded on the X chromosome (see Shashidharan et al., 1994). This isoenzyme differs in its kinetic and regulatory properties from the major form of GDH (see e.g., Cho et al., 1995; Shashidharan et al., 1997). The possible functions of these two GDH isoenzymes in brain have been discussed by Plaitakis and Zaganas (2001). NOTE: Unless otherwise stated, all required chemicals can be obtained from SigmaAldrich or BDH. They should be of the highest purity available.
BASIC PROTOCOL 1

PURIFICATION OF OX BRAIN OR LIVER GLUTAMATE DEHYDROGENASE BY AFFINITY CHROMATOGRAPHY ON GTP-SEPHAROSE The initial steps in this protocol involve the classical procedures of salt precipitation and ion-exchange chromatography. These are then followed by affinity chromatography on GTP Sepharose. GTP is an allosteric inhibitor of glutamate dehydrogenase when NAD+ is used as the substrate (see McCarthy and Tipton, 1985; Coue and Tipton, 1989a). The procedure described here is that of McCarthy et al. (1980). The steps involved are summarized in Figure 1.4.1. Materials Fresh ox (beef) brain (400 g) or liver (50 g) from slaughterhouse 0.32 M sucrose (optional; if tissue samples are to be stored before use) Homogenization buffer (see recipe), 4C Ammonium sulfate [(NH4)2SO4] 20 mM and 200 mM sodium phosphate buffer, pH 7.4 (APPENDIX 2E), 4C Affinity chromatography buffer (see recipe) 400 mM KCl in affinity chromatography buffer (see recipe for buffer) Glycerol Scissors Waring blender or kitchen liquidizer Refrigerated centrifuge (e.g., Sorvall RC-5C) Dounce (hand-held) homogenizer with loosely-fitting pestle (clearance, 0.03 cm) 250 ml or 500 ml polycarbonate centrifuge tubes Dialysis tubing, 12,000 molecular weight cut-off (MWCO) Conductivity meter Gradient mixer (e.g., UNIT 8.2) Chromatography columns (see Support Protocol 5 for equilibration; column lengths may be varied somewhat according to availability): 40 3cm packed to height of 30 cm with DEAE-cellulose (DE-52, Whatman) and equilibrated with 20 mM sodium phosphate buffer, pH 7.4 (see APPENDIX 2E for buffer) 30 2.5cm packed to height of 22 cm with Sephadex G-25 (Pharmacia Biotech) and equilibrated with affinity chromatography buffer (see recipe for buffer)

Purification of Glutamate Dehydrogenase from Liver and Brain

1.4.2
Supplement 29 Current Protocols in Protein Science

ox brain (400 g)

ox liver (50 g)

homogenize for 1 min in 3 volumes (w/v) of 4 mM sodium phosphate buffer, pH 7.4, containing 0.5mM EDTA and 0.1 mM PMSF

homogenize for 1 min in 500 ml of 4 mM sodium phosphate buffer, pH 7.4, containing 0.5 mM EDTA and 0.1 mM PMSF

stage 1 (homogenate)
add solid (NH4)2 SO4 (113 g/liter), stir for 20 min

stage 2 (NH4)2 SO4 supernatant

centrifuge discard pellet


add solid (NH4)2 SO4 (188 g/liter), stir for 20 min

stage 3 (dialyzed pellet)

centrifuge discard supernatant dialyse against 20 mM sodium phosphate buffer, pH 7.4 and apply to DEAE-cellulose column elute with 20 to 200 mM sodium phosphate, pH 7.4, gradient
gel-filter into affinity chromatography buffer; pass through hydrazide-Sepharose; and apply to GTP Sepharose column

stage 4 (DEAE eluate)

stage 5 (GTP-Sepharose eluate)

elute with 0 to 15 mM KCl gradient in affinity-chromatography buffer concentrate by ultrafiltration dialyze against 20 mM sodium phosphate buffer, pH 7.4
purified glutamate dehydrogenase

Figure 1.4.1 Flow chart for the purification of ox liver and brain glutamate dehydrogenase according to the Basic Protocol 1. Full details of the steps involved are given in the text.

6 2cm packed to height of 4 cm with 10 ml of hydrazide-Sepharose 4B and equilibrated with affinity chromatography buffer (see Support Protocol 1, steps 1 to 3 for resin; see recipe for buffer) 6 2cm packed to height of 4 cm with GTP-Sepharose 4B and equilibrated with affinity chromatography buffer (see Support Protocol 1, steps 1 to 7 for resin; see recipe for buffer)

Strategies of Protein Purification and Characterization

1.4.3
Current Protocols in Protein Science Supplement 29

Additional reagents and equipment for dialysis (UNIT 4.4 and APPENDIX 3B), determining protein concentration (see Basic Protocol 2 and UNIT 3.4), assaying GDH activity (see Basic Protocol 3), and concentrating protein solutions by ultrafiltration (see Support Protocol 6) NOTE: Unless indicated otherwise, all steps are performed at 0 to 4C in a cold room or refrigerated cabinet. Prepare and homogenize the tissue 1. Obtain one ox brain (400 g) or liver (50 g) from a freshly slaughtered animal transported to the laboratory on ice to be used as the starting material for the purification.
The brain may be used fresh or stored at 70C, using the procedure as follows: Place the fresh tissue in ice-cold 0.32 M sucrose solution and keep it in a 20C freezer until it has frozen. Transfer to a 70C freezer until required. Before use place the vessel containing the frozen material in a water bath at 37C and allow it to thaw, with occasional agitation. The larger amount of enzyme in ox liver permits the use of a smaller amount of starting material (e.g., 50 g in the example purification shown here) to be used.

2a. For brain tissue: Divide the brain tissue into three equal portions, weigh, and suspend each portion in 3 volumes (w/v) of cold homogenization buffer. 2b. For liver tissue: Weigh the liver tissue and suspend in 10 volumes (w/v) of cold homogenization buffer. 3. Chop the tissue into smaller pieces using scissors, then homogenize in a Waring blender at full speed for 1 min. Retain a small aliquot of this homogenate for determining protein concentration by the biuret procedure (see Basic Protocol 2) and assaying GDH activity (see Basic Protocol 3).
A kitchen liquidizer can be used instead of a Waring blender. However, extra care is then needed with any biohazard material (see Critical Parameters) since kitchen liquidizers can release aerosols. These are generally less powerful than the Waring blender and it is thus necessary to increase the homogenization time (e.g., 1.5 min using a Kenwood liquidizer at full speed). The volumes given in the subsequent steps refer to the preparation of GDH from brain. The corresponding values for liver are given in Table 1.4.6 (see Critical Parameters and Troubleshooting).

Precipitate with ammonium sulfate 4. Add solid (NH4)2SO4 slowly in four approximately equal amounts to the homogenates while they are still in the blender, homogenizing for 1 min between each addition, to yield a final 20% saturated solution (113 g/liter). Continue homogenization for another 30 sec at full speed after the final addition. For brain tissue, repeat this procedure with the other two tissue homogenates. 5. Combine the ammonium sulfatetreated homogenates in a beaker on ice and stir the mixture for 20 to 30 min. Centrifuge 30 min at 14,000 g, 4C. Carefully decant the supernatants and discard the pellets. Retain a small aliquot of this material for determining protein concentration by the biuret procedure (Basic Protocol 2) and assaying GDH activity (Basic Protocol 3).
Purification of Glutamate Dehydrogenase from Liver and Brain

6. Add 188 g/liter of solid (NH4)2SO4 slowly with continuous stirring. Stir the mixture for an additional 20 to 30 min, then centrifuge again as described in step 5.
This results in 50% saturation with ammonium sulfate.

1.4.4
Supplement 29 Current Protocols in Protein Science

7. Decant and discard the supernatants. Resuspend the pellets in 20 mM sodium phosphate buffer, pH 7.4, using a Dounce homogenizer, to yield a total volume of 200 to 250 ml (160 ml for liver). Retain a small aliquot of this material for determining protein concentration by the biuret procedure (Basic Protocol 2) and assaying GDH activity (Basic Protocol 3).
The ammonium sulfate pellet can be resuspended by careful stirring if a Dounce homogenizer is not available.

8. Dialyze (UNIT 4.4 and APPENDIX 3B) for 18 hr against 20 volumes (v/v) of cold 20 mM sodium phosphate buffer, pH 7.4, with at least two changes of buffer, using an MWCO 12,000 dialysis membrane. 9. Centrifuge the material from the dialysis bag 30 min at 40,000 g, 4C, and retain the supernatants. Resuspend the pellets in the 20 mM sodium phosphate buffer, pH 7.4, and centrifuge again for 30 min at 40,000 g, 4C. Combine the supernatants with those from the first centrifugation and discard the pellets. Retain a small aliquot of this material for determining protein concentration by the biuret procedure (Basic Protocol 2) and assaying GDH activity (Basic Protocol 3).
Extraction by resuspension and recentrifugation of the pellet after dialysis and centrifugation increases the yield of enzyme, since otherwise some glutamate dehydrogenase activity is lost in the pellet. As is common with salt precipitation in enzyme purification, this step does not give a high degree of purification of glutamate dehydrogenase. However, it is an effective way of reducing the volume of material for the subsequent steps, and it also removes some contaminants.

Perform chromatography on DEAE-cellulose 10. Pour the supernatant carefully onto the top surface of a 40 3cm chromatography column packed to 30 cm with DEAE-cellulose, equilibrated with 20 mM sodium phosphate buffer, pH 7.4 (Support Protocol 5). After the protein solution has run into the column, continue to pass buffer through it and determine the absorbance of aliquots of the effluent at 280 nm (A280). Continue washing the column until the A280 of the effluent is <0.1. 11. Pass a 2-liter linear gradient from 20 to 200 mM sodium phosphate buffer, pH 7.4, through the column. Collect fractions (10 ml) and determine their A280 (see Basic Protocol 2), conductivity, and enzyme activities (see Basic Protocol 3). Pool the fractions containing glutamate dehydrogenase activity. Retain a small aliquot of this pooled material for determining protein concentration by the biuret procedure (Basic Protocol 2) and assaying GDH activity (Basic Protocol 3).
The combined active fractions require concentration before the next step; this can be done either by ultrafiltration or ammonium sulfate precipitation. The authors have used both of these procedures and they work equally well.

12a. Concentrate protein by ultrafiltration: Concentrate the combined fractions containing GDH activity in an Amicon ultrafiltration cell using either an Ultracel Amicon YM 30 or Ultracel PLTK membrane, as described in Support Protocol 6, until the volume remaining in the cell is reduced to 20 ml. 12b. Concentrate protein by ammonium sulfate precipitation: Add 312 g/liter solid (NH4)2SO4 slowly, over a period of 30 min with continuous stirring (this brings the saturation to 50%). Continue stirring for a further 20 min, then centrifuge 20 min at 14,000 g, 4C. Discard the supernatant and resuspend the pellet in 20 ml of 20 mM sodium phosphate buffer, pH 7.4.

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Perform chromatography on GTP-Sepharose 13. Desalt the sample by passing the concentrated enzyme solution through a 30 2.5cm chromatography column packed to 22 cm with Sephadex G-25, equilibrated with affinity chromatography buffer.
The next chromatographic steps should be performed as quickly as possible, since the enzyme is slightly unstable in the affinity chromatography buffer.

14. Apply the enzyme solution to a 6 2cm chromatography column packed to 4 cm with hydrazide-Sepharose 4B equilibrated with affinity chromatography buffer. Elute by passing an additional 50 ml of affinity chromatography buffer through the column.
Glutamate dehydrogenase activity is not appreciably retarded by this material, and essentially complete recovery of the applied activity is obtained when the applied volume plus an additional 50 ml of buffer has been passed through the column. This procedure removes any components which bind nonspecifically to the Sepharose or to the spacer arm.

15. Apply the enzyme solution eluted from the above column to a 6 2cm chromatography column packed to 4 cm with GTP-Sepharose 4B, equilibrated with affinity chromatography buffer. Use a flow rate of not more than 1 ml/min. Pass affinity chromatography buffer through the column until the A280 of the effluent has fallen to <0.01 (150 ml of buffer should be required).
Once the enzyme solution has passed into the column the flow rate may be increased to 1.5 ml/min, and this flow rate should be used for step 16.

16. Elute the enzyme activity with a linear 400-ml gradient from 0 to 400 mM KCl in affinity chromatography buffer. Collect fractions (10 ml) and monitor the A280 (see Basic Protocol 2), conductivity, and enzyme activity (see Basic Protocol 3) in each. 17. Combine fractions containing glutamate dehydrogenase activity and concentrate by ultrafiltration, followed by osmotic dehydration, as described in Support Protocol 6, to yield a protein concentration of at least 3 mg/ml. 18. Dialyze (UNIT 4.4 and APPENDIX 3B) overnight against 3 liters of 20 mM sodium phosphate buffer, pH 7.4, using MWCO 12,000 dialysis membrane. Add glycerol to the dialyzed enzyme solution to yield a final concentration of 30% (v/v) and store in a sealed container, which has been flushed with N2, at 4C.
There may be some small variations between different preparations of the GTP-Sepharose in the sharpness of the peak of activity that emerges and in the salt concentration necessary to elute the enzyme. This may reflect varying concentrations of bound ligand, but does not affect the degree of purification obtained. SUPPORT PROTOCOL 1

PREPARATION OF GTP-SEPHAROSE Although GTP-agarose is available from Sigma, the preparation described in this protocol generally yields more satisfactory and reproducible results than commercial preparations. The steps involved are outlined in Figure 1.4.2. GTP is coupled to Sepharose 4B by the method of Jackson et al. (1973) as modified by Godinot et al. (1974), where L-glutamic acid -methyl ester is used as the spacer instead of -aminohexanoic acid methyl ester. After the spacer is coupled to the activated Sepharose and the ester group is treated with hydrazine (Godinot et al., 1974), a portion of the gel (10 ml) is retained and used as the hydrazide gel in the purification procedure.

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OH OH Sepharose CNBr

O C O

NH
CH2OPOPOP

HO
L-glutamic acid -methyl ester

HO GTP

O G

COOH

NHCHCH2 CH2COCH3 OH

NaIO4

H2NNH2 COOH O O O

CH2OPOPOP

NHCHCH2 CH2CNHNH2 OH hydrazine derivative

CH2OPOPOP

COOH

O O G

NHCHCH2 CH2CNHN OH GTP-Sepharose

Figure 1.4.2 The chemical processes involved in the synthesis of GTP-Sepharose according to the method described in Support Protocol 1. The activation of Sepharose by cyanogen bromide (Support Protocol 3) is also shown in simplified form; alternative activated products are formed in this step.

Materials Cyanogen bromideactivated Sepharose 4B (Pharmacia Biotech, or see Support Protocol 3). Sodium bicarbonate buffer: prepare 0.1 M NaHCO3 and adjust pH to 9.0 with NaOH L-glutamic acid -methyl ester Hydrazine hydrate Guanosine 5-triphosphate (GTP) 0.1 M disodium hydrogen phosphate adjusted to pH 5.0 with 1 M citric acid Sodium periodate Ethylene glycol

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Nitrogen source 50 mM sodium phosphate buffer, pH 6.8 (APPENDIX 2E) containing 5 mM EDTA Butanol Sintered-glass filter funnel (Pyrex, porosity G2) 70C water bath Prepare the hydrazide gel 1. Wash 4.5 g of cyanogen bromideactivated Sepharose 4B in a sintered-glass filter funnel with ice-cold water, then wash with 100 ml ice-cold sodium bicarbonate buffer. Finally, suspend the resin in sodium bicarbonate buffer to a volume of 16 ml.
The cyanogen bromideactivated Sepharose 4B contains dextran and lactose as stabilizers. It is necessary to remove these by washing. It is possible to activate Sepharose with cyanogen bromide in the laboratory as described in Support Protocol 3. This is a less expensive alternative, but cyanogen bromide is toxic and should be handled with great care.

