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 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. has provided buffers in the physiologically relevant range (6.1-10.4) of most enzymes, which have limited side effects with most enzymes. 2 It has been found that these buffers are nontoxic 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 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 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) B (base) + H + ~ B H ÷ (conjugate acid)

(1) (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.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.

BUFFERS; PRINCIPLES AND PRACTICE

25

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

(3)

[4]

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 different purification techniques. Table I 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. 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 carboxypeptidase, urease, as well as many kinases and dehydrogenases. 5 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. 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 potential problem. Where metal chelation presents a problem, the Good buffers are useful since they have been shown to have low metal-binding capabilities. 2 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.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]

27

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

Trivial name

Buffer name

PKa

d pKa/dt

Phosphate (pK0 Malate ( p K l ) Formate Succinate (pKI) Citrate (pK2) Acetate Malate Pyridine Succinate (pK2) MES Cacodylate Dimethylglutarate Carbonate (pK0 Citrate (pK3) Bis-Tris

--

2.15 3.40 3.75 4.21 4.76 4.76 5.13 5.23 5.64 6.10 6.27 6.34 6.35 6.40 6.46

0.0044 -0.0 -0.0018 -0.0016 0.0002 --0.014 0.0 -0.011 -0.0060 -0.0055 0.0 0.0

6.59 6.60 6.65 6.80

-0.011 ----

6.76 6.78

-0.0085 -0.020

6.95

-0.015

6.95 7.09

-0.020 -0.016

7.20 7.20

0.015 -0.0028

7.23 7.40

--0.020

7.48

-0.014

7.60

-0.015

7.76 7.85

-0.020 -0.013

8.00

--

ADA Pyrophosphate EDPS (pK0 B i s - T r i s propane PIPES ACES MOPSO Imidazole BES MOPS Phosphate (pK2) EMTA TES HEPES DIPSO TEA POPSO EPPS, HEPPS

--

-------2-(N-Morpholino)ethanesulfonic acid Dimethylarsinic acid 3,3-Dimethylglutarate (pK2) --[Bis(2-hydroxyethyl)imino]tris(hydroxymethyl)methane N-2-Acetamidoiminodiacetic acid --

N,N'-Bis(3-sulfopropyl)ethylenediamine 1,3-Bis[tris(hydroxymethyl)methylamino] propane Piperazine-N,N'-bis(2-ethanesulfonic acid) N-2-Acetamido-2-hydroxyethanesulfonic acid 3-(N-Morpholino)-2-hydroxypropanesulfonic acid -N,N-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid 3-(N-Morpholino)propanesulfonic acid -3,6-Endomethylene-1,2,3,6-tetrahydrophthalic acid 2-[Tris(hydroxymethyl)methylamino]ethanesulfonic acid N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid 3- [N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid Triethanolamine Piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) N-2-Hydroxyethylpiperazine-N'-3-propanesulfonic acid

(continued)

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GENERAL METHODS FOR HANDLINGPROTEINSAND ENZYMES

[4]

TABLE I (continued) Trivial name Tris Tricine Glycinamide PIPPS Glycylglycine Bicine TAPS Morpholine PIBS AES Borate Ammonia Ethanolamine CHES Glycine (pK2) EDPS APS Carbonate (pK2) CAPS Piperidine Phosphate (pK3)

Buffer name

pKa

d pKa/dt

Tris(hydroxymethyl)aminomethane N-[Tris(hydroxymethyl)methyl]glycine -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' -Bis(3-sulfopropyl)ethylenediamine 3-Aminopropanesulfonic acid -3-(Cyclohexylamino)propanesulfonic acid ---

8.06 8.05 8.06 8.10 8.25 8.26 8.40

-0.028 -0.021 - 0.029 --0.025 -0.018 0.018

8.49 8.60 9.06 9.23 9.25 9.50 9.55 9.78 9.80 9.89 10.33 10.40 11.12 12.33

---0.022 -0.008 - 0.031 -0.029 0.029 -0.025 ---0.009 0.032 --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 utilization, 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 temperature, and in glass bottles (plastic bottles can leach UV-absorbing material). 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 addition of all c o m p o n e n t s . Choosing a buffer for protein purification requires some special considerations. 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 appreciably 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 titrated to the desired pH. Before titrating a buffer solution the pH meter must be calibrated. Calibration should be done using commercially available pH standards, bracketing the desired pH. If monovalent cations interfere, or are being investigated, then titration with tetramethylammonium hydroxide can be done to avoid mineral cations. Similarly, the substitution 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 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 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 87 ml Glacial acetic acid + 25 ml 88% HCOOH in 11 liters 25 ml 88% HCOOH in 1 liter Pyridine-formic acid Trimethylamine-formic acid Triethylamine-formic (or acetic) acid 5 ml Pyridine + 100 ml glacial acetic acid in 1 liter 5 ml Pyridine + 50 ml glacial acetic acid in 1 liter Trimethylamine-acetic acid 25 ml Pyridine + 25 mi glacial acetic acid in 1 liter Collidine-acetic acid 100 ml Pyridine + 4 ml glacial acetic acid in 1 liter Triethanolamine-HC1 Ammonia-formic (or acetic) acid Trimethylamine-C02 Triethylamine-CO2 24 g NH4HCO3 in 1 liter Ammonium carbonate-ammonia Ethanolamine-HCl 20 g (NH4)2CO3 in 1 liter a

pH range 1.9 2.1 2.3-3.5 3.0-5.0 3-6 3.1 3.5 4.0-6.0 4.7 5.5-7.0 6.5 6.8-8.8 7.0-10.0 7-12 7-12 7.9 8.0-10.5 8.5-10.5 8.9

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 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

[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 6.4 8.2

3.6 3.4 3.2

16.8 24.2 32.4

2.8 2.6

. 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 43.7 40.0 37.0 35.0 33.0 31.5

3.5 6.3 10.0 13.0 15.0 17.0 18.5

3.0 3.2 3.4 3.6 3.8 4.0 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 x

y

pH

28.0 25.5 23.0 20.5 18.0 16.0 13.7 11.8 9.5 7.2

22.0 24.5 27.0 29.5 32.0 34.0 36.3 38.2 41.5 42.8

4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2

[4]

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 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

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 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).

of

[4]

BUFFERS: PRINCIPLES AND PRACTICE

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

y

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

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 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

13 G. Gomori, unpublished observations.

33

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 4.2 6.3 9.3 13.3 18.3 13.8

7.4 7.2 7.0 6.8 6.6 6.4 6.2

29.6 34.8 39.2 43.0 45.0 47.0

6.0 5.8 5.6 5.4 5.2 5.0

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 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

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 2.5 4.0 6.0 9.0 2.7 17.5 22.5 27.5 32.5 39.0 43.0 45.0

9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 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 8.1 12.2 16.5

9.0 8.8 8.6 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 x

pH

21.9 26.8 32.5 38.4 41.4 ~.2

8.4 8.0 7.8 7.6 7.4 7.2

[4]

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 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

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 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

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 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

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 7.0 11.0 17.6 23.0 29.O 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

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 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

[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 preparation. 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 classification 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.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.

Buffers: principles and practice.

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...
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