24 GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES [4]

[4] B u f f e r s : P r i n c i p l e s a n d P r a c t i c e
By VINCENT S. STOLL and JOHN S. BLANCHARD

The necessity for maintaining a stable pH when studying enzymes is

well established.I Biochemical processes can be severely affected by mi-
nute 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. has provided buffers in the physiologically
relevant range (6.1-10.4) of most enzymes, which have limited side ef-
fects with most enzymes. 2 It has been found that these buffers are non-
toxic to cells at 50 m M concentrations and in some cases much higher. 3

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". 4 Buffers
consist of an acid and its conjugate base, such as carbonate and bicarbon-
ate, 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 basc), 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:
H A (acid) ~- H ÷ + A - (conjugate base) (1)
B (base) + H + ~ B H ÷ (conjugate acid) (2)
The pH of a solution of a weak acid or base may be calculated from the
Henderson-Hasselbalch equation:

R. J. J o h n s o n and D. E. Metzler, this series, Vol. 22, p. 3; N. E. Good and S. lzawa, Vol.
24, p. 53.
2 N. E. Good, G. D. Winget, W. Winter, T. N. Connolly, S. Izawa, and R. M. M. Singh,
Biochemistry 5, 467 (1966).
3 W. J. F e r g u s o n et al., Anal. Biochem. 104, 300 (1980).
4 D. D. Perrin a n d B. D e m p s e y , " B u f f e r s for p H and Metal Ion C o n t r o l . " C h a p m a n & Hall,
L o n d o n , 1974.

Copyright © 1990by AcademicPress, Inc.

METHODS IN ENZYMOLOGY, VOL. 182 All rights of reproduction in any form reserved.
[4] BUFFERS; PRINCIPLES AND PRACTICE 25

pH = pK" + log[basic species]/[acidic species] (3)

The pKa of a buffer is that pH where the concentrations of basic and acidic
species are equal, and in this basic form the equation is accurate between
the pH range of 3 to 11. Below pH 3 and above pH 11 the concentrations
of the ionic species of water must be included in the equation. 4 Since the
pH range of interest here is generally in the pH 3-11 range, this will be
ignored•
From the Henderson-Hasselbalch equation an expression for buffer
capacity may be deduced. If at some concentration of buffer, c, the sum
[A-] + [HA] is constant, then the amount of strong acid or base needed to
cause a small change in pH is given by the relationship

dpH t(Ka + [H+]) 2 + [H÷] + [--ffq (4)

In this equation Kw refers to the ionic product of water, and the second
and third terms are only significant below pH 3 or above pH 11. In the pH
range of interest (pH 3-11) this equation yields the following expression:
timex = 2.303c/4 = 0.576c (5)
which represents a maximum value for d [B]/d pH when pH = pKa. The
buffer capacity of any buffer is dependent on the concentration, c, and
may be calculated over a buffer range of - 1 pH unit around the pK to
determine the buffer capacity, as shown in Fig. 1 for one of the Good
buffers, HEPES. It can be seen that the buffer capacity is greatest at its

0.025

0.015

0,005
_d
•0 -0.5 0~.0 O'.5 1.0
ApH
FIG. 1. Buffercapacity(/3)versus ApH over the range -+ 1 pH unitof the pKafor HEPES
(0.05 M). Points calculatedusingEq. (5), and data fromD. D. Perrin and Dempsey,"Buffers
for pH and Metal Ion Control" (Chapmanand Hall, London, 1974).
26 GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES [4]

pK, and drops off quickly I pH unit on either side of the pK. In practice,
buffers should not be used beyond these values.

