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[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
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.
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
TABLE I
SELECTED BUFFERS AND THEm p K VALUES AT 25 °
(continued)
28 GENERAL
METHODS FOR HANDLINGPROTEINSAND ENZYMES [4]
TABLE I (continued)
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
TABLE II
TYPES OF SYSTEMS FOR USE AS VOLATILE BUFFERSa
System pH range
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
. 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
. 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
x y pH
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
. 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)
x y pH
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
. 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
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
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
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
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
x pH x pH
x pH x pH
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
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
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)
x y pH
[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.