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 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 effects with most enzymes. 2 It has been found that these buffers are non- toxic to cells at 50 mM 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) (1) B (base) + H + ~ BH ÷ (conjugate acid) (2)

The pH of a solution of a weak acid or base may be calculated from the Henderson-Hasselbalch equation:

R. J. Johnson 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. Fe rguson et al., Anal. Biochem. 104, 300 (1980). 4 D. D. Perrin and B. Dempsey , "Buf fe r s for pH and Metal Ion Cont ro l . " Chapman & Hall,

London , 1974.

Copyright © 1990 by Academic Press, 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

d p H 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. Buffer capacity (/3) versus ApH over the range -+ 1 pH unit of the pKa for HEPES

(0.05 M). Points calculated using Eq. (5), and data from D. D. Perrin and Dempsey, "Buffers for pH and Metal Ion Control" (Chapman and Hall, London, 1974).


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 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 potential 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 pK 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 - - Bis-Tris 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)


TABLE I (continued)

Trivial name Buffer name pKa d pKa/dt

T r i s Tris(hydroxymethyl)aminomethane 8.06 -0.028 T r i c i n e 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 B i c i n e N,N-Bis(2-hydroxyethyl)glycine 8.26 -0.018 T A P S 3-{[Tris(hydroxymethyl)methyl]amino}pro- 8.40 0.018

panesulfonic acid Morpholine - - 8.49 - - P I B S 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 C A P S 3-(Cyclohexylamino)propanesulfonic acid 10.40 0.032 Piperidine - - 11.12 - - Phosphate (pK3) - - 12.33 -0.026

a low (10-20 mM) concentrat ion 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 concentrat ion should be raised to 50 mM. In cases where protons are consumed or released stoichiometrically with substrate utili- zation, 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 mate- rial). 4 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 con- siderations. Large 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 glycine 6 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 available 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.

V o l a t i l e B u f f e r s

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).


TABLE II TYPES OF SYSTEMS FOR USE AS VOLATILE BUFFERS a

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." Chapman and 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

R e c i p e s fo r B u f f e r S t o c k S o l u t i o n s

. 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°

Stock 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 C6HsO7Na3" 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.


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

Na2HPO4.7H20 or 71.7 g of Na2HPO4" 12H20 in 1000 ml) g o f

ii G. S. Walpole, J. Chem. Soc. 105, 2501 (1914). t2 T. C. McIlvaine, J. Biol. Chem. 49, 183 (1921).



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


x pH x pH

7.5 10.0 13.3 16.7 20.0 23.5

3.8 26.7 5.0 4.0 30.3 5.2 4.2 34.2 5.4 4.4 37.5 5.6 4.6 40.7 5.8 4.8 43.5 6.0

13 G. Gomori, unpublished observations.


. 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


x pH x pH

2.7 7.4 4.2 7.2 6.3 7.0 9.3 6.8

13.3 6.6 18.3 6.4 13.8 6.2

29.6 6.0 34.8 5.8 39.2 5.6 43.0 5.4 45.0 5.2 47.0 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 y pH x y pH

93.5 6.5 92.0 8.0 90.0 10.0 87.7 12.3 85.0 15.0 81.5 18.5 77.5 22.5 73.5 26.5 68.5 31.5 62.5 37.5 56.5 43.5 51.0 49.0

5.7 45.0 5.8 39.0 5.9 33.0 6.0 28.0 6.1 23.0 6.2 19.0 6.3 16.0 6.4 13.0 6.5 10.5 6.6 8.5 6.7 7.0 6.8 5.3

55.0 6.9 61.0 7.0 67.0 7.1 72.0 7.2 77.0 7.3 81.0 7.4 84.0 7.5 87.0 7.6 90.5 7.7 91.5 7.8 93.0 7.9 94.7 8.0

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


8. Barbital Buffer 15

Stock Solutions

A: 0.2 M solut ion 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 o f 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


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.


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)


x pH x pH

2.0 7.6 22.5 3.1 7.8 30.0 4.9 8.0 42.5 7.3 8.2 59.0

11.5 8.4 83.0 17.5 8.6 115.0

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


x pH x pH

2.0 10.0 22.0 3.7 9.8 29.5 5.7 9.6 34.0 8.5 9.4 37.7

12.5 9.2 41.0 16.7 9.0 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).


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


x pH x pH

4.0 8.6 6.0 8.8 8.8 9.0

12.0 9.2 16.8 9.4

22.4 9.6 27.2 9.8 32.0 10.0 38.6 10.4 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).



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 Act iv i ty

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 classifica- tion scheme (see below).

i E. F. R o s s o m a n d o , " H i g h Per formance Liquid Chromatography in Enzymat ic Ana ly s i s . " Wiley, N e w York, 1987.

Copyright © 1990 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 182 All rights of reproduction in any form reserved.

buffers- principles and practice

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