2. Dissolve 1.12 g of L-glutamic acid -methyl ester in 4 ml sodium bicarbonate buffer. Add this solution to the 16 ml of the gel suspension and stir this mixture for 16 to 18 hr at 4C. Finally, filter the mixture and wash it with cold water in a sintered-glass filter funnel before suspending it in water to a volume of 30 ml.
The following steps should be performed in a fume hood.

3. Add 28 ml of hydrazine hydrate (99% to 100%) slowly to 30 ml of the suspended gel and heat the reaction mixture to 70C in a water bath for 15 min. Allow the mixture to cool and then filter and wash with cold water on a sintered-glass filter.
This material, the hydrazide gel, may be overlaid with butanol to impede bacterial growth, and stored at 4C. Retain a 10-ml portion of this hydrazide gel for use in the purification procedure. After use, the hydrazide gel can be recycled as described in Support Protocol 3.

Prepare GTP-Sepharose 4. Dissolve 14.2 mg of GTP in 5 ml of 0.1 M disodium hydrogen phosphate that has been adjusted to pH 5.0 with 1 M citric acid. Dissolve 8.6 mg of sodium periodate in 0.4 ml of the same buffer. Mix these two solutions and incubate in the dark for 30 min at room temperature. Add 8 l of ethylene glycol to remove the excess periodate, and incubate the mixture for a further 15 min. Bubble nitrogen through the solution for 5 to 10 min.
Bubbling with N2 is effected by connecting a Pasteur pipet to a nitrogen cylinder by plastic tubing. The Pasteur pipet is placed in the tube containing the reacted mixture and the nitrogen is allowed to bubble through slowly. This procedure removes any formaldehyde produced during the reaction, which would compete with the oxidized GTP for reaction with the hydrazide gel.

5. Dilute the mixture from step 4 (oxidized GTP solution) to 10 ml with 0.1 M disodium hydrogen phosphate that has been adjusted to pH 5.0 with 1 M citric acid. If necessary, remove the butanol layer from the hydrazide gel (from step 3) by aspiration and wash the gel with cold water, then suspend it in 20 ml of cold water. Mix 10 ml of this gel suspension with the oxidized GTP solution and stir the mixture at room temperature for 2 hr.
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6. Filter the gel on a sintered-glass funnel and wash with at least 100 ml of cold 50 mM sodium phosphate buffer, pH 6.8, containing 5 mM EDTA. Retain washings if step 8 is to be performed. Store the resultant GTP-Sepharose at 4C with an overlay of butanol to retard bacterial growth. Remove this butanol layer by aspiration and wash the gel with buffer prior to use of the gel.
After use, the GTP-Sepharose gel can be recycled as described in Support Protocol 4.

ASSESSMENT OF INCORPORATION OF GTP INTO THE HYDRAZIDE GEL The following modification to Support Protocol 1 may be applied if there are any problems using the GTP-Sepharose medium prepared in Support Protocol 1 in the affinity purification steps (see Basic Protocol 1, steps 13 to 16). It is generally not necessary to resort to this procedure. CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer (also see APPENDIX 2B). Additional Materials (also see Support Protocol 1) [8-14C]GTP (Amersham Pharmacia Biotech) 250:500:1 (v/v/w) Triton X-100/toluene/2,5-diphenyloxazole (PPO) Carry out Support Protocol 1 with the following modifications at the indicated steps. 4a. Add 2.7 Ci of [8-14C]GTP to the solution of unlabeled GTP before periodate oxidation. 7b. Determine radioactivity in a sample of the gel washings by scintillation counting in a mixture by scintillation counting in 250:500:1 Triton X-100/toluene/PPO.
About 35% of the added GTP should be found to be tightly bound to the gel, corresponding to 0.6 mol of GTP per ml of settled gel (see Table 1.4.1).

SUPPORT PROTOCOL 2

Table 1.4.1 Recovery of [8-14C]GTP After Coupling to Sepharose 4B

Aqueous phase 10 M of initial 14C-GTP solution + 1 ml H2O 1 ml gel washings (from 475 ml) 10 M of initial 14C-GTP solution + 1 ml gel washings
radioactivity determined in a liquid scintillation counter.

Radioactivity (cpm)a,b 14760 5167 20100

Total radioactivity (cpm)a,b 3.77 106 2.44 106

a9 ml of 250:500:1 (v/v/w) Triton X-100:toluene:PPO were mixed with 1 ml of the aqueous phase and the bSince the cpm obtained with the 10 l 14C-GTP solution + 1 ml gel washings equals the sum obtained for the

two solutions separately, there is no significant difference in the quenching for the two solutions and therefore it is unnecessary to convert the results to dpm.

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SUPPORT PROTOCOL 3

ACTIVATION OF SEPHAROSE 4B WITH CYANOGEN BROMIDE This protocol is given as an alternative to the purchase of preprepared cyanogen-bromideactivated Sepharose. Materials Sepharose 4B (Pharmacia Biotech) Cyanogen bromide Acetonitrile 5 M sodium hydroxide Sodium bicarbonate buffer: prepare 0.1 M NaHCO3 and adjust pH to 9.0 with NaOH CAUTION: Because cyanogen bromide is very toxic, it should be handled with extreme care. See APPENDIX 2A for general precautions. 1. Wash 10 ml Sepharose 4B with water and suspend in 40 ml water. 2. Stir this mixture slowly in an ice-bath in a fume-hood. Add 2.4 g cyanogen bromide dissolved in 1.7 ml acetonitrile and monitor the pH of the resultant mixture constantly, maintaining it at pH 11 by the addition of 5 M sodium hydroxide.
Use extreme caution as hydrocyanic acid may be formed in this step.

3. After hydrogen ion release has subsided (i.e., the pH ceases to fall), filter the gel on a sintered-glass funnel. Wash with at least 100 ml of ice-cold sodium bicarbonate and resuspend in 16 ml of that buffer.
SUPPORT PROTOCOL 4

REUSE OF CHROMATOGRAPHY MEDIA Hydrazide-Sepharose (prepared as in Support Protocol 1, steps 1 to 3) Wash the resins after use with 500 ml of 2 M KCl, followed by 1 liter of water. Store at 4C with an overlay of butanol, to retard bacterial growth. Remove the butanol layer by aspiration and equilibrate with affinity chromatography buffer (see recipe) immediately before use. GTP Sepharose (prepared as in Support Protocol 1, steps 1 to 7) Follow the same procedure as described for the hydrazide-Sepharose, above. Over a period of several months, the properties of the GTP-Sepharose change and the enzyme activity is eluted earlier in the gradient, probably as a result of instability of the bound GTP. The gel should therefore be discarded after 6 months. DEAE-Cellulose The manufacturers provide full instructions for recycling this material for reuse. After use, remove tightly bound material from the ion-exchange material by passing 2 column volumes of 2 M NaCl through the column. Equilibrate the column with the buffer to be used and then pass 1 column volume of the same buffer containing 0.02% sodium azide through the column. Before use, wash the column with azide-free buffer. If a small portion at the top of the column becomes discolored as a result of tightly bound material, that portion may be removed and replaced by fresh DEAE-cellulose. Removal of the top few centimeters from the column may also be effective if the flow rate through the column becomes excessively slow. After removing the discolored top resin, transfer the remaining resin from the column to a beaker. Add the appropriate amount of fresh

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DEAE-cellulose from which the fines have been removed and which has been equilibrated with 20 mM sodium phosphate buffer, pH 7.4 (see Support Protocol 5), and repack the column. If the characteristics of the DEAE-cellulose change after protracted use, it may be regenerated by the following procedure. 1. Empty the contents of the column into a beaker. Stir with 15 volumes of 0.5 M HCl for 30 min.
Stirring can be continued for up to, but no longer than, 2 hr if convenient.

2. Filter the DEAE-cellulose on a sintered-glass funnel and wash it with water until the pH of the effluent reaches 4. 3. Stir the DEAE-cellulose with 15 volumes of 0.5 N NaOH for 30 min. 4. Allow to settle, remove the supernatant by decantation, and repeat step 3. 5. Filter on a sintered glass funnel and wash with water until the pH of the effluent is close to neutrality. The DEAE-cellulose is then ready for re-equilibration and reuse, although it may be necessary to remove the fines (see Support Protocol 5). For longer-term storage, remove the DEAE-cellulose from the column and store it in a saturated solution of NaCl, in a tightly-sealed container. Sephadex G-25 Sephadex G-25 is supplied in dry form and must be swollen before use. Full instructions for swelling and recycling this material are provided in the literature from the manufacturers. EQUILIBRATION OF CHROMATOGRAPHIC MEDIA The chromatographic medium must never be allowed to dry. When using it in columns there should always be a layer of the appropriate buffer above the surface. Equilibration of DEAE-Cellulose DEAE-cellulose (Whatman DE-52) may contain small amounts of relatively small-sized particles (fines) which can slow down the rate of flow through a column if not removed. The following steps are appropriate for the DEAE-cellulose chromatography performed in Basic Protocol 1 and the Alternate Protocol. For the DEAE-cellulose chromatography performed in Support Protocol 7 (i.e., step 15 of that protocol), the procedure is the same as that described below except that 1 M NH4HCO3 may be used for removing the fines. A total of 10 column volumes of 1 M NH4HCO3 should then be used to equilibrate the column, which is then washed with 8 liters of water. Materials DEAE-cellulose (Whatman DE-52) 200 mM and 20 mM sodium phosphate buffer, pH 7.4 (APPENDIX 2E) Chromatography column; 40 3cm (length diameter) Conductivity meter (e.g., Linton, type WPA CMD80) 1. Suspend the DEAE-cellulose resin in 5 volumes of 200 mM sodium phosphate buffer, pH 7.4. Allow the mixture to settle and remove the buffer and fines by decantation. Repeat this process at least three times, after which the supernatant, upon settling, should be clear.
Strategies of Protein Purification and Characterization

SUPPORT PROTOCOL 5

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2. Pour a suspension of this material into a 40 3cm chromatography column to produce a final bed volume of 30 3 cm. Allow 20 mM sodium phosphate buffer, pH 7.4, to flow through the column until the effluent has the same pH and conductivity as the in-flowing buffer.
The column is now ready for use.

Equilibration of Dowex AG 1X2 Dowex AG 1X2 medium is used in Support Protocol 7. Suspend the Dowex AG 1X2 (200 to 400 mesh, Cl form) in 3 M HCl and pour into a column of 4 cm diameter to give a settled bed volume of 10 cm. Wash the column before use with 1 liter of 3 M HCl, followed by exhaustive washing with at least 20 liters of water, until the pH of the effluent reaches neutrality. Equilibration of Sephadex G-25 Pass at least 2.5 column volumes of the appropriate buffer through the packed column before use.
SUPPORT PROTOCOL 6

CONCENTRATION OF PROTEIN-CONTAINING SOLUTIONS Some of the purification procedures used in Basic Protocol 1 and the Alternate Protocol result in some degree of concentration. Precipitation with ammonium sulfate followed by resuspension of the pellet in a smaller volume, as well as ion-exchange chromatography, can also be effective concentration procedures. In contrast, however, dialysis and gel filtration tend to dilute samples. The procedures described below are specifically for concentration purposes. For additional information, see UNIT 4.4. It is important to note that in both of the procedures described below, inattention may result in the concentration proceeding until all the fluid has been removed. It is essential to monitor the progress of the procedure and stop when the desired volume has been reached, because attempts to recover enzyme activity from material that has been taken to dryness by either of these procedures will result in significant losses. Ultrafiltration The Amicon ultrafiltration cell uses pressure of nitrogen gas to force the solution through a membrane that retains molecules above a pre-determined size. Prepare the apparatus according to the manufacturers instructions using the ultrafiltration cell from Amicon either an Amicon Ultracel YM 30 or Ultracel PLTK membrane. Slowly stir the contents during the concentration process, which is carried out at 4C. Continue until the required volume remains in the cell. Earlier work used Amicon Diaflo XM-50 membranes for this procedure, but these are no longer readily available. The Ultracel Amicon YM 30 or Ultracel PLTK membranes are appropriate substitutes. Centriplus concentrators (Amicon) may be an alternative for concentrating the enzyme from volumes of less than 60 to 70 ml. They rely on centrifugation to force solvent through a membrane (e.g., Centriplus 50, MWCO 50 kDa). The sample containing enzyme is retained by the membrane. The authors have found this procedure to be successful with several other dehydrogenases.

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Osmotic Dehydration Although ultrafiltration is a highly effective procedure for concentrating relatively large volumes, it may be less effective and convenient with small volumes and high protein concentrations. Osmotic dialysis relies on the use of a high concentration of material outside a dialysis tube to draw water and small-molecule solutes from its contents by osmosis, leaving a more concentrated protein solution within the dialysis tubing. Either sucrose or polyethylene glycol (PEG; Mr = 15,000 to 20,000) may be used. However the purity of PEG can be variable and it is important to use preparations that are as pure as possible to avoid contamination of the enzyme solution from low-Mr impurities that may be inhibitory. Put the enzyme solution in a length of dialysis tubing (see APPENDIX 3B for preparation) that has been knotted at the lower end, and then tie a knot in the other end of the tubing. Surround the resulting tube with solid sucrose or PEG. This may be done by heaping the sucrose or PEG on a sheet of cooking foil, embedding the dialysis tubing in the sucrose, and then folding the foil to form a parcel around the sucrose or PEG-covered dialysis tube. Alternatively, the dialysis tubing may be buried in the material within a plastic box of appropriate dimensions. Leave at 4C until the solution in the dialysis bag has reached the required volume; this will take several hours. Rinse the outside of the dialysis tube with water or buffer to remove adhering sucrose or PEG. This also dampens the dialysis membrane, which becomes brittle during the concentration procedure. Whereas with ordinary dialysis it is necessary to leave an empty space in the tubing to allow for the osmotic volume increase, this need not be done for osmotic dehydration where a decrease in volume occurs. In Basic Protocol 1 and the Alternate Protocol, this concentration procedure is followed by dialysis. In order to avoid too great a degree of dilution occurring at this stage, reknot the dialysis tube to confine the enzyme solution in a smaller volume of the tubing. DETERMINATION OF PROTEIN CONCENTRATION The first two methods presented under this heading are the Biuret and Bio-Rad protein assays. A spectrophotometer and 1-cm light-path cuvettes (either glass or plastic) will be required for each of these determinations. In the case of the third method presented below, determination of protein concentration by absorbance at 280 nm (A280), the cuvettes must be quartz or some other UV-transmitting material. Other procedures for determining protein concentration may also be used, including the Lowry-Folin method, which is described in adequate detail in Dawson et al. (1989). UNIT 3.4 also provides additional information on determination of protein concentration. Biuret Protein Assay Materials Biuret reagent (see recipe). 1.5% (w/v) sodium deoxycholate (1.5 g sodium deoxycholate in 100 ml distilled H2O) 50 mg/ml bovine serum albumin (BSA), or alternative protein calibration standard 1. Prepare a series of standards in triplicate according to the scheme described in Table 1.4.2.
Strategies of Protein Purification and Characterization

BASIC PROTOCOL 2

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Table 1.4.2

Preparation of Standard Curve for Biuret Protein Assay

mg protein 0 0.25 0.5 0.75 1.0 1.5 2.0

50 mg/ml BSA (l) 0 5 10 15 20 30 40

1.5% (w/v) sodium deoxycholate (l) 100 100 100 100 100 100 100

H2O (l) 900 895 890 885 880 870 860

Biuret reagenta (ml) 4 4 4 4 4 4 4

aSee recipe in Reagents and Solutions.

