S I X
1. Introduction
2. Theory
3. Buffer Selection
4. Buffer Preparation
5. Volatile Buffers
6. Broad-Range Buffers
7. Recipes for Buffer Stock Solutions
References
1. Introduction
The necessity for maintaining a stable pH when studying enzymes is
well established (Good and Izawa, this series; Johnson and Metzler, this
series). Biochemical processes can be severely affected by minute changes in
hydrogen ion concentrations. At the same time, many protons may be
consumed or released during an enzymatic reaction. It has become increasingly important to find buffers to stabilize hydrogen ion concentrations
while not interfering with the function of the enzyme being studied. The
development of a series of N-substituted taurine and glycine buffers by
Good et al. (1966) has provided buffers in the physiologically relevant range
(6.110.4) of most enzymes, which have limited side effects with most
enzymes (Good et al., 1966). It has been found that these buffers are
nontoxic to cells at 50 mM concentrations and in some cases much higher
(Ferguson et al., 1980).
43
44
2. Theory
The observation that partially neutralized solutions of weak acids or
weak bases are resistant to pH changes on the addition of small amounts of
strong acid or strong base leads to the concept of buffering (Perrin and
Dempsey, 1974). Buffers consist of an acid and its conjugate base, such as
carbonate and bicarbonate, or acetate and acetic acid. The quality of a buffer
is dependent on its buffering capacity (resistance to change in pH by
addition of strong acid or base), and its ability to maintain a stable pH
upon dilution or addition of neutral salts. Because of the following equilibria, additions of small amounts of strong acid or strong base result in the
removal of only small amounts of the weakly acidic or basic species;
therefore, there is little change in the pH:
HAacid H A conjugate base
6:1
6:2
basic species
acidic species
6:3
45
0.025
0.015
0.005
1.0
0.5
0.0
pH
0.5
1.0
Figure 6.1 Buffer capacity (b) versus DpH over the range 1 pH unit of the pKa for
HEPES (0.05 M). Points calculated using Eq. (6.5), and data from Perrin and Dempsey
(1974).
3. Buffer Selection
There are many factors that must be considered when choosing a buffer.
When studying an enzyme one must consider the pH optimum of the enzyme,
nonspecific buffer effects on the enzyme, and interactions with substrates or
metals. When purifying a protein, cost becomes an important consideration, as
does the compatibility of the buffer with different purification techniques.
Table 6.1 lists a wide variety of buffers covering a broad pH range.
Determining the pH optimum of a protein is a first step in determining the
best buffer to employ (Blanchard, this series). Since the buffering capacity is
maximal at the pK, buffers should be used close to this value. When determining the pH optimum for an enzyme, it is useful to use a series of related buffers
that span a wide pH range. Once an optimal pH has been approximated,
different buffers within this pH range can be examined for specific buffer effects.
The Good buffers have been shown to be relatively free of side effects.
However, inorganic buffers do have a high potential for specific buffer
effects. Many enzymes are inhibited by phosphate buffer, including carboxypeptidase, urease, as well as many kinases and dehydrogenases
(Blanchard, this series). Borate buffers can form covalent complexes with
mono- and oligosaccharides, the ribose moieties of nucleic acids, pyridine
nucleotides, and other gem-diols. Tris and other primary amine buffers may
form Schiff base adducts with aldehydes and ketones.