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 dif-
ferent purification techniques. Table I lists a wide variety of buffers cov-
ering a broad pH range.
Determining the pH optimum of a protein is a first step in determining
the best buffer to employ. 5 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 car-
boxypeptidase, urease, as well as many kinases and dehydrogenases. 5
Borate buffers can form covalent complexes with mono- and oligosac-
charides, 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.
Buffer complexation with metals may present additional problems. In
this respect inorganic buffers can prove problematic in that they may
remove, by chelation, metals essential to enzymatic activity (e.g., Mg 2÷
for kinases, Cu 2÷ or Fe 2÷ for hydroxylases). Release of protons upon
chelation or precipitation of metal-buffer complexes may also be a poten-
tial problem. Where metal chelation presents a problem, the Good buffers
are useful since they have been shown to have low metal-binding capa-
bilities. 2
Once a suitable buffer has been found (noninteracting, with an appro-
priate pK), a concentration should be chosen. Since high ionic strength
may decrease enzyme activity, the buffer concentration should be as low
as possible.5 A reasonable way to determine how low a concentration may
be used is to examine the properties (reaction rate, or protein stability) at

5 j. S. Blanchard, this series, Vol. 104, p. 404.

[4] BUFFERS" PRINCIPLES AND PRACTICE 27

TABLE I
SELECTED BUFFERS AND THEm p K VALUES AT 25 °

Trivial name Buffer name PKa d pKa/dt

Phosphate (pK0 -- 2.15 0.0044

Malate ( p K l ) -- 3.40 --
Formate -- 3.75 0.0
Succinate (pKI) -- 4.21 -0.0018
Citrate (pK2) -- 4.76 -0.0016
Acetate -- 4.76 0.0002
Malate -- 5.13 --
Pyridine -- 5.23 -0.014
Succinate (pK2) -- 5.64 0.0
MES 2-(N-Morpholino)ethanesulfonic acid 6.10 -0.011
Cacodylate Dimethylarsinic acid 6.27 --
Dimethylglutarate 3,3-Dimethylglutarate (pK2) 6.34 0.0060
Carbonate (pK0 -- 6.35 -0.0055
Citrate (pK3) -- 6.40 0.0
Bis-Tris [Bis(2-hydroxyethyl)imino]tris(hydroxy- 6.46 0.0
methyl)methane
ADA N-2-Acetamidoiminodiacetic acid 6.59 -0.011
Pyrophosphate -- 6.60 --
EDPS (pK0 N,N'-Bis(3-sulfopropyl)ethylenediamine 6.65 --
B i s - T r i s propane 1,3-Bis[tris(hydroxymethyl)methylamino] 6.80 --
propane
PIPES Piperazine-N,N'-bis(2-ethanesulfonic acid) 6.76 -0.0085
ACES N-2-Acetamido-2-hydroxyethanesulfonic 6.78 -0.020
acid
MOPSO 3-(N-Morpholino)-2-hydroxypropane- 6.95 -0.015
sulfonic acid
Imidazole -- 6.95 -0.020
BES N,N-Bis-(2-hydroxyethyl)-2-amino- 7.09 -0.016
ethanesulfonic acid
MOPS 3-(N-Morpholino)propanesulfonic acid 7.20 0.015
Phosphate (pK2) -- 7.20 -0.0028
EMTA 3,6-Endomethylene-1,2,3,6-tetrahydro-
phthalic acid 7.23 --
TES 2-[Tris(hydroxymethyl)methylamino]ethane- 7.40 -0.020
sulfonic acid
HEPES N-2-Hydroxyethylpiperazine-N'-2-ethane- 7.48 -0.014
sulfonic acid
DIPSO 3- [N-Bis(hydroxyethyl)amino]-2-hydroxy- 7.60 -0.015
propanesulfonic acid
TEA Triethanolamine 7.76 -0.020
POPSO Piperazine-N,N'-bis(2-hydroxypropane- 7.85 -0.013
sulfonic acid)
EPPS, HEPPS N-2-Hydroxyethylpiperazine-N'-3-propane- 8.00 --
sulfonic acid

(continued)
28 GENERAL
METHODS FOR HANDLINGPROTEINSAND ENZYMES [4]

TABLE I (continued)