2. For enzyme protein determination use 20 to 100 l of appropriately diluted sample and prepare as described in Table 1.4.2. 3. Mix each sample and allow the reaction to proceed at room temperature for 30 min. 4. Measure absorbance at 540 nm using the samples containing no protein as blanks. 5. Prepare a standard curve by plotting absorbance at 540 nm against the protein concentration. 6. Use the standard curve to determine the protein concentration of the enzyme sample from its absorbance. Determine the absorbance of the sample against a blank containing the same medium. Dilute, if necessary, to ensure that the absorbance is <1.0. Bio-Rad Protein Assay Materials Bio-Rad Dye Reagent Concentrate 0.1 mg/ml bovine serum albumin (BSA), or alternative protein calibration standard (this can be conveniently prepared by diluting some of the stock solution prepared for the biuret assay; see above) Method 1. Prepare a series of standards in triplicate according to the scheme described in Table 1.4.3. 2. For enzyme protein determination use 20 to 100 l of appropriately diluted sample and prepare as described in Table 1.4.3.
Table 1.4.3 Preparation of Standard Curve for Bio-Rad Protein Assay

g protein 0 2 5 10 15 20

0.1 mg/ml BSA (l) 0 20 50 100 150 200

H2O (l) 800 780 750 700 650 600

Bio-Rad Dye Reagent Concentrate (l) 200 200 200 200 200 200

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3. After mixing, leave for a period of 5 to 60 min, then determine the absorbance at 595 nm using the samples containing no protein as blanks. 4. Prepare a standard curve by plotting absorbance at 595 nm against the protein concentration. 5. Use the standard curve to determine the protein concentration of the enzyme sample from its absorbance. Determine the absorbance of the sample against a blank containing the same medium. Dilute, if necessary, to ensure that the absorbance is <1.0.
Note that the Bio-Rad assay involves determination of the absorbance of 1 ml of mixture. The optical arrangements of some spectrophotometers may not permit this in standard 1-cm2 cross-section cuvettes. In such cases microcuvettes should be used. However, some older spectrophotometers may not focus the light beam to pass through the narrower path of a microcuvette. It is a good idea to check the instructions supplied with the spectrophotometer in regard to this. If all else fails, the volumes shown for this procedure can all be increased proportionally.

Absorbance at 280 nm Using quartz or other non-UV-absorbing cuvettes, determine the absorbance at 280 nm of the sample against a blank composed of the medium in which the sample is dissolved. A UV-Vis spectrophotometer is required. Use the rough approximation that a 1 mg/ml protein solution has an absorbance of 1.0 at this wavelength. ASSAY OF GLUTAMATE DEHYDROGENASE ACTIVITY Glutamate dehydrogenase catalyzes the reaction:
2-oxoglutarate + NH 3 + NADH + H + U L-glutamate + H 2 O + NAD +
BASIC PROTOCOL 3

The assay described here determines the decrease of absorbance at 340 nm as NADH is oxidized to NAD+ concomitant with the reductive amination of 2-oxoglutarate to L-glutamate. Materials Enzyme preparation (see appropriate steps of Basic Protocol 1 or Alternate Protocol) 0.4% (v/v) Triton X-100 in 50 mM sodium phosphate buffer, pH 7.4 100 mM potassium cyanide (KCN) 50 mM sodium phosphate buffer, pH 7.4 (APPENDIX 2E) 2.5 M ammonium sulfate in 50 mM sodium phosphate buffer, pH 7.4 4 mM NADH in 50 mM sodium phosphate buffer, pH 7.4 (prepared fresh on day of use) 0.25 M 2-oxoglutarate (-ketoglutarate), in 50 mM sodium phosphate buffer, pH 7.4 Spectrophotometer with cuvette holder set at 37C 1-cm path-length cuvettes (quartz or plastic with good light transmittance at 340 nm) NOTE: All GDH enzyme assays are performed at 37C. In order to ensure rapid temperature equilibration, the assay buffer should be prewarmed to that temperature.
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Other hints on the performance and validation of enzyme assays can be found in Tipton (2002). 1. For crude enzyme preparations (see Basic Protocol 1, step 1): Incubate 50 l of the enzyme solution with 2.68 ml of 0.4% Triton X-100 in 50 mM sodium phosphate buffer, pH 7.4, and 30 l of 100 mM KCN (which will give a final KCN concentration of 1 mM in the complete assay) for 2 min at 37C before proceeding to step 2.
For assaying the enzyme at later steps of the purification, begin with step 2. Triton X-100 serves to disrupt particulate material, and KCN decreases any contaminating NADH oxidase activity. It has been shown that neither Triton X-100 nor KCN affect the activity of purified GDH at these concentrations. These assays are performed in phosphate buffer. Do not attempt to perform them in Tris buffer since the enzyme is unstable in Tris and either no activity or a rapid decrease in activity may be observed.

2. Add reagents to the cuvettes in the order listed, in a final volume of 3 ml: 2.71 ml 50 mM sodium phosphate buffer, pH 7.4 60 l 2.5 M ammonium sulfate (final concentration 50 mM) 120 l 4 mM NADH (final concentration 160 M) 50 l enzyme preparation. 3. Cover the cuvette with Parafilm, mix the contents by inversion and incubate in the spectrophotometer for 3 min. Initiate the reaction by the addition of 60 l of 0.25 M 2-oxoglutarate (final concentration 5 mM), invert to mix and allow the reaction to proceed for 10 min in the spectrophotometer. 4. Determine the initial (linear) rate of decrease in absorbance as NADH is oxidized to NAD+, subtracting any blank rate occurring before the addition of the 2-oxoglutarate (see Critical Parameters).
The initial absorbance resulting from the NADH present will be 1.0, although impurities in the enzyme solution may add to this. The assay has been found to proceed in a linear fashion until the absorbance has decreased by 0.15 absorbance units. In all cases it will be necessary to dilute the enzyme preparation in order to obtain a rate that is sufficiently slow to measure with accuracy. See Critical Parameters for a detailed discussion of dilution considerations.

5. Calculate the specific activity.


Since the molar absorbance coefficient of NADH in a 1-cm path-length cuvette is 6220 M-1cm-1, the production of 1 mol of glutamate, or the reductive amination of 1 mol 2-oxoglutarate in an assay volume of 3.0 ml will give an absorbance change of 2.073. Thus, for a 1-cm path-length cuvette, the specific activity of the enzyme (mol product formed.per min per mg protein) will be related to the rate of decrease in absorbance per min (A/min), which will be given by:

specific activity =

A /min assay volume (ml) 6.22 protein (mg)

It is most common to express enzyme activities in these terms. 1 unit (U) of enzyme activity is defined as the amount catalyzing the production of 1 mol of product (or the disappearance of 1 mol of substrate) in 1 min under stated conditions.
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PURIFICATION OF RAT OR OX LIVER GDH INVOLVING AFFINITY PRECIPITATION WITH bis-NAD+ Affinity precipitation of enzymes was developed by Mosbach and co-workers (see Flygare et al., 1983; Larsson et al., 1984). The bifunctional ligand bis-NAD+ (N2, N2-adipodihydrazido-bis-(N6-carbonyl-methyl)NAD+) comprises two molecules of NAD+ joined by a spacer arm (see Fig. 1.4.5). This separates the NAD+ groups by a sufficient distance (1.7 nm) to allow them to bind to the coenzyme-binding sites of two different molecules of glutamate dehydrogenase. Mammalian GDH is a polymer, so extensive cross-linking of the enzyme molecules occurs and hence they precipitate from solution, as illustrated in Figure 1.4.3. Since there are many oligomeric dehydrogenases

ALTERNATE PROTOCOL

+
glutamate dehydrogenase

bis-NAD+

Figure 1.4.3 A diagrammatic view of the cross-linking of glutamate dehydrogenase hexamers by bis-NAD+, which results in the formation of an insoluble lattice. The locking-on in the presence of glutarate is not shown. Further details of the process are given in the Alternate Protocol.

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in the cell, this process might be expected to be somewhat nonspecific, causing the precipitation of a number of enzymes containing binding sites for NAD+. However, the procedure can be made specific for the precipitation of a selected dehydrogenase by the use of a compound that is a competitive inhibitor of the enzyme with respect to its other substrate. Thus, in the case of GDH, glutarate, which is a competitive inhibitor towards glutamate, is used. This strengthens the binding between the bis-NAD+ and glutamate dehydrogenase, allowing it to be used at concentrations below those that would be necessary to precipitate other dehydrogenases.

ox liver (46 g)

rat liver (10g)

homogenize for 1 min in 10 volumes (w/v) of 4 mM sodium phosphate buffer, pH 7.4, containing 0.5 mM EDTA and 0.1 mM PMSF

Stage 1 (homogenate)
add (NH4)2 SO4 (113 g/liter), stir for 20 min

centrifuge and discard pellet add (NH4)2 SO4 (188 g/liter), stir for 20 min
centrifuge and discard pellet dialyze against 20 mM sodium phosphate buffer, pH 7.4

Stage 2 [dialyzed (NH4)2 SO4 pellet]


apply to DEAE-cellulose column

Stage 3 (DEAE eluate)

elute with 20 to 200 mM sodium phosphate, pH 7.4, gradient and concentrate to ~10 ml
affinity precipitate with the appropriate amount determined from pilot precipitation experiment (Support Protocol 9) of bis-NAD+ in the presence of 79 mM glutaric acid and keep on ice overnight

centrifuge discard supernatant redissolve pellet by stirring for 6 hr in 1 ml of buffer containing 0.6 mM NADH dialyze against 20 mM sodium phosphate buffer, pH 7.4
purified glutamate dehydrogenase

Stage 4 (purified enzyme)


Purification of Glutamate Dehydrogenase from Liver and Brain

Figure 1.4.4 Outline scheme for the purification of ox and rat liver glutamate dehydrogenase according to the Alternate Protocol. Full details of the steps involved are given in the text.

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This behavior, which was termed the locking-on effect by OCarra (see OCarra et al., 1996; OFlaherty et al., 1999; Mulcahy and OFlaherty, 2001), was originally developed for increasing the strength of enzyme binding to an immobilized ligand. It depends on the analog of the substrate that is oxidized by NAD+ binding to the enzymeNAD+ binary complex and thus displacing the coenzyme-binding equilibrium by ternary complex formation:
NAD glutarate ZZZZZX Z ZZZZZZZ X Z GDH YZZZZ GDH < NAD+ YZZZZZ GDH< NAD+ < glutarate Z Z
+

This locking-on behavior occurs only if the oxidizable substrate, or a competitive inhibitor that is an analog of the oxidizable substrate, binds to the enzymeNAD+ binary complex and not to the free enzyme. The basic concept is that, although several enzymes bind NAD+, only the GDHNAD+ complex binds glutarate. The majority of dehydrogenases have ordered sequential kinetic mechanisms in which the coenzyme binds before the second substrate. To date, the technique has been found to work well for the purification of yeast alcohol dehydrogenase and lactate dehydrogenase (see Irwin and Tipton, 1996), and the synthesis of the corresponding bis-ATP analog has extended the application of bis-coenzymes to kinases (Beattie et al., 1987). The precipitated dehydrogenase can then be dissolved by incubation in the presence of NADH, which effectively competes with bis-NAD+ for binding to the enzyme. The procedure described here is that of Graham et al. (1985), as modified by Irwin and Tipton (1996). The steps involved are summarized in Figure 1.4.4. Additional Materials (also see Basic Protocol 1) Bis-NAD+ (see Support Protocol 7) 0.7 M glutaric acid, adjusted to pH 7.0 with NaOH Additional reagents and equipment for pilot-scale precipitation (see Support Protocol 9) NOTE: Unless indicated otherwise all steps are performed at 0 to 4C. 1. Homogenize the sample and perform ammonium sulfate precipitation and DEAEcellulose chromatography steps (see Basic Protocol 1, steps 1 to 12), concentrating the eluate from the DEAE-cellulose column to 10 ml at step 12a or b.
Although it is possible to perform affinity precipitation with preparations that have not been subjected to chromatography on DEAE-cellulose, the yield is much lower and the precipitated material is not completely pure.

2. Dialyze the enzyme preparation overnight at 4C against 200 ml of sodium phosphate buffer, pH 7.4, with at least one change of buffer. Carry out a pilot-scale precipitation as described in Support Protocol 9 to determine the optimal amount of bis-NAD+ to use. Add that amount of bis-NAD+ to the remaining dialyzed enzyme preparation and add 0.7 M glutaric acid (preadjusted to pH 7) to a final concentration of 79 mM glutarate.
Keep the mixture on ice overnight.

3. Centrifuge 15 min at 10,000 g, 4C. Redissolve the pellet by stirring for 6 hr in 1 ml of 20 mM sodium phosphate buffer, pH 7.4, containing 0.6 mM NADH. Dialyze the redissolved pellet overnight (using an MWCO 12,000 dialysis membrane) against 200 ml of sodium phosphate buffer, pH 7.4. Add glycerol and store as described in Basic Protocol 1, step 18.

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SUPPORT PROTOCOL 7

SYNTHESIS OF N2, N2-ADIPODIHYDRAZIDO-bis(N6-CARBONYLMETHYL)NAD+ (bis-NAD) This reagent is used in the Alternate Protocol. The steps involved are outlined in Figure 1.4.5. Materials Iodoacetic acid (fresh) 2 M and 1 M lithium hydroxide (LiOH) NAD+ (98%, free acid) 2 M and 6 M HCl 96% (v/v) ethanol 0.24 M NaHCO3 1 M NaOH Nitrogen source Sodium dithionite 2 M Tris (base) Acetaldehyde, redistilled Yeast alcohol dehydrogenase (crystalline, Sigma-Aldrich or Roche Diagnostics) 5 mM CaCl2, pH 2.7, and 50 mM CaCl2, pH 2.0 2 M Ca(OH)2 Adipic acid dihydrazide dichloride (Sigma-Aldrich) 0.5 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC; Sigma-Aldrich) 0.25, 1, and 2 M ammonium bicarbonate (NH4HCO3) Sintered-glass funnel (10 cm diameter) Rotary evaporator Vacuum desiccator 75C water bath 4 10cm Dowex AG 1X2 anion-exchange column (Support Protocol 5) Gradient mixer (e.g., UNIT 8.2) 30 2.5cm chromatography column packed with Whatman DE-52 resin and equilibrated (see Support Protocol 5) with 1 M NH4HCO3, pH 8.0 Additional reagents and equipment for resolution of NAD+ derivatives (see Support Protocol 8) Synthesize N(1)-carboxymethyl-NAD+ 1. Dissolve 9 g fresh iodoacetic acid in 10 ml of water, neutralize the solution with 2 M LiOH, and add 3 g of NAD+. Adjust the pH to 6.5 with 2 M HCl (the total volume should be 30 ml) and leave the solution in darkness for 7 days at room temperature (20C), or alternatively, at 37C for 2 days. Check the pH daily (or every 4 to 6 hr at the higher temperature) and readjust to 6.5 with 2 M HCl as required.
The progress of the reaction should also be followed by TLC and/or HPLC (see Support Protocol 8). The solid iodoacetic acid should be white; if it is discolored as a result of decomposition it should not be used. Solutions of iodoacetic acid should be prepared fresh.