46
pKa
dpKa/dt
2-(N-Morpholino)
ethanesulfonic acid
Cacodylate
Dimethylarsinic acid
Dimethylglutarate 3,3-Dimethylglutarate (pK2)
Carbonate (pK1)
Citrate (pK3)
BisTris
[Bis(2-hydroxyethyl)imino]tris
(hydroxymethyl)methane
ADA
N-2-Acetamidoiminodiacetic
acid
Pyrophosphate
EDPS (pK1)
N,N 0 -Bis(3-sulfopropyl)
ethylenediamine
BisTris propane 1,3-Bis[tris(hydroxymethyl)
methylamino]propane
PIPES
Piperazine-N,N 0 -bis(2ethanesulfonic acid)
ACES
N-2-Acetamido-2hydroxyethanesulfonic acid
MOPSO
3-(N-Morpholino)-2hydroxypropanesulfonic acid
Imidazole
BES
N,N-Bis-(2-hydroxyethyl)-2aminoethanesulfonic acid
MOPS
3-(N-Morpholino)
propanesulfonic acid
Phosphate (pK2)
EMTA
3,6-Endomethylene-1,2,3,6tetrahydrophthalic acid
TES
2-[Tris(hydroxymethyl)
methylamino]ethanesulfonic
acid
2.15
3.40
3.75
4.21
4.76
4.76
5.13
5.23
5.64
6.10
0.0044
0.0
0.0018
0.0016
0.0002
0.014
0.0
0.011
6.27
6.34
6.35
6.40
6.46
0.0060
0.0055
0.0
0.0
6.59
0.011
Trivial name
Phosphate (pK1)
Malate (pK1)
Formate
Succinate (pK1)
Citrate (pK2)
Acetate
Malate
Pyridine
Succinate (pK2)
MES
6.60
6.65
6.80
6.76
0.0085
6.78
0.020
6.95
0.015
6.95
7.09
0.020
0.016
7.20
0.015
7.20
7.23
0.0028
7.40
0.020
x
47
Buffer name
pKa
dpKa/dt
HEPES
1,4-Bis(3-sulfopropyl)piperazine
N,N-Bis(2-hydroxyethyl)glycine
3-{[Tris(hydroxymethyl)methyl]
amino}propanesulfonic acid
1,4-Bis(4-sulfobutyl)piperazine
2-Aminoethylsulfonic acid,
taurine
Cyclohexylaminoethanesulfonic
acid
N,N 0 -Bis(3-sulfopropyl)
ethylenediamine
3-Aminopropanesulfonic acid
3-(Cyclohexylamino)
propanesulfonic acid
7.48
0.014
7.60
0.015
7.76
7.85
0.020
0.013
DIPSO
TEA
POPSO
EPPS, HEPPS
Tris
Tricine
Glycinamide
PIPPS
Glycylglycine
Bicine
TAPS
Morpholine
PIBS
AES
Borate
Ammonia
Ethanolamine
CHES
Glycine (pK2)
EDPS
APS
Carbonate (pK2)
CAPS
Piperidine
Phosphate (pK3)
8.00
8.06
0.028
8.05
0.021
8.06
8.10
8.25
8.26
8.40
0.029
0.025
0.018
0.018
8.49
8.60
9.06
0.022
9.23
9.25
9.50
9.55
0.008
0.031
0.029
0.029
9.78
9.80
0.025
9.89
10.33
10.40
0.009
0.032
11.12
12.33
0.026
48
Cu2 or Fe2 for hydroxylases). Release of protons upon chelation or precipitation of metalbuffer complexes may also be a potential problem. Where
metal chelation presents a problem, the Good buffers are useful since they have
been shown to have low metal-binding capabilities (Good et al., 1966).
Once a suitable buffer has been found (noninteracting, with an appropriate pK ), a concentration should be chosen. Since high ionic strength may
decrease enzyme activity, the buffer concentration should be as low as
possible (Blanchard, this series). A reasonable way to determine how low
a concentration may be used is to examine the properties (reaction rate or
protein stability) at a low (1020 mM) concentration of buffer. The pH
prior to, and an adequate time after, addition of protein should not vary
more than 0.05 pH. If the pH changes too drastically (greater than 0.1
pH unit), then the buffer concentration should be raised to 50 mM. In cases
where protons are consumed or released stoichiometrically with substrate
utilization, pH stability becomes increasingly important.
Buffers may be made up in stock solutions, then diluted for use. When
stock solutions are made, it should be done close to the working temperature, and in glass bottles (plastic bottles can leach UV-absorbing material)
(Perrin and Dempsey, 1974). Buffers have temperature-sensitive pK values,
particularly amine buffers. The carboxylic acid buffers are generally the least
sensitive to temperature, and the Good buffers have only a small inverse
temperature dependence on pK. The effects of dilution of stock solutions,
or addition of salts, on pH should be checked by measurement of the pH
after addition of all components.
Choosing a buffer for protein purification requires some special considerations. Large amounts of buffer will be needed for centrifugation,
chromatographic separations, and dialysis, which makes cost a concern.
Tris and many inorganic buffers are widely used since they are relatively
inexpensive. Although buffers like Tris are inexpensive, and have been
widely used in protein purification, they do have disadvantages. Tris is a
poor buffer below pH 7.5 and its pK is temperature dependent (a solution
made up to pH 8.06 at 25 C will have a pH of 8.85 at 0 C). Many primary
amine buffers such as Tris and glycine (Bradford, 1976) will interfere with
the Bradford dye-binding protein assay. Some of the Good buffers, HEPES,
EPPS, and Bicine, give false-positive colors with Lowry assay.