Trivial name Buffer name pKa d pKa/dt

Tris Tris(hydroxymethyl)aminomethane 8.06 -0.028

Tricine N-[Tris(hydroxymethyl)methyl]glycine 8.05 -0.021
Glycinamide -- 8.06 - 0.029
PIPPS 1,4-Bis(3-sulfopropyl)piperazine 8.10 --
Glycylglycine -- 8.25 -0.025
Bicine N,N-Bis(2-hydroxyethyl)glycine 8.26 -0.018
TAPS 3-{[Tris(hydroxymethyl)methyl]amino}pro- 8.40 0.018
panesulfonic acid
Morpholine -- 8.49 --
PIBS 1,4-Bis(4-sulfobutyl)piperazine 8.60 --
AES 2-Aminoethylsulfonic acid, taurine 9.06 -0.022
Borate -- 9.23 -0.008
Ammonia -- 9.25 - 0.031
Ethanolamine -- 9.50 -0.029
CHES Cyclohexylaminoethanesulfonic acid 9.55 0.029
Glycine (pK2) -- 9.78 -0.025
EDPS N, N' -Bis(3-sulfopropyl)ethylenediamine 9.80 --
APS 3-Aminopropanesulfonic acid 9.89 --
Carbonate (pK2) -- 10.33 -0.009
CAPS 3-(Cyclohexylamino)propanesulfonic acid 10.40 0.032
Piperidine -- 11.12 --
Phosphate (pK3) -- 12.33 -0.026

a low (10-20 m M ) concentration of buffer. The p H prior to, and an

adequate time after, addition o f protein should not vary more than -+ 0.05
pH. If the p H changes too drastically (greater than - 0.1 p H unit), then
the buffer concentration should be raised to 50 mM. In cases where
protons are c o n s u m e d or released stoichiometrically with substrate utili-
zation, p H stability b e c o m e s increasingly important.
Buffers m a y be made up in stock solutions, then diluted for use. When
stock solutions are made, it should be done close to the working tempera-
ture, and in glass bottles (plastic bottles can leach UV-absorbing mate-
rial). 4 Buffers have temperature-sensitive p K values, particularly amine
buffers. The carboxylic acid buffers are generally the least sensitive to
temperature, and the G o o d buffers have only a small inverse temperature
d e p e n d e n c e on pK. The effects of dilution of stock solutions, or addition
of salts, on p H should be c h e c k e d by measurement of the pH after addi-
tion of all c o m p o n e n t s .
Choosing a buffer for protein purification requires some special con-
siderations. L a r g e amounts of buffer will be needed for centrifugation,
[4] BUFFERS:PRINCIPLES AND PRACTICE 29

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° will have a pH of 8.85 at 0°). Many primary
amine buffers such as Tris and glycine6 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 apprecia-
bly in the spectral region of interest. The Good buffers, and most buffers
listed in Table I, can be used above 240 nm.

Buffer Preparation
Once a suitable buffer has been chosen it must be dissolved and ti-
trated to the desired pH. Before titrating a buffer solution the pH meter
must be calibrated. Calibration should be done using commercially avail-
able pH standards, bracketing the desired pH. If monovalent cations
interfere, or are being investigated, then titration with tetramethylammo-
nium hydroxide can be done to avoid mineral cations. Similarly, the sub-
stitution of the most commonly used counteranion, chloride, with other
anions such as acetate, sulfate, or glutamate, may have significant effects
on enzyme activity or protein-DNA interactions. 7 Stock solutions should
be made with quality water (deionized and double-distilled, preferably)
and filtered through a sterile ultrafiltration system (0.22/zm) to prevent
bacterial or fungal growth, especially with solutions in the pH 6-8 range.
To prevent heavy metals from interfering, EDTA (10-100/zM) may be
added to chelate any contaminating metals.

Volatile Buffers

In certain cases it is necessary to remove a buffer quickly and com-

pletely. Volatile buffers make it possible to remove components that may
interfere in subsequent procedures. Volatile buffers are useful in electro-
phoresis, ion-exchange chromatography, and digestion of proteins fol-
lowed by separation of peptides or amino acids. Most of the volatile

6 M. M. Bradford, Anal. Biochem. 22, 248 (1976).

7 S. Leirmo, C. Harrison, D. S. Cayley, R. R. Burgess, and M. T. Record, Biochemistry 26,
2095 (1987).
30 GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES [4]