2. When reaction is complete, adjust pH to 3.0 with 6 M HCl and add 2 volumes of 96% (v/v) ethanol to produce a milky, pink-tinged suspension. Add a further 10 volumes of cold 96% ethanol to complete the precipitation.
Purification of Glutamate Dehydrogenase from Liver and Brain

The water/ethanol ratio is important; using a wet vessel can give rise to the formation of a brown, sticky substance which is water-soluble.

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3. Filter the crude N(1)-carboxymethyl-NAD+ on a sintered funnel, wash with ethanol and diethyl ether, and dry in a rotary evaporator at 35C under vacuum or by lyphilization (average yield 2.9 g). Store the product at 20C under vacuum. Carry out rearrangement of N(1)-carboxymethyl-NAD+ to N6-carboxymethyl-NAD+ 4. Dissolve the crude N(1)-carboxymethyl-NAD+ in 0.24 M NaHCO3 (90 ml), which gives a pale orange solution, and adjust the pH to 8.5 with 1 M NaOH. 5. Deoxygenate the solution by bubbling N2 gas through it for 2 min, add 1.5 g of sodium dithionite, and leave the solution in the dark until maximum reduction is achieved. Monitor progress of reduction by taking samples, diluting them 1:50 or 1:100, and measuring the increase in absorbance at 340 nm (A340). 6. Terminate the reaction by stirring vigorously for 10 min to oxygenate the solution and then bubble N2 gas through it for 2 min. Adjust the pH to 11.5 with 1 M NaOH and leave in a water bath at 75C to allow the rearrangement from the N(1)- to the N6-substituted derivative to occur (Dimroth rearrangement; see Engel, 1975). Monitor the rearrangement by HPLC or TLC (see Support Protocol 8). 7. Cool the reaction mixture to room temperature and add 6 ml of 2 M Tris (base) and 1.5 ml of redistilled acetaldehyde. Adjust the pH to 7.5 with 1 M HCl and add 8 to 24 mg (2500 to 7500 U) of yeast alcohol dehydrogenase. 8. Monitor the reaction at 340 nm as in step 5. When a minimum A340 is reached, add 1 volume of 96% ethanol and pour the milky flocculent precipitate into 10 volumes of vigorously stirred 96% ethanol. Leave for 30 min (or overnight, if desired) and collect the precipitate by filtration. Store the crude N6-carboxymethyl-NAD+ at 20C in a vacuum desiccator for up to a month (yield 2.6 g). Purify the crude N6-carboxymethyl-NAD+ 9. Dissolve the crude powder (2.6 g, from step 7) in 30 ml water, adjust the pH to 8.0 with 1 M LiOH, and apply this solution to a 2.5 30cm Dowex AG 1X2 column.
See Support Protocol 5 for details of packing and equilibrating the Dowex column.

10. After applying the coenzyme solution, wash the column with 0.5 liter of water, followed by 1 liter of 5 mM CaCl2, pH 2.7, until the pH of the effluent is 2.8. Apply a 2-liter linear gradient from 5 mM CaCl2, pH 2.7, to 50 mM CaCl2, pH 2.0. Collect 10 to 20 ml fractions and monitor the absorbance at 260 nm.
The composition of the fractions can be monitored by TLC (see Support Protocol 8).

11. Pool the fractions containing the N6 derivative, adjust the pH to 7.0 with 2 M Ca(OH)2, and concentrate to 5 to 10 ml by rotary evaporation at 40C. 12. Precipitate with 96% ethanol as in step 2, above, and lyophilize.
This gives a pale yellow compound (yield 0.82 g, 25%); = 19,300 M1cm1 at 266 nm.

Synthesis of bis-NAD+ 13. Dissolve 0.82 g of purified N6-carboxymethyl-NAD+ and 105 mg of adipic acid dihydrazide dihydrochloride in 20 ml water, to produce a brownish solution. 14. Adjust the pH to 4.6 with 1 M HCl and then add 0.5 M EDC at 0C in 15- to 100-l aliquots over a period of 35 min. Monitor the pH and readjust it to 4.6 before each addition. 15. Add water (2 liters) to dilute the solution 100-fold, adjust the pH to 8.0 with 2 M NH4HCO3, and apply the solution to a 30 2.5cm DEAE-cellulose (DE-52) column equilibrated with 1 M NH4HCO3, pH 8.0 (Support Protocol 5). Wash the column with
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water until the A260 is <0.1 and apply a 2-liter linear gradient from 0 to 0.25 M NH4HCO3, pH 8.0. 16. Monitor the Rf values of the fractions by TLC (Support Protocol 8).
The yield from pure N6-carboxymethyl-NAD+ is 14%. = 42,800 M1 cm1 at 266 nm.

17. Concentrate the product-containing fractions to 3 to 5 ml by rotary evaporation at 35C and store at 20C.
For longer-term storage (>7 to 10 days) the material may be lyophilized and stored in a vacuum desiccator over silica gel at 20C.

NH2

NH2
ICH2COO pH 6.5, 25C, 5 to 7 days

N N R N

N N R N

+ N

CH2COO

NAD+

N(1) carboxymethyl-NAD+ Sodium dithionite pH 8.5, 2 to 4 hr

NHCH2 COO

NH2
Dimroth rearrangement
pH 11.5, 75C, 2 hr

N N N

N
N

+ N

CH2COO

RH N 6-carboxymethyl-NADH

N RH N(1) carboxymethyl-NADH

alcohol dehydrogenase, acetaldehyde, pH 7.5 NHCH2 COO + N H

CONH2 O H OH

N N

N R

OH OH O H

O
H

H OH

N 6-carboxymethyl-NAD+ H2NOC(CH2)4 CONH2 + EDC


pH 4.6, 0C, 35 min 0
HN CH2

C NH NH C (CH2)4 C NH NH C CH2 NH

N N R N

N N R

bis-NAD+

EDC= CH3CH2-N
Purification of Glutamate Dehydrogenase from Liver and Brain

C N

(CH2)3 NH(CH2)2 Cl-

1-ethyl-3-(3)-dimethylaminopropyl) carbodiimide hydrochloride

Figure 1.4.5 Outline of the steps involved in the synthesis of bis-NAD+. Full details of the steps involved are given in Support Protocol 7.

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R=

CH2O P O P OCH2

O
H H OH

N N

RESOLUTION OF NAD+ DERIVATIVES Thin-layer Chromatography (TLC) The techniques of TLC are not described in detail here since they are adequately described in many textbooks (e.g., Wilson and Walker, 2000; Hahn-Deinstrop, 2000). Two different TLC solvent systems have been used for monitoring the products of the synthesis of bis-NAD+: System A: 60:100:2 (w/v/v) (NH4)2SO4/0.1 M potassium phosphate, pH 6.8/1-propanol. System B: 5:3 (v/v) isobutyric acid/1 M aqueous NH3, saturated with disodium EDTA. Apply the samples as spots to TLC plates (aluminium backed, silica gel 60, fluorescent indicator F254, layer thickness 0.2 mm; Merck), dry, and develop by ascending chromatography in a TLC developing tank. After developing the plates and allowing them to dry, the spots can be visualized by their fluorescence under UV light (wavelength 254 nm). They appear purple on a green background. The Rf values of bis-NAD+ and the compounds involved as intermediates in its synthesis are shown in Table 1.4.4. High-Performance Liquid Chromatography (HPLC) Inject the samples onto the HPLC column (4 mm 30-cm C18 reversed-phase; e.g., Waters Bondapak C18) and elute isocratically with 0.1 M KH2PO4, pH 6.0, containing 10% methanol, at a flow rate of 0.7 to 1.0 ml/min. The elution profile is determined by continuously monitoring the absorbance of the eluate at 259 to 266 nm using a UV detector flow cell. Good separation between NAD+ and N(1)-carboxymethyl-NAD+ can be achieved, and N6-carboxymethyl-NAD+ and bis-NAD+ can be well separated, but this system does not resolve N(1)-carboxymethyl-NAD+ and N6-carboxymethyl-NAD+ satisfactorily. Retention times for NAD+ derivatives are given in Table 1.4.4. The retention times are decreased if buffer containing 20% methanol is used. A gradient of 0% to 20% methanol may be used to separate the N(1)- and N6-substituted NAD+ derivatives. The advantage of HPLC over TLC is that it is easier to quantify the levels of any impurities present in the product.

SUPPORT PROTOCOL 8

Table 1.4.4

Some Properties of bis-NAD+ and its Precursors

Compound NAD+ N(1)-carboxymethyl-NAD+ N6-carboxymethyl NAD+ Bis- NAD+

Retention timeb Molar absorbance (M-1cm-1) Solvent Ac Solvent Bd (HPLC) 0.41 0.31 0.22 0.09 0.44 0.27 0.22 0.05 4.6 min 3.7 min 3.6 min 6.9 min 16,900 (at 259 nm) 17,100 (at 259 nm) 19,300 (at 266 nm) 42,800 (at 266 nm)

Rfa (TLC)

aThe R values given for TLC of coenzyme derivatives are not exact. They may vary by up to 0.05. This may result from f

slight changes in the solvent composition over time, possibly as a result of evaporation.
bThe HPLC retention times were obtained for samples chromatographed on a reversed-phase Bondapak C-18 column

(4 mm 30 cm) and eluted with 0.1 M KH2PO4 containing 10% methanol. cSolvent A: 60:100:2 (w/v/v) (NH ) SO ;/0.1 M potassium phosphate, pH 6.8/1-propanol. 42 4 d5:3 (v/v) isobutyric acid/1 M aqueous NH , saturated with disodium EDTA. 3

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SUPPORT PROTOCOL 9

PILOT-SCALE AFFINITY PRECIPITATION STUDY In order to obtain optimal results from the purification procedure described in the Alternate Protocol, it is necessary to perform a small-scale (pilot) study to determine the optimum concentrations of protein and bis-NAD+ to use (see Fig. 1.4.6). This will vary somewhat between different enzyme preparations, owing to differences in enzyme concentration and the presence of other contaminants that may bind the bis-coenzyme. The optimum ratio of NAD+ equivalents/enzyme subunit may vary with the preparation used; for example, with a preparation of beef liver enzyme that had a specific activity of 2.7 U/mg (see Basic Protocol 3), about half the activity was found to precipitate at a ratio of 2 NAD+ equivalents/GDH subunit. However, a preparation with the lower specific activity of 0.8 U/mg required 8 NAD+ equivalents/GDH subunit to precipitate half the enzyme activity. The precipitation yield also depends on the protein concentration, as shown, for example, in Figure 1.4.7. Materials Sample of the enzyme preparation (Alternate Protocol) Bis-NAD+ (see Support Protocol 7) 700 mM glutaric acid, pH 7.0 Additional reagents and equipment for assaying protein concentration (see Basic Protocol 2) and GDH activity (see Basic Protocol 3) 1. Take an aliquot of the enzyme-containing solution from the DEAE-cellulose purification step (see Alternate Protocol and Basic Protocol 1) and assay it for GDH activity (see Basic Protocol 3) and protein concentration (see Basic Protocol 2) to determine its specific activity.

100

Precipitation (%)

50

0 0 5 10 15 20
[bis-NAD+ ] (mol/mol GDH subunit)
Purification of Glutamate Dehydrogenase from Liver and Brain

Figure 1.4.6 An example of a pilot-scale affinity precipitation of ox liver glutamate dehydrogenase purified according to steps 1 to 3 of the Alternate Protocol. The experimental details are described in Support Protocol 9. The precise behavior will vary between preparations. The affinity precipitation is expressed as a percentage of the activity remaining in the control sample.

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100

Precipitation (%)

50

0.05 0.1 0.2 0.3 0.4

10

[Protein] (mg/ml)

Figure 1.4.7 An example of the effects of protein concentration on the pilot-scale affinity precipitation of ox liver glutamate dehydrogenase by bis-NAD+. These results are from a sample experiment performed as described in Support Protocol 9 in which the enzyme-containing sample was diluted to the indicated protein concentration before precipitation with a constant amount of bis-NAD+.

2. Calculate the approximate concentration of enzyme, based on the specific activity and subunit relative molecular mass.
Assuming each subunit to have a relative molecular mass of 56,700 and the pure enzyme to have a specific activity of 40 mol/min/mg protein, this corresponds to 2267 mol product/min/mol enzyme subunit. Thus:

mol of subunit =

activity (mol/min ) 2267 10 6

3. Measure effects of bis-NAD+ concentration as follows: a. Add a 100-l sample of the enzyme preparation to each of 10 microcentrifuge tubes. b. To each tube, add 12.7 l of 700 mM glutaric acid, pH 7.0, and varying amounts of bis-NAD+ within the range 0 to 10 coenzyme equivalents/enzyme subunit. c. Also prepare sample containing no bis-NAD+ (to which water is added in place of the coenzyme derivative) as well as a control tube that contains no bis-NAD+ or glutarate, so that the percentage inhibition resulting from the presence of the glutarate can be calculated.
The concentration of bis-NAD+ can be calculated from the absorbance at 266 nm and the molar absorbance coefficient given in Table 1.4.4. A 1 M solution of bis-NAD+ will give an absorbance, in a 1-cm light-path cuvette, of 42,800, and each mole of bis-NAD+ contains 2 NAD+ equivalents. Thus:

2( A266 ) = coenzyme equivalent concentration in mol/liter 42,800

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4. Allow the tubes to stand at 4C for at least 30 min (or overnight, if required), then microcentrifuge 5 min at maximum speed (13,000 g), 4C. 5. Assay the GDH activity remaining in the supernatant (see Basic Protocol 3) of each sample and express it as a percentage of the activity remaining in the control sample.
The maximum affinity precipitation has occurred in the tube with the minimal residual activity and thus gives the appropriate concentration of bis-coenzyme to add to obtain maximum affinity precipitation. These experiments should be performed at least in duplicate.

REAGENTS AND SOLUTIONS


Use Milli-Q-purified water or equivalent for the preparation of all buffers. For common stock solutions, see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.

Affinity chromatography buffer 100 mM Tris acetate 1 mM KH2PO4 0.1 mM EDTA Adjust pH to 7.15 at 4C Store at 0 to 4C up to 1 week; check pH before use and adjust if necessary Biuret reagent Dissolve 1.5 g copper sulfate (CuSO45H2O) and 6.0 g sodium potassium tartrate (C4H4KNaO64H2O) in 500 ml distilled H2O. Add 300 ml of 10% (w/v) sodium hydroxide and dilute the mixture to 1 liter with distilled water. Homogenization buffer 4 mM sodium phosphate buffer, pH 7.4 (APPENDIX 2A) 0.5 mM EDTA Store with above components at 0 to 4C up to 1 week; check pH before use and adjust if necessary 0.1 mM phenymethanesulfonyl fluoride (PMSF; add immediately before use as described below)
PMSF is unstable in aqueous solution and must be added to the buffer immediately before use. This is done by adding a suitable volume of a 100 mM PMSF solution in acetone to a small quantity (approximately one-tenth of the final volume) of boiling buffer, and immediately mixing this solution with the remainder of the ice-cold buffer.