Spectroscopic measurement of enzyme rates is a commonly applied
method. It may be important to use a buffer that does not absorb appreciably
in the spectral region of interest. The Good buffers, and most buffers listed
in Table 6.1, can be used above 240 nm.
4. Buffer Preparation
Once a suitable buffer has been chosen it must be dissolved and titrated to
the desired pH. Before titrating a buffer solution, the pH meter must be
calibrated. Calibration should be done using commercially available pH
49
5. Volatile Buffers
In certain cases, it is necessary to remove a buffer quickly and
completely. Volatile buffers make it possible to remove components that
may interfere in subsequent procedures. Volatile buffers are useful in electrophoresis, ion-exchange chromatography, and digestion of proteins followed by separation of peptides or amino acids. Most of the volatile buffers
(Table 6.2) are transparent in the lower UV range except for the buffers
Table 6.2 Types of systems for use as volatile buffersa
System
pH range
1.9
2.1
2.33.5
3.05.0
36
3.1
3.5
4.06.0
4.7
5.57.0
6.5
6.88.8
7.010.0
712
712
7.9
8.010.5
8.510.5
8.9
50
containing pyridine (Perrin and Dempsey, 1974). An important consideration is interference in amino acid analysis (i.e., reactions with ninhydrin).
Most volatile buffers will not interfere with ninhydrin if the concentrations
are not too high (e.g., triethanolamine less than 0.1 M does not interfere).
6. Broad-Range Buffers
There may be occasions where a single buffer system is desired that can
span a wide pH range of perhaps 5 or more pH units. One method would be
a mixture of buffers that sufficiently covers the pH range of interest. This
may lead to nonspecific buffer interactions for which corrections must be
made. Another common approach is to use a series of structurally related
buffers that have evenly spaced pK values such that each pK is separated by
approximately 1 pH unit (the limit of buffering capacity). The Good
buffers are ideal for this approach since they are structurally related and have
relatively evenly spaced pK values. As the pH passes the pK of one buffer it
becomes nonparticipatory and therefore has no further function. These
nonparticipating buffer components may show nonspecific buffer effects
as well as raising the ionic strength with potential deleterious effects.
A detailed description of buffer mixtures which provide a wide range of
buffering capacity with constant ionic strength is available (Ellis and
Morrison, this series).
pH
pH
5.0
6.4
8.2
3.6
3.4
3.2
16.8
24.2
32.4
2.8
2.6
51
pH
46.5
43.7
40.0
37.0
35.0
33.0
31.5
28.0
25.5
23.0
20.5
18.0
16.0
13.7
11.8
9.5
7.2
3.5
6.3
10.0
13.0
15.0
17.0
18.5
22.0
24.5
27.0
29.5
32.0
34.0
36.3
38.2
41.5
42.8
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0
6.2
pH
46.3
44.0
41.0
36.8
30.5
25.5
14.8
10.5
8.8
4.8
3.7
6.0
9.0
13.2
19.5
24.5
35.2
39.5
41.2
45.2
3.6
3.8
4.0
4.2
4.4
4.6
5.0
5.2
5.4
5.6
52
pH
44.6
42.2
39.8
37.7
35.9
33.9
32.3
30.7
29.4
27.8
26.7
25.2
24.3
23.3
22.2
21.0
19.7
17.9
16.9
15.4
13.6
9.1
6.5
5.4
7.8
10.2
12.3
14.1
16.1
17.7
19.3
20.6
22.2
23.3
24.8
25.7
26.7
27.8
29.0
30.3
32.1
33.1
34.6
36.4
40.9
43.6
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0
pH
pH
7.5
10.0
13.3
16.7
20.0
23.5
3.8
4.0