TABLE II
TYPES OF SYSTEMS FOR USE AS VOLATILE BUFFERSa

System pH range

87 ml Glacial acetic acid + 25 ml 88% HCOOH in 11 liters 1.9

25 ml 88% HCOOH in 1 liter 2.1
Pyridine-formic acid 2.3-3.5
Trimethylamine-formic acid 3.0-5.0
Triethylamine-formic (or acetic) acid 3-6
5 ml Pyridine + 100 ml glacial acetic acid in 1 liter 3.1
5 ml Pyridine + 50 ml glacial acetic acid in 1 liter 3.5
Trimethylamine-acetic acid 4.0-6.0
25 ml Pyridine + 25 mi glacial acetic acid in 1 liter 4.7
Collidine-acetic acid 5.5-7.0
100 ml Pyridine + 4 ml glacial acetic acid in 1 liter 6.5
Triethanolamine-HC1 6.8-8.8
Ammonia-formic (or acetic) acid 7.0-10.0
Trimethylamine-C02 7-12
Triethylamine-CO2 7-12
24 g NH4HCO3 in 1 liter 7.9
Ammonium carbonate-ammonia 8.0-10.5
Ethanolamine-HCl 8.5-10.5
20 g (NH4)2CO3 in 1 liter 8.9

a From D. D. Perrin and Boyd Dempsey, "Buffers for pH and Metal

Ion Control." Chapmanand Hall, London, 1974.

buffers (Table II) are transparent in the lower UV range except for the
buffers containing pyridine. 4 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).

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 cor-
rections 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
[4] BUFFERS: PRINCIPLES AND PRACTICE 31

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. 8

Recipes for Buffer Stock Solutions

. Glycine-HCl Buffer 9

Stock Solutions
A: 0.2 M solution of glycine (15.01 g in 1000 ml)
B: 0.2 M HCI
50 ml of A + x ml of B, diluted to a total of 200 ml:

x pH x pH

5.0 3.6 16.8 2.8

6.4 3.4 24.2 2.6
8.2 3.2 32.4

. Citrate Buffer 1°

S t o c k Solutions
A: 0.1 M solution of citric acid (21.01 g in 1000 ml)
B: 0.1 M solution of sodium citrate (29.41 g C 6 H s O 7 N a 3 " 2H20 in
1000 ml)
x ml of A + y ml of B, diluted to a total of I00 ml:

x y pH

46.5 3.5 3.0

43.7 6.3 3.2
40.0 10.0 3.4
37.0 13.0 3.6
35.0 15.0 3.8
33.0 17.0 4.0
31.5 18.5 4.2

s K. J. Ellis and J. F. Morrison, this series, Vol. 87, p. 405.

9 S. P. L. Sorensen, Biochem. Z. 21, 131 (1909); 22, 352 (1909).
10 R. D. Lillie, "Histopathologic Technique." Blakiston, Philadelphia, Pennsylvania, 1948.
32 GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES [4]

x y pH

28.0 22.0 4.4

25.5 24.5 4.6
23.0 27.0 4.8
20.5 29.5 5.0
18.0 32.0 5.2
16.0 34.0 5.4
13.7 36.3 5.6
11.8 38.2 5.8
9.5 41.5 6.0
7.2 42.8 6.2

3. Acetate Buffer 11

Stock Solutions
A: 0.2 M solution of acetic acid (11.55 ml in I000 ml)
B : 0.2 M solution of sodium acetate (16.4 g of C2H302Na or 27.2 g of
C2H302Na" 3H20 in 1000 ml)
x ml of A + y ml of B, diluted to a total of 100 ml:

x y pH

46.3 3.7 3.6

44.0 6.0 3.8
41.0 9.0 4.0
36.8 13.2 4.2
30.5 19.5 4.4
25.5 24.5 4.6
14.8 35.2 5.0
10.5 39.5 5.2
8.8 41.2 5.4
4.8 45.2 5.6

. Citrate-Phosphate Buffer 12

Stock Solutions
A: 0.1 M solution of citric acid (19.21 g in 1000 ml)
B: 0.2 M solution of dibasic sodium phosphate (53.65 g of
Na2HPO4.7H20 or 71.7 g of Na2HPO4" 12H20 in 1000 ml)

ii G. S. Walpole, J. Chem. Soc. 105, 2501 (1914).