COMMENTARY Background Information


There are several factors that must be considered in the choice of a purification procedure. The major factors are how much enzyme is needed for the planned studies, how long the procedure will take, and how much it will cost. To these should be added the question of whether the purified enzyme is different from the material that was in the tissues to start with. As discussed below, GDH purified by some older procedures has been shown to have undergone limited proteolysis, which alters its kinetic and regulatory behavior. Clearly, such preparations can give misleading information about the behavior of the enzyme. Several alternative procedures have been used to purify GDH from different mammalian sources. The procedures described in this unit are preferable to the earlier procedures that involved precipitation with ethanol or acetone and/or detergent extraction followed by fractional precipitation with ammonium sulfate and/or sodium sulfate (see e.g., Fahien et al., 1969). Although that procedure resulted in a crystalline preparation of the enzyme, it has been shown that limited proteolysis results in a loss of a tetrapeptide from the amino-terminal end of the protein (McCarthy et al., 1980). This modification, which results in a slightly faster mobility on SDS (0.1% w/v) PAGE in 13%

Purification of Glutamate Dehydrogenase from Liver and Brain

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polyacrylamide gels (McCarthy et al., 1980), significantly affects the kinetic behavior of the enzyme (McCarthy and Tipton, 1984) and its in teraction s with allosteric effectors (McCarthy and Tipton, 1985; Coue and Tipton, 1989a) and with antipsychotic drugs such as perphenazine (Coue and Tipton, 1990a,b). An obvious alternative to the hard work of purifying an enzyme is to buy it from a commercial source. Purified preparations of ox (beef) liver GDH have been available from several commercial sources: BoehringerMann heim (n ow Ro ch e Diagn ostics, Mannheim), Calbiochem, PL Biochemicals (now Pharmacia P-L Biochemicals), Sigma, and United States Biochemical (USB), although some of these sources no longer supply purified preparations of GDH. However, the authors have found the enzyme from all these sources to have suffered the same, or more, proteolytic modification as that suffered by the enzyme prepared by the earlier procedures discussed above (see McCarthy et al., 1980). Cloning and expression of GDH is an alternative source of enzyme (Shashidharan et al., 1994; Plaitakis et al., 2000). An expression system has the advantage that it permits site-directed mutagenesis studies (see Fang et al., 2002; Zaganas and Plaitakis, 2002). It is also, of course, useful in cases where there are rather small quantities of enzyme in the chosen source material. This is not the case with the major isoform of GDH (GLUD1) in liver, but it is the case for the so-called nerve tissuespecific form (GLUD2) which is present in much lower abundance in the brain (see Zaganas and Plaitakis, 2002). It is still necessary to purify such an expressed enzyme if one needs to work with a homogeneous preparation. In some cases, this can be facilitated by the insertion of a tag into the gene to be expressed. Such tags range from oligohistidine tails to another protein, such as glutathione-S-transferase. It is common not to remove a tag after purification, and indeed in some cases it is not easy to do so. The assumption that the presence of such a tag will not affect the behavior of the expressed enzyme may sometimes be true, but it is certainly not always so (see e.g., Halliwell et al., 2001; Hiser et al., 2001; Ledent et al., 1997). In the case of GDH, as already discussed, modification at the amino-terminal end results in alterations in its behavior, and the C-terminal region appears to be involved in its allosteric regulation (Peterson and Smith, 1999). This suggests that expression of the protein with a tag at either end would be

likely to produce artefactual results. Indeed, it is necessary to use conventional procedures, involving a GTP affinity column, to purify the enzyme after cloning and expression (see Fang et al., 2002; Zaganas and Plaitakis, 2002). To assess the purity of the protein, polyacrylamide gel electrophoresis (PAGE) in the presence and absence of sodium dodecyl sulfate (SDS) remains the accepted standard for assessing protein purity (UNITS 10.2-10.4). The hexameric glutamate dehydrogenase from ox and some other mammalian species forms higher aggregates at higher protein concentrations (see, e.g., McCarthy et al.,1981). Although this process is reversible, with a dissociation constant of 1 mg/ml (Cohen et al., 1976), it hampers attempts to determine the behavior on nondenaturing PAGE. However PAGE in the presence of SDS reveals that the enzyme prepared as described here contains only a few minor impurities (see McCarthy et al., 1980).

A critical aspect of protein purification procedures is to have everything that will be needed prepared in advance. For example, it is a recipe for disaster to finish one step and then find that the column needed for the next has not yet been equilibrated. Many enzymes are relatively unstable and susceptible to degradation by contaminating peptidases. Generally, to avoid losses of enzyme activity, the procedure should be completed as rapidly as possible. The essence of any robust purification procedure is that any minor problems with one step are corrected by the others. Thus, for example, a somewhat lower degree purification on the DEAE-cellulose column may be compensated for in the affinity-chromatographic step. Some problems that are common to many purification procedures are listed below. Failure to use the highest purity reagents. Analytical grade chemicals should be used for such procedures as preparing buffers and ammonium sulfate precipitation. Less pure materials may contain compounds that inhibit enzyme activity. Working at the wrong temperature. Most of the procedures involved should be executed at 0 to 4C. This is most easily accomplished in a cold room, although cooling the material in an ice bucket is satisfactory for some steps. Performing those steps at higher temperatures will result in enzyme inactivation. Overzealous stirring. This can cause frothing of protein solutions, which results in some

Critical Parameters and Troubleshooting

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Purification of Glutamate Dehydrogenase from Liver and Brain

denaturation. Stirring speeds should be fast enough to ensure adequate mixing, but no faster; if frothing does occur the stirring is too fast. Note that some frothing does occur during steps 1 and 2 of Basic Protocol 1, but this does not result in significant losses of GDH activity, probably because of the high protein concentrations at those stages. Failure to equilibrate chromatographic columns. Equilibration can be judged to have occurred when the pH and conductivity of the out-flowing buffer is the same as that of the in-flowing equilibration buffer. To use a column before complete equilibration has occurred will usually result in poor resolution of the sample components. Using a chromatographic column that is too old. The degradation of GTP-Sepharose after 6 months of storage is discussed in Support Protocol 4. DEAE-cellulose is quite stable if stored under the conditions described in Support Protocol 4. However, with time, the accumulation of tightly-bound material may affect the performance of columns, in which case the recycling procedure outlined in Support Protocol 4 should be used, or, alternatively, fresh DEAE cellulose can be used. Failure to check that the pH of buffers is correct. The pH should be adjusted at the temperature at which it will be used. Some buffers contain additional components, such as EDTA, and the pH should be checked and, if necessary, adjusted after all ingredients have been added. Failure to desalt samples before chromatographic steps. The presence of high concentrations of salt may prevent compounds of interest from binding to the column. In all cases where the enzyme is meant to bind to the column, it is important to retain the material that passes unretarded through the column and to assay it for enzyme activity as soon as possible. If material that should have bound passes straight through the column, it may be possible to rescue it by further desalting and application to a freshly equilibrated column. If in doubt about effective desalting, check the conductivity. The flow rate through the column is very slow, or there is apparently no flow at all. Do not load the enzyme-containing material onto a column that is running more slowly than specified. Very slow flow rates may occur if the material in the column has become compacted. This can arise if the column is run at too high a pressure. Always follow the manufacturers instructions about the maximum hydrostatic pressure that can be applied, and do not exceed that value. The accumulation of denatured material at the top of

a column can also slow the flow rate. This may be rectified by removing some of the chromatographic material from the top of the column, which is often identifiable by discoloration, adding an equivalent amount of fresh material, and repacking the column (see Support Protocol 4). In the case of DE-52 DEAE-cellulose, failure to remove the fines before using the resin (see Support Protocol 5) can result in inadequate flow rates. Flow rates through columns that are flowing well are likely to slow considerably if the sample applied contains appreciable amounts of particulate material. If particulate material is present, it should be removed by centrifugation, or, where appropriate, filtration, before the sample is applied to the column. Air bubbles or cracks are present in columns. This can result in the applied material not coming into adequate contact with the chromatographic material. Air bubbles can occur all too easily in gel-filtration media (e.g., Sephadex and Sepharose, including GTP-Sepharose) unless the manufacturers instructions for packing the material into columns are heeded. Cracks can occur if the material in the column is allowed to dry out. If that occurs, the only solution is to remove the material from the column, rehydrate and reequilibrate it, and then repack the column. The following subsections discuss only those steps where difficulties may be encountered through causes other than those discussed above. Should the enzyme lose activity or the final product have a specific activity significantly lower than expected, comparison of the data for each step with those shown in Tables 1.4.5 and 1.4.6 for Basic Protocol 1, or Tables 1.4.7 and 1.4.8 for the Alternate Protocol, should indicate where the problem occurred. Basic Protocol 1: purification of GDH involving affinity chromatography on GTP-Sepharose Ammonium sulfate precipitation. Too rapid addition of solid ammonium sulfate can result in high local concentrations of the salt, causing excess precipitation of material that does not all redissolve. In the present procedure, homogenization between additions minimizes this problem. If the solid ammonium sulfate salt contains lumps, break these up using a pestle and mortar before using it. It is possible to perform the precipitation by continuously stirring the enzyme solution in a beaker and adding the ammonium sulfate to it. If this approach is used, the salt should be added very slowly, over a period of 1 to 2 hr, to minimize the above problem. In either proce-

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Supplement 29 Current Protocols in Protein Science

Table 1.4.5 Protocol 1

Purification of Glutamate Dehydrogenase From Ox Brain According to Basic

Stage (see Fig. 1.4.1) 1 2 3 4 5

Volume (ml) 1500 1100 250 600 90

Total protein Total activity (mg) (mol/min) 75,000a 23,000a 6200a 320b 12.5b 2280 1150 925 775 500

Specific activity Purification (mol/min/mg) (x-fold) 0.03 0.05 0.15 2.4 40 1 2 5 80 1300

Yield (%) 100 50 41 33 22

aProtein concentration determined by the biuret procedure. bProtein concentration determined from the absorbance at 280 nm.

Table 1.4.6 Purification of Glutamate Dehydrogenase From Ox Liver According to Basic Protocol 1

Stage (see Fig. 1.4.1) 1 2 3 4 5

Volume (ml) 500 450 160 500 90

Total protein Total activity (mg) (mol/min) 6000a 2500a 880a 150b 25b 1800 15000 1410 1200 1000

Specific activity Purification (mol/min/mg) (x-fold) 0.3 0.6 1.6 8.0 40 1 2 5 25 120

Yield (%) 100 85 75 67 55

aProtein concentration determined by the biuret procedure. bProtein concentration determined from the absorbance at 280 nm.

Table 1.4.7 Protocol 1

Purification of Glutamate Dehydrogenase From Ox Liver According to Alternate

Stage (see Fig. 1.4.4) 1 2 3 4

Volume (ml) 370 75 8.4 1.2

Total protein Total activity (mg)a (mol/min) 8580 2010 139 9.4 1920 1640 408 376

Specific activity Purification (mol/min/mg) (x-fold) 0.2 0.3 2.9 40 1 4 13 180

Yield (%) 100 86 21 20

aProtein concentration determined by the Lowry method.

Table 1.4.8 Purification of Glutamate Dehydrogenase From Rat Liver According to Alternate Protocol 1

Stage (see Fig. 1.4.4) 1 2 3 4

Volume (ml) 90 35 9.8 1.0

Total protein Total activity (mg)a (mol/min) 2720 980 130 9.4 760 505 195 190

Specific activity Purification (mol/min/mg) (x-fold) 0.3 0.5 1.5 37 1 2 5 140

Yield (%) 100 67 26 25

aProtein concentration determined by the Lowry method.

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1.4.29
Current Protocols in Protein Science Supplement 29

dure, it is important to continue to stir for 20 to 30 min after the last ammonium sulfate addition, to ensure that any overprecipitated material has a chance to redissolve and that any material that should precipitate at that salt concentration does so. The use of a homogenizer during this stage has proven to be effective, but it is not recommended for ammonium sulfate fractionation of purer material, since excessive frothing can result in denaturation. Addition of ammonium sulfate will result in a decrease in pH. For GDH purification, it is not necessary to adjust the pH to the starting value between each addition of ammonium sulfate, although such readjustment is necessary for many other enzymes. Dialysis and concentration. Provided that the dialysis tubing is pretreated as described in UNIT 4.4 and APPENDIX 3B and that it has been tested for leaks, there should be no difficulties with these procedures. It should be stressed again that an air-free space should be left at the top of the knotted dialysis tube, because volume increases during dialysis can otherwise cause the bag to rupture. The concentration procedures should not cause difficulties if the directions given in Support Protocol 6 are followed. Chromatography on DEAE cellulose. Common difficulties with this step have been discussed in the general points at the beginning of this section. Chromatography on GTP-Sepharose. If the GDH enzyme does not bind to the column and the troubleshooting points at the beginning of this section have been taken into account, the likely causes are either that the affinity resin has degraded (see Support Protocol 4) or that there was a problem with its preparation. In first case, the GTP-Sepharose should be replaced by fresh material. If there has been a problem with its preparation, a fresh batch should be prepared using the procedure described in Support Protocol 1 to check for effective GTP coupling. Choice of reaction buffer. As indicated in Basic Protocol 1, the enzyme is unstable in the Tris/acetate buffer and losses of activity can occur if the enzyme is left in that buffer for extended periods. Basic Protocol 3: assay of glutamate dehydrogenase activity In all cases, it will be necessary to dilute the enzyme preparation in order to obtain a rate that is sufficiently slow to measure with accuracy. The amount of dilution will depend on the state of purification of the enzyme, its protein concentation, and also the scale expansion used on

the spectrophotometer. For example, the crude homogenate of liver GDH has a specific activity of 0.3 mol/min/mg protein (see Table 1.4.6). Since the production of 1 mol of glutamate in the assay will give an absorbance change of 2.073 (see Basic Protocol 3, annotation to step 5) , 1 mg of this material will give an absorbance decrease, in 1 min, of 0.3 2.073 0.62. The protein concentration of this crude homogenate is 12 mg/ml (see Table 1.4.6); therefore, 50 l of this would contain 0.6 mg protein, and would be expected to give an absorbance change of 0.373 absorbance units per min. This would mean that the reaction would be finished in <3 min, which would be too fast for accurate initial-rate determinations. See, for example, Tipton (2002) for a fuller treatment of enzyme assays and initial-rate determinations. Dilution of this material 20 with the assay buffer before adding 50 l to the assay would give an absorbance change of 0.01865 absorbance units per min, which would mean that it would take a little over 8 min for the absorbance to fall by 0.15 absorbance units. Similar calculations from the values in Table 1.4.6 for the purified liver GDH show that a dilution factor of 600 would be necessary so that a 50-l volume added to the assay would give a rate similar to that given by the crude enzyme. The appropriate dilution factors for the other stages of purification and for the brain enzyme can be estimated from the date given in Tables 1.4.6 and 1.4.5, respectively. It should be noted that these numbers are only a rough guide, since the degree of purification and protein concentration may vary for individual steps of the purification. The addition of smaller volumes of the enzyme preparation to the assay will reduce the degree of dilution necessary. Alternate Protocol: purification of GDH involving affinity precipitation with bis-NAD+ The initial steps in this procedure are the same as those of Basic Protocol 1. Affinity precipitation. A pilot-scale precipitation, as described in Support Protocol 9, should always be carried out to determine the optimum concentration of bis-NAD+ to use. Figure 1.4.6 shows an example of a concentration-precipitation relationship, but it should be remembered that the optimum bis-NAD+ concentration may vary with protein concentration. Failure to obtain affinity precipitation could result from: 1. A fault in the synthesis of the bifunctional ligand. Monitoring the success of each step in the preparatory procedure using the pro-

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cedures outlined in Support Protocol 8 and the data in Table 1.4.2, should reveal any problems in this respect and where they occur. 2. Attempting to precipitate from a solution containing too low a concentration of GDH (see Fig. 1.4.7). Further concentration of the enzymecontaining sample should deal with this problem. Stability The enzyme, stored as described in Basic Protocol 1, is stable for at least 6 months without significant loss of activity or alteration of its mobility on a 13% polyacrylamide gel in the presence SDS. This is important in view of the relatively large amounts of purified GDH that can be prepared. However, changes can occur in its responses to allosteric effectors when the enzyme is stored in an atmosphere of air (Coue and Tipton, 1991). These changes, which may only become apparent after 2 to 3 months, are prevented by storing the enzyme solution under an atmosphere of nitrogen, and tubes containing stored GDH preparations should be routinely flushed with N2 each time they are opened. Safety All personnel involved should have received training in safe laboratory procedures and in the use of radioactive materials if Support Protocol 2 (involving radiolabeled GTP) is to be used. National and local regulations for the disposal of such materials as radiochemical waste and or-

ganic solvents should be followed. Several of the compounds used in these procedures are highly toxic and should be handled with great care. Toxicity information on these compounds is available from the manufacturers (e.g., Sigma-Aldrich at https://www.sigma-aldrich. com) and should be consulted in advance. For the use of animal materials, precautions against infectious and transmissible diseases are necessary. It is advisable that personnel be immunized against such diseases where appropriate. Although the ox tissue samples that the authors have used recently have been certified as being BSEfree, the initial steps of the purification are still performed under biohazard containment, protection and disposal conditions. It is essential that those steps indicated in Support Protocols 1 and 3 be performed under an adequately functioning, biohazard-containment level II fume hood. Safety glasses should be worn, and, in connection with the TLC procedure in Support Protocol 8, these should provide protection from UV light.