4.2
4.4
4.6
4.8
26.7
30.3
34.2
37.5
40.7
43.5
5.0
5.2
5.4
5.6
5.8
6.0
53
pH
pH
2.7
7.4
29.6
6.0
4.2
7.2
34.8
5.8
6.3
7.0
39.2
5.6
9.3
6.8
43.0
5.4
13.3
6.6
45.0
5.2
18.3
6.4
47.0
5.0
13.8
6.2
7. Phosphate buffer (Sorensen, 1909a,b) stock solutions
A: 0.2 M solution of monobasic sodium phosphate (27.8 g in 1000 ml)
B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HPO4
7H2O or 71.7 g of Na2HPO4 12H2O in 1000 ml)
x ml of A y ml of B, diluted to a total of 200 ml:
x
pH
pH
93.5
92.0
90.0
87.7
85.0
81.5
77.5
73.5
68.5
62.5
56.5
51.0
6.5
8.0
10.0
12.3
15.0
18.5
22.5
26.5
31.5
37.5
43.5
49.0
5.7
5.8
5.9
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
45.0
39.0
33.0
28.0
23.0
19.0
16.0
13.0
10.5
8.5
7.0
5.3
55.0
61.0
67.0
72.0
77.0
81.0
84.0
87.0
90.5
91.5
93.0
94.7
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
pH
pH
1.5
2.5
4.0
6.0
9.0
2.7
17.5
9.2
9.0
8.8
8.6
8.4
8.2
8.0
22.5
27.5
32.5
39.0
43.0
45.0
7.8
7.6
7.4
7.2
7.0
6.8
54
pH
5.0
9.0
8.1
8.8
12.2
8.6
16.5
8.4
21.9
8.4
26.8
8.0
32.5
7.8
38.4
7.6
41.4
7.4
44.2
7.2
10. Boric acidborax buffer (Holmes, 1943) stock solutions
A: 0.2 M solution of boric acid (12.4 g in 1000 ml)
B: 0.05 M solution of borax (19.05 g in 1000 ml; 0.2 M in terms of
sodium borate)
50 ml of A x ml of B, diluted to a total of 200 ml:
x
pH
pH
2.0
3.1
4.9
7.3
11.5
17.5
7.6
7.8
8.0
8.2
8.4
8.6
22.5
30.0
42.5
59.0
83.0
115.0
8.7
8.8
8.9
9.0
9.1
9.2
55
pH
pH
2.0
3.7
5.7
8.5
12.5
16.7
10.0
9.8
9.6
9.4
9.2
9.0
22.0
29.5
34.0
37.7
41.0
43.5
8.8
8.6
8.4
8.2
8.0
7.8
pH
pH
4.0
6.0
8.8
12.0
16.8
8.6
8.8
9.0
9.2
9.4
22.4
27.2
32.0
38.6
45.5
9.6
9.8
10.0
10.4
10.6
pH
0.0
7.0
11.0
17.6
23.0
29.0
34.0
38.6
43.0
46.0
9.28
9.35
9.4
9.5
9.6
9.7
9.8
9.9
10.0
10.1
56
pH
4.0
7.5
9.5
13.0
16.0
19.5
22.0
25.0
27.5
30.0
33.0
35.5
38.5
40.5
42.5
45.0
46.0
42.5
40.5
37.0
34.0
30.5
28.0
25.0
22.5
20.0
17.0
14.5
11.5
9.5
7.5
5.0
9.2
9.3
9.4
9.5
9.6
9.7
9.8
9.9
10.0
10.1
10.2
10.3
10.4
10.5
10.6
10.7
REFERENCES
Blanchard, J. S. (this series). Vol. 104, p. 404.
Bradford, M. M. (1976). Anal. Biochem. 22, 248.
Clark, W. M., and Lubs, H. A. (1917). J. Bacteriol. 2, 1.
Delory, G. E., and King, E. J. (1945). Biochem. J. 39, 245.
Ellis, K. J., and Morrison, J. F. (this series). Vol. 87, p. 405.
Ferguson, W. J., et al. (1980). Anal. Biochem. 104, 300.
Gomori, G. (1946). Proc. Soc. Exp. Biol. Med. 62, 33.
Gomori, G. Unpublished observations.
Good, N. E., and Izawa, S. (this series). Vol. 24, p. 53.
Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., and Singh, R. M. M.
(1966). Biochemistry 5, 467.
Hayaishi, O. (this series). Vol. 1, p. 144.
Holmes, W. (1943). Anat. Rec. 86, 163.
Johnson, R. J., and Metzler, D. E. (this series). Vol. 22, p. 3.
Leirmo, S., Harrison, C., Cayley, D. S., Burgess, R. R., and Record, M. T. (1987).
Biochemistry 26, 2095.
Lillie, R. D. (1948). Histopathologic Technique. Blakiston, Philadelphia, PA.
McIlvaine, T. C. (1921). J. Biol. Chem. 49, 183.
Michaelis, L. (1930). J. Biol. Chem. 87, 33.
Perrin, D. D., and Dempsey, B. (1974). Buffers for pH and Metal Ion Control.
Chapman & Hall, London.
Plumel, M. (1949). Bull. Soc. Chim. Biol. 30, 129.
Sorensen, S. P. L. (1909a). Biochem. Z. 21, 131.
Sorensen, S. P. L. (1909b). Biochem. Z. 22, 352.
Walpole, G. S. (1914). J. Chem. Soc. 105, 2501.