t2 T. C. McIlvaine, J. Biol. Chem. 49, 183 (1921).
[4] BUFFERS: PRINCIPLES AND PRACTICE 33

x ml of A + y ml of B, diluted to a total of 100 ml:

x y pH

44.6 5.4 2.6

42.2 7.8 2.8
39.8 10.2 3.0
37.7 12.3 3.2
35.9 14.1 3.4
33.9 16.1 3.6
32.3 17.7 3.8
30.7 19.3 4.0
29.4 20.6 4.2
27.8 22.2 4.4
26.7 23.3 4.6
25.2 24.8 4.8
24.3 25.7 5.0
23.3 26.7 5.2
22.2 27.8 5.4
21.0 29.0 5.6
19.7 30.3 5.8
17.9 32.1 6.0
16.9 33.1 6.2
15.4 34.6 6.4
13.6 36.4 6.6
9.1 40.9 6.8
6.5 43.6 7.0

5. Succinate Buffer 13

Stock Solutions
A: 0.2 M solution of succinic acid (23.6 g in 1000 ml)
B: 0.2 M NaOH
25 ml of A + x ml of B, diluted to a total of 100 ml:

x pH x pH

7.5 3.8 26.7 5.0

10.0 4.0 30.3 5.2
13.3 4.2 34.2 5.4
16.7 4.4 37.5 5.6
20.0 4.6 40.7 5.8
23.5 4.8 43.5 6.0

13 G. Gomori, unpublished observations.

34 GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES [4]

. Cacodylate Buffer 14

Stock Solutions
A: 0.2 M solution of sodium cacodylate (42.8 g of Na(CH3)2AsO2 • 3H20
in 1000 ml)
B: 0.2 M NaOH
50 ml of A + x ml of B, diluted to a total of 200 ml:

x pH x 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 9
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 • 7H20 or 71.7 g of Na2HPO4.12H20 in 1000 ml)
x ml of A + y ml of B, diluted to a total of 200 ml:

x y pH x y pH

93.5 6.5 5.7 45.0 55.0 6.9

92.0 8.0 5.8 39.0 61.0 7.0
90.0 10.0 5.9 33.0 67.0 7.1
87.7 12.3 6.0 28.0 72.0 7.2
85.0 15.0 6.1 23.0 77.0 7.3
81.5 18.5 6.2 19.0 81.0 7.4
77.5 22.5 6.3 16.0 84.0 7.5
73.5 26.5 6.4 13.0 87.0 7.6
68.5 31.5 6.5 10.5 90.5 7.7
62.5 37.5 6.6 8.5 91.5 7.8
56.5 43.5 6.7 7.0 93.0 7.9
51.0 49.0 6.8 5.3 94.7 8.0

14 M. Plumel, Bull. Soc. Chim. Biol. 311, 129 (1949).

[4] BUFFERS: PRINCIPLES AND PRACTICE 35

8. Barbital Buffer 15

Stock Solutions
A: 0.2 M solution o f sodium barbital (veronal) (41.2 g in 1000 ml)
B: 0.2 M HC1
50 ml o f A + x ml o f B, diluted to a total of 200 ml:

x pH

1.5 9.2
2.5 9.0
4.0 8.8
6.0 8.6
9.0 8.4
2.7 8.2
17.5 8.0
22.5 7.8
27.5 7.6
32.5 7.4
39.0 7.2
43.0 7.0
45.0 6.8

Solutions more concentrated than 0.05 M may crystallize on standing,

especially in the cold.

. Tris(hydroxymethyl)aminomethane (Tris) Buffer 16

Stock Solutions
A: 0.2 M solution of tris(hydroxymethyl)aminomethane (24.2 g in 1000
ml)
B: 0.2 M HC1
50 ml of A + x ml of B, diluted to a total of 200 ml:

x pH

5.0 9.0
8.1 8.8
12.2 8.6
16.5 8.4

15 L. Michaelis, J. Biol. Chem. 87, 33 (1930).

16 O. Hayaishi, this series, Vol. 1, p. 144.
36 GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES [4]

x pH

21.9 8.4
26.8 8.0
32.5 7.8
38.4 7.6
41.4 7.4
~.2 7.2

10. Boric Acid-Borax Buffer 17

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 x pH

2.0 7.6 22.5 8.7

3.1 7.8 30.0 8.8
4.9 8.0 42.5 8.9
7.3 8.2 59.0 9.0
11.5 8.4 83.0 9.1
17.5 8.6 115.0 9.2