Anticipated Results
Basic Protocol 1: purification of ox brain or liver glutamate dehydrogenase involving affinity chromatography on GTP-Sepharose Tables 1.4.5 and 1.4.6 summarize the results obtained from typical purifications of the ox brain and liver enzymes using this procedure.

10

A280 nm ( ) conductivity ( ) GDH Activity ( )

6
GDH activity (mol/min/ml)

Conductivity (mS)

8 4 6 2 4

0 50 100 Fraction number 150

Figure 1.4.8 Chromatography of ox liver glutamate dehydrogenase at step 3 of Basic Protocol 1. A typical elution profile is shown. However the exact behavior may vary between preparations. The fraction size was 10 ml. In the left-hand y-axis label, mS stands for millisiemens.

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Current Protocols in Protein Science Supplement 29

2.5

A280 nm ( ) conductivity ( ) GDH Activity ( )

12

8 1.5
A280

30

6 20
Conductivity (mS)

1.0

0.5

10

0 15 30 Fraction number 45

Figure 1.4.9 A typical elution profile of affinity chromatography of ox liver glutamate dehydrogenase on GTP-Sepharose at step 4 of Basic Protocol 1. The exact chromatographic behavior may vary between preparations. The fraction size was 10 ml. In the label for the inset scale, mS stands for millisiemens.

Examples of the elution profiles from DEAE-cellulose and GTP-Sepharose are shown in Figures 1.4.8 and 1.4.9. Alternate Protocol: purification of rat or ox liver GDH involving affinity precipitation with bis-NAD+ Tables 1.4.7 and 1.4.8 summarize the results obtained from typical purifications of the ox and rat liver enzymes using this procedure.

Time Considerations

The time estimates given below do not include the time involved in pouring and equilibrating the chromatographic columns, which should be done before the purification procedure is started. Basic Protocol 1: purification of ox brain or liver glutamate dehydrogenase involving affinity chromatography on GTP-Sepharose The entire purification procedure can easily be performed in 3 days. Day 1: Extraction and ammonium sulfate precipitation, followed by overnight dialysis. Day 2: DEAE-cellulose fractionation and concentration of appropriate column fractions.

Day 3: Gel filtration into the GTPSepharose buffer, passage through the hydrazide gel column, and affinity chromatography on GTP-Sepharose. This schedule excludes the time taken to prepare the GTP-Sepharose. This should take 3 days or less, starting from CNBr-activated Sepharose. However much of this time is taken up by incubations, such as the 16- to 18-hr reaction of L-glutamic acid -methyl ester with the activated gel (see Support Protocol 1). The affinity resin, once made, can be used several times. Alternate Protocol: purification of rat or ox liver GDH involving affinity precipitation with bis-NAD+ This is a somewhat quicker procedure than Basic Protocol 1 because it avoids the final column chromatography steps. However, it still extends into several days. The first two of these are the same as those for Basic Protocol 1, and the third is for the pilot-scale precipitation study and the affinity precipitation itself. The preparation of bis-NAD+ can be completed in 4 days, provided that the reaction

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Supplement 29 Current Protocols in Protein Science

Glutamate dehydrogenase activity ( mol/min/ml)

2.0

10

between iodoacetate and NAD+ is performed at 37C (see Support Protocol 7). Once prepared, the bis-NAD+ may be used for several GDH preparations, as well as for the purification of some other dehydrogenases (see Irwin and Tipton, 1996)

chromatography on GTP-sepharose. Anal. Biochem. 61:264-270. Graham, L.D., Griffin, T.O., Beattie, R..E., McCarthy, A..D., and Tipton, K.F. 1985. Purification of liver glutamate dehydrogenase by affinity precipitation and studies on its denaturation. Biochim. Biophys. Acta 828:266-269. Hahn-Deinstrop, E. 2000. Applied Thin-Layer Chromatography: Best Practice and Avoidance of Mistakes. Wiley-VCH, Weinheim, Germany. Halliwell, C.M., Morgan, G., Ou, C.P., and Cass, A.E. 2001. Introduction of a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active and inactive molecules. Anal. Biochem. 295:257-261. Hiser, C., Mills, D.A., Schall, M., and FergusonMiller, S. 2001. C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping. Biochemistry 40:1606-1615. Irwin, J.A., and Tipton, K.F. 1996. Affinity precipitation methods. In Methods in Molecular Biology 59: Protein Purification Protocols (S. Doonan, ed.) pp. 217-238. Humana Press, Totowa, N.J. Jackson, R.J., Wolcott, R.M., and Shiota, T. 1973. The preparation of a modified GTP-sepharose derivative and its use in the purification of dihydroneopterin triphosphate synthetase, the first enzyme in folate biosynthesis. Biochem Biophys. Res. Commun. 51:428-435. Larsson, P.O., Flygare, S., and Mosbach, K. 1984. Affinity precipitation of dehydrogenases. Methods Enzymol. 104:364-349. Ledent, P., Duez, C., Vanhove, M., Lejeune, A., Fonze, E., Charlier, P., Rhazi-Filali, F., Thamm, I., Guillaume, G., Samyn, B., Devreese, B., Van Beeumen, J., Lamotte-Brasseur, J., and Frere J.M. 1997. Unexpected influence of a C-terminal-fused His-tag on the processing of an enzyme and on the kinetic and folding parameters. FEBS Lett. 413:194-196. Lee, W.K., Shin, S., Cho, S.S., and Park, J.S. 1999. Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum. J. Cell. Biochem. 76:244-253. McCarthy, A.D. and Tipton, K.F. 1984. The effects of magnesium ions on the interactions of ox brain and liver glutamate dehydrogenase with ATP and GTP. Biochem. J. 220:853-855. McCarthy, A.D. and Tipton, K.F. 1985. Ox liver glutamate dehydrogenase: Comparison of the kinetic properties of native and proteolysed preparations. Biochem. J. 230:95-99. McCarthy, A.D., Walker, J.M., and Tipton, K.F. 1980. Purification of glutamate dehydrogenase from ox brain and liver: Evidence that commercially available preparations of the enzyme have suffered proteolytic cleavage. Biochem. J. 191:605-611.

Literature Cited

Beattie. R.E., Buchanan, M.,. and Tipton K.F. 1987. The synthesis of N2, N2-adipodihydrazido-bis(N-carboxymethyl-ATP) and its use in the purification of phosphofructokinase. Biochem. Soc. Trans. 15:1043-1044. Cho, S.W., Lee, J., and Choi, S.Y. 1995. Two soluble forms of glutamate dehydrogenase isoproteins from bovine brain. Eur. J. Biochem. 233:340-346. Cohen, R.J., Jedziniak, J.A., and Benedek, G.B. 1976. The functional relationship between polymerization and catalytic activity of beef liver glutamate dehydrogenase. II. Experiment. J. Mol. Biol. 108:179-199. Coue, I. and Tipton, K.F. 1989a. Activation of glutamate dehydrogenase by L-leucine. Biochim. Biophys. Acta 995:97-101. Coue, I. and Tipton, K.F. 1989b. The effects of phospholipids on the activation of glutamate dehydrogenase by L-leucine. Biochem. J. 261:921-925. Coue, I. and Tipton, K.F. 1990a. The inhibition of glutamate dehydrogenase by some antipsychotic drugs. Biochem. Pharmacol. 39:827-832. Coue, I. and Tipton, K.F. 1990b. Inhibition of ox brain glutamate dehydrogenase by perphenazine Biochem. Pharmacol. 39:1167-1173. Coue, I. and Tipton, K.F. 1991. The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities.. Neurochem. Res. 16:773-780. Dawson, R.M.C., Elliott, D.C., Elliott ,W.H., and Jones, K.M. 1989. Data for Biochemical Research, 3rd ed. Clarendon Press, Oxford. Engel, J.D. 1975. Mechanism of the Dimroth rearrangement in adenine. Biochem. Biophys. Res. Commun. 64:581 - 585. Fahien, L.A., Strmecki, M., and Smith, S. 1969. Studies of gluconeogenic mitochondrial enzymes. I. A new method of preparing bovine liver glutamate dehydrogenase and effects of purification methods on properties of the enzyme. Arch. Biochem. Biophys. 130:449-455. Fang, J., Hsu, B.Y., MacMullen, C.M., Poncz, M., Smith, T.J., and Stanley, C.A. 2002. Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations. Biochem J. 363:81-87. Flygare, S., Griffin, T., Larsson, P.O., and Mosbach, K. 1983. Affinity precipitation of dehydrogenases. Anal. Biochem. 133:409-416. Godinot, C, Julliard, J.H., and Gautheron, D.C. 1974. A rapid and efficient new method of purification of glutamate dehydrogenase by affinity

Strategies of Protein Purification and Characterization

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Current Protocols in Protein Science Supplement 29

McCarthy, A.D., Johnson, P., and Tipton, K.F. 1981. Sedimentation properties of native and proteolysed preparations of ox glutamate dehydrogenase. Biochem. J. 199:235-236. McDaniel, H.G. 1995. Comparison of the primary structure of nuclear and mitochondrial glutamate dehydrogenase from bovine liver. Arch. Biochem. Biophys. 319:316-321. Mulcahy, P. and OFlaherty, M. 2001. Kinetic locking-on strategy and auxiliary tactics for bioaffinity purification of NAD(P)(+)-dependent dehydrogenases. Anal. Biochem. 299:1-18. OCarra, P., Griffin, T., OFlaherty, M., Kelly, N., and Mulcahy, P. 1996. Further studies on the bioaffinity chromatography of NAD(+)-dependent dehydrogenases using the locking-on effect. Biochim. Biophys. Acta 1297:235-243. OFlaherty, M., OCarra, P., McMahon, M., and Mulcahy, P. 1999. A stripping ligand tactic for use with the kinetic locking-on strategy: Its use in the resolution and bioaffinity chromatographic purification of NAD(+)-dependent dehydrogenases. Protein Expr. Purif. 16:424-431. Peterson, P.E. and Smith, T.J. 1999. The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery. Structure Fold. Des. 15:769-782. Plaitakis, A. and Zaganas, I. 2001. Regulation of human glutamate dehydrogenases, implications for glutamate, ammonia and energy metabolism in brain. J. Neurosci. Res. 66:899-908. Plaitakis, A., Metaxari, M., and Shashidharan, P. 2000. Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms: Implications for biologic function. J. Neurochem. 75:1862-1869. Preiss, T., Hall, A.G., and Lightowlers, R.N. 1993. Identification of bovine glutamate dehydrogenase as an RNA-binding protein. J. Biol. Chem. 268:24523-24526. Rajas, F. and Rousset, B. 1993. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity. Biochem. J. 295:447-455. Shashidharan, P., Michaelides, T.M., Robakis, N.K., Kretsovali, A., Papamatheakis, J., and Plaitakis, A. 1994. Novel human glutamate dehydrogenase expressed in neural and testicular tissues and encoded by an X-linked intronless gene. J. Biol. Chem. 269:16971-16976 Shashidharan, P., Clarke, D.D., Ahmed, N., Moschonas, N., and Plaitakis, A. 1997. Nerve tissue-specific human glutamate dehydrogenase that is thermolabile and highly regulated by ADP. J. Neurochem. 68:1804-1811. Smith, T.J., Peterson, P.E., Schmidt, T., Fang, J., and Stanley, C.A. 2001. Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation. J. Mol. Biol. 307:707-720. Tipton K.F. 2002. Principles of enzyme assay and kinetic studies. In Enzyme Assays: A Practical

Approach (R. Eisenthal and M.J. Danson, eds.) pp. 1-47. Oxford University Press, Oxford, U.K. Tipton, K.F. and Coue, I. 1988. Glutamate dehydrogenase. In Glutamine and Glutamate in Mammals. (E. Kvamme, ed.) pp.81-100. CRC Press, Baton Roca, Fla. Wilson, K. and Walker, J. (eds.) 2000. Principles and Techniques of Practical Biochemistry, 5th ed.. Cambridge University Press, Cambridge, U.K. Yoon, H.Y., Hwang, S.H., Lee, E.Y., Kim, T.U., Cho, E.H., and Cho, S.W. 2001. Effects of ADP on different inhibitory properties of brain glutamate dehydrogenase isoproteins by perphenazine. Biochimie 83:907-913. Zaganas, I. and Plaitakis, A. 2002. A single amino acid substitution (Gly456Ala) in the vicinity of the GTP binding domain of human housekeeping glutamate dehydrogenase markedly attenuates GTP inhibition and abolishes the enzymes cooperative behavior. J. Biol. Chem. 277:2642226428.

Key References
Glutamate dehydrogenase
Plaitakis, A. and Zaganas, I. 2001. See above. Tipton, K.F. and Coue, I. 1988. See above. There have been few comprehensive reviews of glutamate dehydrogenase in recent years. The references cited above cover some fundamental aspects.

Affinity chromatography
Matejtschuk, P. (ed.) 1997. Affinity Separations: A Practical Approach. Oxford University Press, U.K. A collection of chapters on the theory and practice of different aspects of affinity chromatography.

Affinity precipitation
Irwin, J.A. and K.F. Tipton, K.F. 1995. Affinity precipitation: A novel approach to protein purification. Essays Biochem. 29:137-156. Describes the theory underlying affinity precipitation and reviews its applications.

