C H A P T E R

S I X

Buffers: Principles and Practice1
Vincent S. Stoll and John S. Blanchard
Contents
43
44
45
48
49
50
50
56

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
(6.1–10.4) 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).

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York
Reprinted from Methods in Enzymology, Volume 182 (Academic Press, 1990)

1

Methods in Enzymology, Volume 463
ISSN 0076-6879, DOI: 10.1016/S0076-6879(09)63006-8

Copyright # 1990 by Academic Press
All rights reserved.

43

The buffer capacity of any buffer is dependent on the concentration. 1974). the sum [A] þ [HA] is constant. there is little change in the pH: HAðacidÞ Ð Hþ þ A ðconjugate baseÞ ð6:1Þ BðbaseÞ þ Hþ Ð BHþ ðconjugate acidÞ ð6:2Þ The pH of a solution of a weak acid or base may be calculated from the Henderson–Hasselbalch equation: pH ¼ pKa0 þ log ½basic species ½acidic species ð6:3Þ The pKa of a buffer is that pH where the concentrations of basic and acidic species are equal. c. and in this basic form the equation is accurate between the pH range of 3–11. In the pH range of interest (pH 3–11). Since the pH range of interest here is generally in the pH 3–11 range. 1974). From the Henderson–Hasselbalch equation an expression for buffer capacity may be deduced. 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. Kw refers to the ionic product of water. Below pH 3 and above pH 11 the concentrations of the ionic species of water must be included in the equation (Perrin and Dempsey. Buffers consist of an acid and its conjugate base. Because of the following equilibria. this equation yields the following expression: 2:303c ¼ 0:576c. c. If at some concentration of buffer. The quality of a buffer is dependent on its buffering capacity (resistance to change in pH by addition of strong acid or base). this will be ignored. and may bmax ¼ . therefore. ð6:5Þ 4 which represents a maximum value for d[B]/dpH when pH ¼ pKa. then the amount of strong acid or base needed to cause a small change in pH is given by the relationship: ( ) d½B ½Ka0 c½Hþ  K w ð6:4Þ þ ½Hþ  þ þ ¼ 2:303 H dpH ðKa0 þ ½Hþ Þ2 In this equation. such as carbonate and bicarbonate. Stoll and John S. Blanchard 2. and its ability to maintain a stable pH upon dilution or addition of neutral salts. and the second and third terms are only significant below pH 3 or above pH 11. or acetate and acetic acid. 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.44 Vincent S.

When determining the pH optimum for an enzyme. HEPES. When studying an enzyme one must consider the pH optimum of the enzyme. urease. Points calculated using Eq. 3. as shown in Fig. the ribose moieties of nucleic acids.5 0. Many enzymes are inhibited by phosphate buffer. Buffer Selection There are many factors that must be considered when choosing a buffer. 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. this series). 6. When purifying a protein.005 −1. it is useful to use a series of related buffers that span a wide pH range. nonspecific buffer effects on the enzyme. cost becomes an important consideration.015 0. However. this series). buffers should be used close to this value.45 Buffers: Principles and Practice b 0. Table 6.0 Figure 6. It can be seen that the buffer capacity is greatest at its pK. be calculated over a buffer range of  1 pH unit around the pK to determine the buffer capacity.and oligosaccharides.5). including carboxypeptidase. pyridine nucleotides. buffers should not be used beyond these values. Since the buffering capacity is maximal at the pK.0 ΔpH 0. In practice.025 0.05 M). as does the compatibility of the buffer with different purification techniques. and data from Perrin and Dempsey (1974). as well as many kinases and dehydrogenases (Blanchard. Tris and other primary amine buffers may form Schiff base adducts with aldehydes and ketones. Determining the pH optimum of a protein is a first step in determining the best buffer to employ (Blanchard. and other gem-diols. Once an optimal pH has been approximated.5 1. .1 lists a wide variety of buffers covering a broad pH range.1 for one of the Good buffers. Borate buffers can form covalent complexes with mono.1 Buffer capacity (b) versus DpH over the range  1 pH unit of the pKa for HEPES (0. and drops off quickly 1 pH unit on either side of the pK.0 −0. and interactions with substrates or metals. (6. inorganic buffers do have a high potential for specific buffer effects.

