Determination of the Relative Protein Concentrations of Cell Free and Stage 1 Extracts from the Purification of Alkaline Phosphatase.

A stract In order to quantify total protein for any stage in the purification of an enzyme or protein, a rapid, sensitive, and reliable method for protein concentration determination is required. In an attempt to measure the relative concentration of the cell free and stage 1 extracts from a purification of E. coli Alkaline Phosphatase, the radford dye binding assay !as used. A standard curve !as developed using a series of ovine "erum Albumin # "A$ standards in the 1%% g&ml to 1,'%% g&ml range. (he absorbance of each sample !as measured at ')' nm and plotted versus * "A+. (he resulting line !as fit by the linear least squared method. "amples from the cell free and stage 1 extracts !ere sub,ected to the radford assay in the same fashion. In theory, the measured absorbance of each together !ith the equation for the line generated in the "A standard curve should allo! determination of the relative protein concentrations in each sample. (he relative protein concentration for the cell free extract !as determined to be 1,-.' g&ml, ho!ever, the apparent non/linearity of the standard suggests that this number may be inaccurate. (he relative protein concentration for stage 1 !as determined to be negative suggesting that the sample measured !as too dilute for the sensitivity range of the radford method. In order to address these problems so that protein concentrations can be reliably determined, the range of "A standards used !ill be reduced to span 1%%/1,%%% g&ml. In addition, a larger volume of stage 1 !ill be used in the assay in an attempt to provide sufficient protein !ith respect to the sensitivity of the method. Alternatively, the sample could be concentrated several fold, thereby allo!ing the lo!er sample volume to be used. (he successful establishment of a reliable standard curve !ill greatly facilitate analysis of future protein preparations as !ell as provide quantitative measures of the purified enzyme that !ill be required for characterization of the enzyme.

!ntroduction Alkaline phosphatases #orthophosphoric monoester phsphohydrolases, 01 -.1.-.1$ are phosphate hydrolases that catalyze the follo!ing general reaction at high p2 #3..%$4 5/6/P6-2/ 7 286 5/62 7 28P69/

(hese enzymes perform important functions at the cellular level in a !ide variety of organisms, both eukaryotic and prokaryotic. (he physiological role of alkaline phosphatase in E. coli is to provide the cell !ith a source of inorganic phosphate #Pi$. It accomplishes this task by cleaving phosphoryl groups from a !ide variety of phosphorylated compounds. (hese reactions take place in the periplasmic space bet!een the outer peptidoglycan membrane of gram/negative bacteria and the cytoplasmic membrane of the cell. (he outer membrane is permeable to

many phosphorylated compounds !hile the cytoplasmic membrane is not. Alkaline phosphatase liberates Pi in this space !here it becomes bound by the Pi/binding protein. (his protein delivers phosphate to the high affinity Pst transport system, !hich transports phosphate across the cytoplasmic membrane #1$. ecause enzymes of this class perform such an important function in a !ide variety of eukaryotic and prokaryotic cells, it is of considerable interest to isolate and characterize the physical properties of a representative member of this enzyme class. A thorough characterization of this essential enzyme !ill provide important insight into the catalytic event that provides inorganic phosphate to the bacterium. In addition, this study !ill provide a general kno!ledge base for the study of homologous alkaline phosphatases from eukaryotic sources. In an effort to provide the necessary foundation required for the complete characterization of the ma,or alkaline phosphatase from the bacterium, Escherichia coli, purification of the enzyme is required. :uring the course of protein purification, it is often necessary to measure the concentration of various samples. "uch information allo!s an evaluation of purity to be made as !ell as providing some idea of the amount of protein that is available for further use. ;arious methods for determining protein concentration in a given sample are available, but they vary in ease of use, sensitivity, and cost. <e chose to use the radford method #8$, !hich is a dye binding method. (he dye, 1oomassie brilliant blue =/8'%, has a maximum absorbance at 9>' nm. <hen it binds to a protein under acidic conditions, the maximum shifts to ')' nm. (he binding of the dye to protein involves strong noncovalent interactions including electrostatic, bet!een amino and carboxyl groups, as !ell as van der <aals forces #1$. ecause all proteins possess amino and carboxyl groups and participate in both electrostatic and van der <aals interactions, this method is largely insensitive to protein structure at any level. (he method can be used develop a standard curve for kno!n concentrations of any abundant protein. (he linear relationship bet!een the absorbance and kno!n protein concentration can then be used to determine the relative protein concentration of any given sample. In the current study, the radford dye !ill be mixed !ith kno!n concentrations of a protein, in this case ovine "erum Albumin # "A$. (he absorbance of each standard !ill be measured at ')' nm. (he straight/line relationship bet!een absorbance and concentration !ill be fit by the least squares method. (he concentration of t!o samples, cell free extract and stage 1 from an alkaline phosphatase purification, !ill be determined using the slope and intercept from the derived equation. "aterials and "ethods Preparation of "tandards "A !as made purchased in crystalline form from "igma 1hemical #"t. ?ouis, @6$. %.1 g of "A !as dissolved in 1% ml of !ater at room temperature.