Contributed by Martha Motherway and Keith F. Tipton Department of Biochemistry Trinity College Dublin, Ireland Alun D. McCarthy GlaxoSmithKline Middlesex, United Kingdom Ivan Coue Centre National de la Recherche Scientifique Rennes, France Jane Irwin University College Dublin Dublin, Ireland

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Overview of the Physical State of Proteins Within Cells


The word protein comes from the Greek word proteios, meaning primary. And, indeed, proteins are of primary importance in the study of cell function. It is difficult to imagine a cellular function not linked with proteins. Almost all biochemical catalysis is carried out by protein enzymes. Proteins participate in gene regulation, transcription, and translation. Intracellular filaments give shape to a cell while extracellular proteins hold cells together to form organs. Proteins transport other molecules, such as oxygen, to tissues. Antibody molecules contribute to host defense against infections. Protein hormones relay information between cells. Moreover, protein machines, such as actin-myosin complexes, can perform useful work, including cell movement. Thus, studying proteins is a prerequisite in understanding cell structure and function. The physical characterization of proteins began well over 150 years ago with Mulders characterization of the atomic composition of proteins. In the latter half of the nineteenth century Hoppe-Seyler (1864) crystallized hemoglobin and Khn (1876) purified trypsin. A variety of physical methods have been developed over the years to increase convenience and precision in the characterization and isolation of proteins. These include ultracentrifugation, chromatography, electrophoresis, and others. In many instances our understanding of cell proteins parallels the introduction and use of new techniques to examine their structure and function.

UNIT 1.5

PROTEIN CLASSIFICATIONS
All proteins are constructed as a linear sequence(s) of various numbers and combinations of 20 -amino acids joined by peptide bonds to form structures from thousands to millions of daltons in size. Proteins are the most complex and heterogeneous molecules found in cells, where they account for >50% of the dry weight of cells and 75% of tissues. Proteins can be classified into three broad groups: globular, fibrous, and transmembrane (Fig. 1.5.1; Table 1.5.1). Globular proteins are, by definition, globe-shaped, although in prac-

globular

fibrous

Figure 1.5.1 General classifications of proteins. In these schematic representations of globular, fibrous, and transmembrane proteins, hydrophobic regions are shaded. Note that the disposition of hydrophobic residues often reflects the protein class.

transmembrane

Strategies of Protein Purification and Characterization Contributed by Howard R. Petty


Current Protocols in Protein Science (2002) 1.5.1-1.5.10 Copyright 2002 by John Wiley & Sons, Inc.

1.5.1
Supplement 31

Table 1.5.1

Broad Classifications for Proteinsa

Type Globular

Location/type Intracellular Extracellular

Examples Hemoglobin, lactate dehydrogenase, cytochrome c Serum albumin, immunoglobulins, lysozyme Intermediate filaments, tropomyosin, lamins Collagen, keratin, elastins Insulin receptor, glycophorin, HLAsb Glucose transporter, rhodopsin, acetylcholine receptor

Fibrous

Intracellular Extracellular

Transmembrane

Single pass Multipass

aAdditional information regarding fibrous and transmembrane proteins can be found in Squire

and Vibert (1987) and Petty (1993). Information concerning globular proteins can be found in numerous books on proteins and enzymes such as Schultz and Schirmer (1979).
bHuman histocompatibility leukocyte antigens.

tice they can be spherical or ellipsoidal. Globular proteins are generally soluble in aqueous environments. Examples of globular proteins are hemoglobin, serum albumin, and most enzymes. Fibrous proteins are elongated linear molecules that are generally insoluble in water and resist applied stresses and strains. Collagen is a physically tough molecule of connective tissue. Just as collagen gives strength to connective tissues, intermediate filaments linked to desmosomes give strength to cells in tissues. The third general class of proteins, transmembrane proteins, contain a hydrophobic sequence buried within the membrane; these proteins are discussed more fully below (see Membrane Proteins). These protein categories are not mutually exclusive. For example, the nominally fibrous intermediate filament proteins also have globular domains. Similarly, transmembrane proteins almost always possess globular domains. Thus, these definitions serve as a useful guide but should not be rigidly applied.

HYDROPATHY PATTERNS OFTEN REFLECT A PROTEINS CLASSIFICATION


A key physical feature of proteins is their hydropathy pattern (i.e., the distribution of hydrophobic and hydrophilic amino acid residues). Indeed, hydrophobic interactions provide the primary net free energy required for protein folding. Figure 1.5.1 illustrates the disposition of hydrophobic amino acids in proteins. In an intact globular protein, hydrophobic

Overview of the Physical State of Proteins Within Cells

amino acids are generally shielded from the aqueous environment by coalescing at the center of the molecule, with the more hydrophilic residues exposed at its surface. However, the linear arrangement of hydrophobic residues fluctuates in an apparently random fashion. The helices within globular proteins may express a hydrophobic face oriented toward the center of the protein. (Within these helices hydrophobic residues are nonrandomly positioned every three or four amino acids to yield a hydrophobic face.) For coiled-coil helixcontaining fibrous proteins, such as tropomyosin and keratin, hydrophobic residues at periodic intervals allow close van der Waals contact of the chains and potentiate assembly as hydrophobic residues are removed from the aqueous environment (Schulz and Schirmer, 1979; Parry, 1987). Secondarily, regularly spaced charged groups can also contribute to the shape of fibrous proteins (Schulz and Schirmer, 1979; Parry, 1987). Transmembrane proteins provide a rather different physical arrangement of hydrophobic residues in which hydrophobic residues are collected primarily into a series of amino acids that is embedded within a cell membrane. One important means of analyzing the hydropathy of a sequenced protein is a hydropathy plot (Kyte and Doolittle, 1982). In this method, each amino acid residue is assigned a hydropathy value, an ad hoc measure that largely reflects its relative aqueous solubility; these values are plotted after being averaged. The successful interpretation of hydropathy plots

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depends on the parameters chosen for averaging. The parameters are the number of residues averaged (amino acid interval or window) and how many amino acids are skipped when calculating the next average (step size). Using this approach with a window of 10 residues, it is often possible to find the positions of hydrophobic residues coalescing near the interior of globular proteins. The method is particularly useful in predicting transmembrane domains of proteins, generally with a window of 20 amino acids. To detect the repetitious pattern of coiled-coil fibrous proteins, however, windows smaller than the repeat length would be required.

MEMBRANE PROTEINS
In addition to their presence in the extracellular and intracellular milieus, proteins are also found in association with biological membranes. Proteins constitute one-half to threequarters of the dry weight of membranes. Membrane proteins perform a broad variety of functions including intermembrane and intercellular recognition, transmembrane signaling, most energy-harvesting processes, and biosynthesis in the endoplasmic reticulum (ER) and Golgi complex. Membrane proteins have been traditionally characterized as integral (or intrinsic) or peripheral (or extrinsic) on the basis of operational criteria. Peripheral membrane proteins are associated with membrane surfaces and can be dislodged from membranes using hypotonic or hypertonic solutions, pH changes, or chela-

tion of divalent cations. Components of the erythrocyte membrane skeleton, for example, are peripheral membrane proteins. Although most peripheral proteins are removed by washing a sample with buffers, integral proteins cannot be removed by such treatments. To isolate integral membrane proteins, which are embedded within the lipid bilayer, one must use detergents that disrupt the bilayer and bind to the proteins, thus solubilizing them. In general, integral membrane proteins have a portion of their peptide sequence buried in the lipid bilayer whereas peripheral proteins do not. However, the discovery of glycosylphosphatidylinositol (GPI)-linked membrane proteins added to the ambiguity of the situation. GPI-linked proteins are globular proteins with no membrane-associated peptide sequence, yet they require harsh conditions for solubilization. As the technology for studying membrane proteins improved, it became necessary to develop a more precise vocabulary to describe membrane proteins. Transmembrane integral membrane proteins have at least one stretch of amino acids spanning a membrane. Membrane proteins are classified as type I, II, III, or IV depending on the nature of their biosynthesis and topology in membranes (Spiess, 1995; Table 1.5.2 and Fig. 1.5.2). The biosynthetic insertion of these proteins in membranes is, in turn, dependent on the presence or absence of a cleavable signal peptide, the relative positions of the hydrophobic transmembrane domain and positively charged topogenic signals, and/or

Table 1.5.2

Definitions of Integral Transmembrane Proteins

Type I

Definition An N-terminalcleavable signal peptide is removed at the luminal face yielding a luminal N terminal during biosynthesis. (Positive charges are found on C-terminal side of first long hydrophobic sequence after the signal peptide.) An N-terminaluncleaved signal peptide leads to a cytoplasmic N terminus. (Positive charges are generally found on N-terminal side of first long hydrophobic sequence.) A long N-terminal hydrophobic sequence is followed by a sequence of positive charges. This leads to a luminal N terminus in the absence of a cleavable signal peptide. A short C terminus is present at the luminal side of membrane. A large N terminus is exposed at the cytoplasmic face.

Examples LDL receptor, insulin receptor, glycophorin A, thrombin receptor

II

Transferrin receptor, sucrase/isomaltase, band 3

III

-Adrenergic receptor, cytochrome P450

IV

Synaptobrevin, UBC6

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nontransmembrane type I N exterior or lumen


bilayer

transmembrane type II C type III N C type IV

cytosol

+ + +

+ + +

+ + +

Figure 1.5.2 Membrane proteins containing hydrophobic anchors. A nontransmembrane or monotopic membrane protein is anchored to the membrane via a hydrophobic amino acid sequence. Transmembrane proteins are classified as types I, II, III, and IV (Table 1.5.2). The first transmembrane segment of a multispanning membrane protein can be inserted as in type I, II, or III proteins. This segment functions as a start-transfer peptide. Subsequent transmembrane segments will function as stop-transfer and start-transfer sequences, resulting in a multispanning membrane topology.

Overview of the Physical State of Proteins Within Cells

the mechanism of nascent protein delivery to the ER. Type I membrane proteins are synthesized with an amino-terminal signal sequence that is inserted into the ER membrane. When the signal sequence is proteolytically removed in the ER lumen, a new luminal amino terminus is exposed. A series of positively charged residues at the C-terminal side of the first hydrophobic transmembrane domain following the signal sequence generally denotes the end of the first transmembrane domain (von Heijne and Gavel, 1988). Although a hydrophobic transmembrane domain followed by a positive sequence of amino acids is sufficient to act as a stop-transfer signal, this motif is not required for stoptransfer events and other, less well-understood regulatory mechanisms are also involved (Andrews and Johnson, 1996). Membrane proteins types I, II, and III are delivered to the ER membrane via a signal recognition particle (SRP)-dependent mechanism. In contrast to type I proteins, type II and III membrane proteins do not have a cleavable N-terminal signal sequence. Instead, they have an internal hydrophobic signal that acts as both a signal sequence for ER delivery and a transmembrane domain in the mature protein. Type II proteins have a cytoplasmic amino terminus and a luminal (or extracellular) carboxyl terminus. In this case a positively charged sequence

of amino acids at the N-terminal side of the first hydrophobic sequence causes the amino terminus to be retained at the cytoplasmic face of the ER membrane. Thus the internal uncleaved signal peptide becomes the transmembrane domain of the mature protein. Type III membrane proteins have the same overall topology as type I proteins, but they are inserted into membranes by a different mechanism. In type III proteins the first hydrophobic sequence of amino acids is immediately followed by a series of positively charged amino acids. Thus, the first hydrophobic sequence becomes the transmembrane domain of the protein, with the amino terminus at the luminal face of the membrane. Type IV membrane proteins are characterized by a large, cytoplasmically exposed amino-terminal domain and a short carboxylterminal domain facing the lumen. Importantly, these proteins are delivered to the ER by an unknown SRP-independent mechanism. In addition to the single-pass membrane proteins just described, integral membrane proteins can display zero, two, three, or more transmembrane domains. Some membrane proteins, such as cytochrome b5, have protein segments buried in the hydrophobic core of membranes but do not cross the membrane. Membrane proteins with multiple membranespanning domains are classified as type I, II, or

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III depending on the topogenic signals in the first transmembrane domain. For example, a multispan membrane protein with a cleavable signal sequence, luminal amino terminal, and a positively charged sequence following the first transmembrane domain from the amino terminal, such as the thrombin receptor, is a type I membrane protein. The remaining transmembrane domains are inserted into the bilayer depending on the orientation of the first transmembrane domain. Multispanning type II and III proteins are similarly defined according to the properties of their single-spanning counterparts. In addition to hydrophobic protein sequences acting as membrane anchors, membrane proteins may also carry bilayer-associated hydrophobic lipid components. These hydrophobic lipid anchors define three broad groups of lipid-modified proteins: fatty acylated, isoprenoid-linked, and GPI-linked (Fig. 1.5.3). Several cytosolic transmembrane proteins have been identified that contain a covalently attached hydrophobic fatty acyl residue. For example, fatty acids, including palmitic, palmitoleic, cis-vaccenic, and cyclopropylene-

hexadecanoic, are covalently linked to the amino terminus and the amino-terminal glycerylcysteine of E. coli lipoprotein. Moreover, palmitate- and myristate-labeled transmembrane proteins have been observed in eukaryotic cells (e.g., Schlesinger et al., 1980). In both isoprenoid-linked and GPI-linked proteins, globular proteins become membranebound due to the addition of a hydrophobic lipid moiety. Certain proteins containing conserved cysteine residues at or near the C-terminus are modified by prenylation, in which a farnesyl or geranylgeranyl isoprenoid tail is added (Zhang and Casey, 1996). This hydrophobic moiety promotes protein association with the cytoplasmic face of cell membranes. Notably, cytosolic G proteins and protein kinases that participate in signal transduction are prenylated. GPI-linked proteins are a major class of membrane proteins (Cardoso de Almeida, 1992; Englund, 1993). In contrast to isoprenoid-modified proteins, GPI-linked proteins are attached to the luminal or extracellular face of membranes via a glycosylphosphatidylinositol anchor of variable structure (e.g.,

EtN P

P exterior or lumen
bilayer

cytosol

C O GPI-linked

S Cys-CH3

fatty acylated

isoprenoid-linked

Figure 1.5.3 Membrane proteins containing lipid moieties. In the simplest case, fatty acids can be covalently attached to transmembrane proteins. Hydrophobic tails are also attached to proteins to form isoprenoid-linked proteins. A third class of lipid-attached proteins are the GPI-linked proteins. Hydrophobic regions are shaded.

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Fig. 1.5.3). Well over 100 GPI-linked proteins have been identified in cells, where they perform numerous functions including acting as enzymes and receptors. The ability of GPIlinked proteins, which possess no transmembrane or cytosolic sequences, to elicit transmembrane signals seems paradoxical. However, studies have suggested that interactions with other proteins, including transmembrane integrins (Petty et al., 1996), contribute to transmembrane signaling of these proteins. In addition, GPI-linked proteins can collect in microdomains called lipid rafts within cell membranes (Rietveld and Simons, 1998). Although GPI-linked proteins must collaborate with other membrane proteins to elicit signals, they do possess certain functional advantages. First, GPI-linked proteins (and isoprenoid-linked proteins as well) diffuse in membranes much faster than transmembrane proteins and thus relay information faster. Second, certain cells, such as leukocytes, can rapidly shed their GPIlinked proteins, thus altering their functional properties in seconds. Although the importance of lipid-linked membrane proteins has only recently been appreciated, the impact of these structures on our understanding of cell properties is growing rapidly.

lows for lock-and-keylike interactions (Petty, 1993). In addition to structural and binding considerations, electrostatic interactions play a regulatory role. For example, the phosphorylation and dephosphorylation of insulin receptors alter electrostatic interations between the active site and a regulatory loop of the kinase domain, thereby changing its three-dimensional shape (Hubbard et al., 1994). This changes the Vmax of the kinase, thus triggering intracellular signals. In addition to the types of physical heterogeneity listed above, >100 distinct chemical modifications of proteins have been observed. These include, for example, glycosylation, ubiquitin attachment, phosphorylation, acetylation, and hydroxylation (Table 1.5.3). Thus, proteins undergo extensive physical-chemical modification.