11. 2-Amino-2-methyi-l ,3-propanediol (Ammediol) Buffer is

Stock Solutions
A: 0.2 M solution of 2-amino-2-methyl-l,3-propanediol (21.03 g in 1000
ml)
B: 0.2 M HC1
50 ml of A + x ml of B, diluted to a total of 200 ml:

x pH x pH

2.0 10.0 22.0 8.8

3.7 9.8 29.5 8.6
5.7 9.6 34.0 8.4
8.5 9.4 37.7 8.2
12.5 9.2 41.0 8.0
16.7 9.0 43.5 7.8

i~ W. Holmes, Anat. Rec. 86, 163 (1943).

is G. Gomori, Proc. Soc. Exp. Biol. Med. 62, 33 (1946).
[4] BUFFERS: PRINCIPLES AND PRACTICE 37

12. Glycine-NaOH Buffer 9

Stock Solutions
A: 0.2 M solution of glycine (15.01 g in 1000 ml)
B: 0.2 M NaOH
50 ml of A + x ml of B, diluted to a total of 200 ml:

x pH x pH

4.0 8.6 22.4 9.6

6.0 8.8 27.2 9.8
8.8 9.0 32.0 10.0
12.0 9.2 38.6 10.4
16.8 9.4 45.5 10.6

13. Borax-NaOH Buffer 19

Stock Solutions
A: 0.05 M solution of borax (19.05 g in 1000 ml; 0.02 M in terms of
sodium borate)
B: 0.2 M NaOH
50 ml o f A + x ml o f B, diluted to a total o f 200 ml:

x pH

0.0 9.28
7.0 9.35
11.0 9.4
17.6 9.5
23.0 9.6
29.O 9.7
34.0 9.8
38.6 9.9
43.0 10.0
46.0 10.1

14. Carbonate-Bicarbonate Buffer 2°

Stock Solutions
A: 0.2 M solution of anhydrous sodium carbonate (21.2 g in 1000 ml)
B: 0.2 M solution of sodium bicarbonate (16.8 g in 1000 ml)

19 W. M. Clark and H. A. Lubs, J. Bacteriol. 2, 1 (1917).

20 G. E. Delory and E. J. King, Biochem. J. 39, 245 (1945).
38 GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES [5]

x ml of A + y ml of B, diluted to a total of 200 ml:

x y pH

4.0 46.0 9.2

7.5 42.5 9.3
9.5 40.5 9.4
13.0 37.0 9.5
16.0 34.0 9.6
19.5 30.5 9.7
22.0 28.0 9.8
25.0 25.0 9.9
27.5 22.5 10.0
30.0 20.0 10.1
33.0 17.0 10.2
35.5 14.5 10.3
38.5 11.5 10.4
40.5 9.5 10.5
42.5 7.5 10.6
45.0 5.0 10.7

[5] M e a s u r e m e n t o f E n z y m e A c t i v i t y
By EDWARD F. ROSSOMANDO

This chapter deals with the development of methods for the assay of
enzyme activity in a cell lysate or in a partially purified enzyme prepara-
tion. They are also applicable during purification and for purified enzymes
as well. Preparations that contain more than one protein will be referred
to as multizymes.

Concepts in the Measurement of Enzyme Activity

Anatomy of Enzyme Assay 1

Dissection of a representative assay reveals several distinct parts (Fig.
1). However, some assays may not require all the components, and the
absence of one or another of these can provide the basis for a classifica-
tion scheme (see below).

i E. F. R o s s o m a n d o , " H i g h P e r f o r m a n c e Liquid C h r o m a t o g r a p h y in E n z y m a t i c A n a l y s i s . "

Wiley, N e w York, 1987.

Copyright © 1990by AcademicPress, Inc.

METHODS IN ENZYMOLOGY, VOL. 182 All rights of reproduction in any form reserved.