(he stock "A solution !as diluted to span the 1%%/1,'%% g&ml range in the follo!ing manner4

#a le 1. "tandard "A solution preparation * "A+ g&ml 1%% 8%% 9%% >%% )%% 1,8%% 1,'%% ;olume #?$ of 1% mg&ml "A "tock ' 1% 8% -% 9' >% .' ;olume #?$ of @illiA !ater 9)' 9)% 9B% 9.% 9'' 99% 98'

>% ? of each standard !as mixed !ith )9% ? of radford reagent. (his !as repeated t!ice for each concentration allo!ing three measurements to be made for each concentration of standard. 0ach sample !as allo!ed to incubate at room temperature for 1% minutes and no longer than C hour before being measured. (he absorbance of each standard !as measured at ')' nm against a blank that !as composed of >% ? of !ater and )9% ? of radford reagent. Absorbance !as plotted against * "A+ and an equation for the line !as generated. A sample from the cell extract and the first stage of a current and ongoing preparation of alkaline phosphatase !as prepared and measured in the same manner as the standards. (hree identical measurements !ere made for each sample.

"pectrophotometer Parameters @easurements !ere made on a 1ary 1 dual beam spectrophotmeter that had been !armed up for at least C hour prior to measurements. @easurements !ere made in the absorbance mode at ')' nm using a 1 second integration time. (he instrument !as zeroed by placing blanks in both the front and rear cavities and instructing the instrument to set the difference in absorbance equal to zero. <hile keeping the blank in the rear cavity, samples !ere placed in the front cavity and their absorbances !ere recorded.

Data$Results #a le %. "A "tandard 1urve Absorbance @easurements A')' %.%. %.81 %.9 %.') %.B1 1.1 1.8 A')' %.%B %.1) %.91 %.> %..) 1.%' 1.1' A')' %.%) %.8% %.-) %.>1 %.B 1.1' 1.8' Average A')' %.%B %.8% %.9 %.> %.B 1.1 1.8

* "A+ g&ml 1%% 8%% 9%% >%% )%% 1,8%% 1,'%%

Bovine Serum Albumin Standard Curve

1.4 Absorbance at 595 nm 1.2 1 0.8 0.6 0.4 0.2 0 0 500 1000 [BSA] ug/ml 1500 2000

Figure 1. In a standard reaction, >% ? of each "A concentration !as mixed !ith )9% l of radford reagent and allo!ed to stand at room temperature for ten minutes. 0ach sample !as measured for absorbance at ')' nm against a blank, !hich contained >% ? of !ater mixed !ith )9% l of the radford reagent.

A linear least square fit of the line produced the follo!ing equation4

y D %.%%%B * "A+ 7 %.%' #a le &. Absorbance of Protein Purification "amples "ample 1ell Eree "tage 1 1alculations "olving the equation for protein concentration using the cell free extract absorbance value4 1.1' D %.%%%B#F$ 7 %.%' F D * "A+ D 1-.' g&ml (he same calculation for "tage 1 gives * "A+ D /8' g&ml A')' 1.1' %.%A')' 1.1 %.%1 A')' 1.8 %.%' Average A')' 1.1' %.%-

Discussion (he absorbance at ')' nm for several concentrations of "A !as measured and averaged. (able 8 clearly sho!s that little variation bet!een measurements at lo! standard concentrations !ith respect to the change in average absorbance observed from standard to standard.!as evident. (his !as not true for the t!o highest concentrations !here the variation, %.%' absorbance units, !as on the order of the average change in absorbance from 1,8%% g&ml to 1,'%% g&ml. (he data graphed in Eigure 1 is not quite linear and the main reason for this appears to be the similarity in absorbance for the highest concentrations of "A. (aken together, these observations indicate that the line derived from the standard curve could be made better by lo!ering the concentration range such that the absorbance change is linear !ith the change in concentration. Analysis of the t!o protein purification samples #(able -$ results in concentrations of 1-.' g&ml for the cell free extract and G8' g&ml for stage 1. Although the absorbance and concentration of the cell free extract are reasonable !ith respect to the standard curve, it is likely not reliable as a measurement for t!o reasons. Eirst, the line derived from the standard curve is non/linear as discussed above so the best estimation of concentration cannot be obtained from this relationship. "econd, the absorbance observed for this sample is on the high end and likely suffers from the same problem as the t!o highest concentration standards, that is that their concentrations are sufficiently high to be out of the linear range of response for this method. (o solve this problem, a dilution of the sample can be made before treating it !ith the radford reagent

so that the measured absorbance falls into the linear range. (he stage 1 sample suffers from the opposite problem. ecause it is not possible to have a negative concentration, there is clearly a problem !ith the measurement of "tage 1. (able - sho!s an absorbance #A')' D %.%-$ for this sample that is lo!er than any of the absorbance values recorded for the range of "A standards. (his value is also close to the reliable detection limit #A D %.%%1$ of the spectrophotometer. (aken together, it appears likely that the sample is too dilute in protein concentration to be measured by this method. A likely solution to the problem is to concentrate the sample or possibly use a larger volume #3>% ?$ of the sample in the assay. In summary, the current method of protein concentration determination has resulted in questionable results. (herefore, reliable concentration values for the t!o protein purification samples cannot be reported. (he method !ill be repeated !ith changes that include lo!ering the span of standard concentrations, diluting the cell free extract sample prior to assay, and finally concentrating or using a larger volume of the stage 1 sample in the assay.

References 1. Hinfa, A.I J allou, :.P. 1))B. Eundamental ?aboratory Approaches for iochemistry and @olecular iology, Eitzgerald "cience Press, Inc., ethesda, @aryland. 8. radford, @. 1).>. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. iochem. '%(89B.