PROTEIN ASSEMBLIES
Proteins can be assembled in a variety of states in both aqueous media and within membranes. Protein assembly into complex supramolecular structures plays vital roles in enzyme regulation, cell skeleton formation, and transmembrane signaling. Both covalent bonds and noncovalent bonds participate in protein assembly. One frequently encountered covalent mechanism of protein assembly is the formation of disulfide bonds. These covalent linkages often form during protein maturation. They can link two separate proteins together or two portions of the same protein. For example, the two chains of insulin molecules are held together by disulfides, as are the two chains of its membrane receptor. However, disulfide bond formation is mostly limited to oxidative environments such as the ER lumen and the exterior face of the cell surface. One of the best-known examples of noncovalent assembly is the formation of hemoglobin tetramers. Polymerization is another frequently encountered mechanism for protein assembly in cells. The globular protein actin polymerizes to form microfilaments in the absence of covalent bond formation. Intermediate filaments are formed by the polymerization of fibrous proteins. Under certain circumstances transmembrane proteins polymerize as well; bacteriorhodopsin, for example, forms two-dimensional pseudocrystals called purple membranes. Protein assemblies formed from various numbers of similar units are homodimers, homooligomers, and homopolymers. Assembly of protein structures from dissimilar subunits is more common than assem-

ADDITIONAL FACTORS AFFECTING THE PHYSICAL HETEROGENEITY OF PROTEINS


Additional factors contributing to the physical-chemical heterogeneity of proteins are size, charge, chemical modifications, and assembly. A typical amino acid has a molecular mass of 110 Da, and a small protein has a molecular mass of a few thousand daltons (e.g., for insulin, Mr = 5733). Large proteins have molecular masses of several hundred thousand daltons. When proteins are assembled to form large multiprotein complexes such as ribosomes, molecular masses are well into the millions. The diameters of these structures range from 4 for an individual amino acid to 30 nm for a ribosome. Electrostatic charge is of major importance in protein structure and function. Charged proteins are more soluble than uncharged proteins. The large number of positive charges on histones allow them to bind DNA. The spatial arrangement of charges on cytochrome c allows it to bind the complementary charges of its oxidase and reductase, thereby orienting the proteins prior to electron transfer. Similarly, the arrangement of charges on the apoprotein and receptor for low-density lipoprotein (LDL) al-

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Table 1.5.3

Common Physical-Chemical Modifications of Proteinsa

Modification Homodimerization Homooligomerization Homopolymerization Heterodimerization Heterooligomerization Heteropolymerization Proteolytic cleavage Prosthetic group addition Oxidation-reduction Glycosylation Phosphorylation Acetylation Ubiquitination Hydroxylation Fatty acylation Isoprenylation GPI addition
Casey (1996).

Example Transferrin receptor S. typhimurium glutamine synthetase Actin Integrins Histones, proteasomes Ribosomes Signal peptide cleavage in ER Heme addition to cytochromes and hemoglobin Disulfide bond formation in ER Glycoprotein maturation Regulation of protein function, such as the tyrosine kinase activity of insulin receptors Blockage of N-termini of certain membrane proteins Ubiquitin-dependent proteolysis via proteasomes, histones Proline hydroxylation on collagen Insulin receptors, E. coli lipoprotein G proteins Alkaline phosphatase, urokinase receptors

aFor details, see Freedman and Hawkins (1980, 1985), Schlesinger et al. (1980), Englund (1993), and Zhang and

bly from identical subunits. For example, heterodimers are formed from the and chains of integrins within cell membranes. Complex heterooligomeric and heteropolymeric structures vary from relatively small structures such as histone octamers, which bind to DNA in the nucleus, to large particles such as ribosomes, found both in the cytosol and attached to nuclear and ER membranes. The signal recognition particle is a relatively small heterooligomeric structure, composed of one RNA subunit and six proteins, that potentiates the delivery of secretory and most membrane proteins to the ER membrane. Membrane-associated heterooligomeric structures have also been observed. One of the best examples of such structures is the components of the electron transport systems in chloroplasts and mitochondria (Petty, 1993). For example, the ubiquinone-cytochrome c reductase is composed of eleven different subunits. Thus, proteins can be assembled in a variety of manners within cells. Although some protein assemblies, such as intermediate filaments, are static structures, many are dynamic structures which provide functional flexibility. For example, microfilaments can rapidly assemble and disassemble. In addition to the physical changes in assembly state, compositional dynamics is also observed.

For example, interferon treatment alters the composition of proteasomes. Developmental changes in protein composition are also observed. As an example, fetal and newborn forms of a component of cytochrome c reductase are expressed in humans. Thus, protein assemblies can be characterized by both physical and compositional dynamics.

ALTERING THE SOLUBILITY OF PROTEINS: PROTEIN EXTRACTION


The in vitro characterization of cellular proteins begins with their extraction from tissues or cells into a buffer. With the exception of globular secretory proteins, such as those found in plasma, proteins are generally not easily accessible for experimental manipulation. For example, many fibrous proteins are not soluble in aqueous buffers. Cellular proteins are entrapped within or on a cell and therefore must be extracted from the cell in a soluble form. A variety of methods including osmotic lysis, enzyme digestion, homogenization using a blender or mortar and pestle, and disruption by French press and sonication have been employed to disrupt cells. For a cytosolic protein such as hemoglobin, no further extraction from the sample is necessary. However, many important cellular proteins, such as those associated

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with membranes, cytoskeletal components, and DNA, remain insoluble. To further solubilize cell proteins, both nonionic (e.g., Triton X-100) and ionic (e.g., sodium dodecyl sulfate) detergents are often employed. Detergents are small amphipathic molecules that interact with both nonpolar and polar environments. Detergents disrupt membranes. They also bind to hydrophobic regions of proteins, such as their transmembrane domains, thereby replacing the unfavorable contacts between hydrophobic protein regions and water with the more favorable hydrophilic domains of the detergent. Thus, instead of the hydrophobic regions of the insoluble protein forming an aggregate in the bottom of a test tube, the protein becomes soluble and can be employed in most in vitro analyses. In addition to detergents, several other solubilization strategies are useful for the extraction and in vitro characterization of proteins (Table 1.5.4). Chaotropic agents enhance the transfer of nonpolar molecules to aqueous environments by their disrupting influence on water structure. Chaotropic agents are generally large molecular ions such as thiocyanate (SCN), perchlorate (ClO4), and trichloroacetate

(CCl3COO). Hydrophobic interactions are also reduced by exposure to organic solvents and low salt concentrations. Electrostatic interactions are reduced by high salt conditions; this decreases the Debye-Hckel screening length and coulombic attraction. To disrupt hydrogen bonds, high concentrations of urea or guanidine are often employed. More vigorous methods of sample denaturation using very low pH or harsh detergents such as sodium dodecyl sulfate are also used to diminish intermolecular contacts. Once proteins are extracted, their size can be characterized by ultracentrifugation on sucrose gradients (UNIT 4.2), gel filtration chromatography (UNIT 8.3), SDS-PAGE (UNIT 10.1), and other methods (Table 1.5.5). The charge characteristics of proteins can be assessed using isoelectric focusing and ion-exchange chromatography. Specific interactions, such as antigen-antibody and biotin-avidin interactions, can also be employed in the characterization and isolation of proteins. These are useful in immunoblotting (UNIT 10.10) and affinity chromatography methods (Chapter 9).

Table 1.5.4

Physical Bases of Common Protein Extraction and/or Elution Methods

Physical property perturbed Hydrogen bonds Ion pair interactions Hydrophobic interactions

Agents Urea or guanidineHCl, pH changes High salt, pH changes Detergents, chaotropic agents, organic solvents, low salt

Table 1.5.5 Physical Bases of Common Protein Characterization and Isolation Methods

Physical property Solubility Size

Method Extraction with salts, detergents, and enzymes Ultracentrifugation on sucrose gradients Gel filtration SDS-PAGE

References to other units Chapter 4, Racker (1985)


UNIT 4.2 UNIT 8.3 UNIT 10.1

Charge Biospecific interaction

UNIT 10.2 Isoelectric focusing Ion-exchange chromatography UNIT 8.2

Immunoblotting Immunoaffinity chromatography

UNIT 10.10

Chapter 9

Overview of the Physical State of Proteins Within Cells

Hydrophobicity

Hydrophobic chromatography UNIT 8.4 Reversed-phase HPLC UNIT 8.7

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LIMITATIONS OF THE IN VITRO MANIPULATION OF PROTEINS


The very act of isolating proteins perturbs their physical environment. Although this is not often a major problem, a few cautionary notes should be made. The most primitive compartment of a cell, the cytosol, is a chemically reducing environment. Consequently, free sulfhydryl groups are observed in the cytosol; in fact, multiple cytosolic pathways help in preserving the proper redox conditions. On the other hand, the extracellular milieu and the luminal side of the ER are oxidative environments. The oxidizing condition within the ER is presumably due to the unidirectional transport of glutathione and cystine. Consequently, disulfides are frequently observed in the ER and extracellular environments. Thus, to prevent disulfide formation during manipulation, sulfhydryl blocking reagents such as iodoacetamide are included in extraction buffers. The cytosol is also a K+-rich and Ca2+-poor solution. These parameters should be considered in designing physiologically relevant experiments. The experimental manipulation of membrane proteins is decidedly more difficult. The exterior face exists in a high Na+ and Ca2+ solution that is oxidative; just the opposite is true for the cytoplasmic face. Since no appropriate solvent exists for such isolated proteins, experimental questions can be directed at properties associated with just one face of the molecule. A second limitation common to all in vitro studies of transmembrane proteins is that they must be solubilized using detergents. In addition to solubilizing a transmembrane protein, detergents can also bind to hydrophobic regions in the globular domain(s) of the protein, thus affecting the properties under study. One means of countering this problem is to test several detergents in the hope of finding one that retains the full biological activity of the purified protein. Protein solubilization can also lead to loss of physiologically relevant protein-protein interactions. This can occur by simple dilution or by disruption of noncovalent interactions among proteins. For example, hemoglobin exists as a supersaturated solution in vivo which cannot be duplicated in vitro. Furthermore, protein-protein associations are generally stronger in the restricted confines of membranes than after solubilization into a buffer. Thus, protein assemblies found in cells may disappear during solubilization. One means of countering these potential difficulties is to co-

valently cross-link protein assemblies prior to disruption and to solubilize proteins using mild detergents (e.g., Brij-58).

CONCLUSIONS
Structural motifs, especially stretches of hydrophobic amino acids, contribute to the shape of a protein and its classification as globular, fibrous, or transmembrane. Proteins are heterogeneous at many different levels including physical attributes, covalent modifications, and supramolecular assembly. The physical properties of proteins are used to characterize and isolate these molecules. For example, the size of a protein is examined by sedimentation on sucrose gradients (UNIT 4.2), gel filtration (UNIT 8.3), and polyacrylamide gel electrophoresis (UNIT 10.1). Its charge is the key physical parameter in isoelectric focusing and ion-exchange chromatography. The units that follow contain detailed protocols describing the characterization of cellular proteins.

LITERATURE CITED
Andrews, D.W. and Johnson, A.E. 1996. The translocon: More than a hole in the ER membrane? Trends Biochem. Sci. 21:365-369. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 1998. Current Protocols in Molecular Biology. John Wiley & Sons, New York. Cardoso de Almeida, M.L. 1992. GPI Membrane Anchors. Academic Press, New York. Englund, P.T. 1993. The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu. Rev. Biochem. 62:121-138. Freedman, R.B. and Hawkins, H.C. 1980. The Enzymology of Post-Translational Modifications of Proteins, Vol. 1. Academic Press, New York. Freedman, R.B. and Hawkins, H.C. 1985. The Enzymology of Post-Translational Modifications of Proteins, Vol. 2. Academic Press, New York. Hoppe-Seyler, F. 1864. ber die chemischen und optischen Eigenschaften des Blutfarbstoffs. Virchows Arch. 29:233-235. Hubbard, S.R., Wei, L., Ellis, L., and Hendrickson, W.A. 1994. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 372:746-754. Khn, W. 1876. ber das Verhalten verschiedner organisirter und sogenannter ungeformter Fermete. ber das Trypsin (Enzym des Pankreas) [Reprint, FEBS Lett. 62:E3-E7 (1976).]. Kyte, J. and Doolittle, R.F. 1982. A simple method for displaying the hydrophobic character of a protein. J. Mol. Biol. 157:105-132. Strategies of Protein Purification and Characterization

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Parry, D.A.D. 1987. Fibrous protein structure and sequence analysis. In Fibrous Protein Structure (J.M. Squire and P.J. Vibert, eds.) pp. 141-171. Academic Press, New York. Petty, H.R. 1993. Molecular Biology of Membranes: Structure and Function. Plenum, New York. Petty, H.R., Worth, R.G., and Todd, R.F. III. 2002. Interactions of integrins with their partner proteins in leukocyte membranes. Immunol. Res. 25:75-95. Racker, E. 1985. Reconstitutions of Transporters, Receptors, and Pathological States. Academic Press, New York. Rietveld, A. and Simons, K. 1998. The differential miscibility of lipids as the basis for the formation of functional membrane rafts. Biochim. Biophys. Acta. 1376:467-479. Schlesinger, M.J., Magee, A.I., and Schmidt, M.F.G. 1980. Fatty acylation of proteins in cultured cells. J. Biol. Chem. 255:10021-10024. Schultz, G.E. and Schirmer, R.M. 1979. Principles of Protein Structure. Springer-Verlag, New York. Spiess, M. 1995. Heads or tailswhat determines the orientation of proteins in the membrane. FEBS Lett. 369:76-79. Squire, J.M. and Vibert, P.J. (eds.) 1987. Fibrous Protein Structure. Academic Press, New York. von Heijne, G. and Gavel, Y. 1988. Topogenic signals in integral membrane proteins. Eur. J. Biochem. 174:671-678. Zhang, F.L. and Casey, P.J. 1996. Protein prenylation: Molecular mechanisms and functional consequences. Annu. Rev. Biochem. 65:241-269.

KEY REFERENCES
Racker, 1985. See above. A wonderful little book on membrane protein manipulation which disproves the hypothesis that scientists cant write. Tanford, C. 1961. Physical Chemistry of Macromolecules. Academic Press, New York. A rigorous introduction to the physical properties of proteins, which remains useful several decades later. Tanford, C. 1980. The Hydrophobic Effect. John Wiley & Sons, New York. A very readable introduction to the hydrophobic effect.

INTERNET RESOURCES
http://www.expasy.ch A user-friendly protein database including two-dimensional PAGE data and 3D protein structures. ftp://ftp.pdb.bnl.gov/ Contains protein crystallography data. http://ww.ncbi.nlm.nih.gov An important protein database.

Contributed by Howard R. Petty Wayne State University Detroit, Michigan

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