0  0.0055 0.020 x .014 0.40 3.0 6.0060  0.016 7.15 3.2.011 Trivial name Phosphate (pK1) Malate (pK1) Formate Succinate (pK1) Citrate (pK2) Acetate Malate Pyridine Succinate (pK2) MES 6.75 4.0016 0.015 7.0002 –  0.3-Bis[tris(hydroxymethyl) methylamino]propane PIPES Piperazine-N.1 Selected buffers and their pK values at 25  C Buffer name pKa dpKa/dt – – – – – – – – – 2-(N-Morpholino) ethanesulfonic acid Cacodylate Dimethylarsinic acid Dimethylglutarate 3.34 6.3-Dimethylglutarate (pK2) Carbonate (pK1) – Citrate (pK3) – Bis–Tris [Bis(2-hydroxyethyl)imino]tris (hydroxymethyl)methane ADA N-2-Acetamidoiminodiacetic acid Pyrophosphate – EDPS (pK1) N.011 6.0 0. Stoll and John S.N 0 -Bis(3-sulfopropyl) ethylenediamine Bis–Tris propane 1.40  0.27 6.6tetrahydrophthalic acid TES 2-[Tris(hydroxymethyl) methylamino]ethanesulfonic acid 2.59  0.95  0.21 4.0  0.020 6.46 0.20 0.64 6.23  0.78  0.0085 6.09  0.40 6.020  0.10 0.76  0.015 6.80 – 6.N-Bis-(2-hydroxyethyl)-2aminoethanesulfonic acid MOPS 3-(N-Morpholino) propanesulfonic acid Phosphate (pK2) – EMTA 3. Blanchard Table 6.76 4.65 – – 6.0028 – 7.N 0 -bis(2ethanesulfonic acid) ACES N-2-Acetamido-2hydroxyethanesulfonic acid MOPSO 3-(N-Morpholino)-2hydroxypropanesulfonic acid Imidazole – BES N.95 7.23 5.6-Endomethylene-1.20 7.13 5.46 Vincent S.0018  0.60 6.3.35 6.76 5.0044 – 0.

g. metals essential to enzymatic activity (e.4-Bis(4-sulfobutyl)piperazine 2-Aminoethylsulfonic acid.33 10.06  0.032 11.80  0.N-Bis(2-hydroxyethyl)glycine 3-{[Tris(hydroxymethyl)methyl] amino}propanesulfonic acid – 1.06 – –  0.022 9.60  0.00 – 8.78 9.026 Buffer complexation with metals may present additional problems.89 10.029 –  0.40  0.40 –  0.05  0. inorganic buffers can prove problematic in that they may remove. Mg2þ for kinases.26 8.76 7.33 –  0.4-Bis(3-sulfopropyl)piperazine – N.025 – 9.12 12. 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) 8.10 8.N 0 -bis(2hydroxypropanesulfonic acid) N-2-HydroxyethylpiperazineN 0 -3-propanesulfonic acid Tris(hydroxymethyl) aminomethane N-[Tris(hydroxymethyl)methyl] glycine – 1.018 8.60 9.50 9.1 (continued) Trivial name Buffer name pKa dpKa/dt HEPES N-2-HydroxyethylpiperazineN 0 -2-ethanesulfonic acid 3-[N-Bis(hydroxyethyl)amino]2-hydroxypropanesulfonic acid Triethanolamine Piperazine-N.49 8.020  0.021 8.48  0.013 DIPSO TEA POPSO EPPS.029 9. In this respect. .N 0 -Bis(3-sulfopropyl) ethylenediamine 3-Aminopropanesulfonic acid – 3-(Cyclohexylamino) propanesulfonic acid – – 7..47 Buffers: Principles and Practice Table 6.55  0.06 8.25 9.85  0.009 0.25 8.031  0. by chelation.029 0.028 8.008  0.018 0.025  0. taurine – – – Cyclohexylaminoethanesulfonic acid – N.014 7.015 7.23 9.

The carboxylic acid buffers are generally the least sensitive to temperature. and an adequate time after. 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 (10–20 mM) concentration of buffer.85 at 0  C). Choosing a buffer for protein purification requires some special considerations. HEPES. pH stability becomes increasingly important. and most buffers listed in Table 6. a concentration should be chosen. 1966). Release of protons upon chelation or precipitation of metal–buffer complexes may also be a potential problem.1 pH unit). Once a suitable buffer has been found (noninteracting. 4. Buffers have temperature-sensitive pK values. Many primary amine buffers such as Tris and glycine (Bradford. it should be done close to the working temperature. Since high ionic strength may decrease enzyme activity. they do have disadvantages. on pH should be checked by measurement of the pH after addition of all components. this series). In cases where protons are consumed or released stoichiometrically with substrate utilization. can be used above 240 nm. and the Good buffers have only a small inverse temperature dependence on pK. EPPS.. Tris is a poor buffer below pH 7. the Good buffers are useful since they have been shown to have low metal-binding capabilities (Good et al. give false-positive colors with Lowry assay. The Good buffers. Stoll and John S. Buffers may be made up in stock solutions.05 pH.48 Vincent S. and Bicine. Where metal chelation presents a problem. then diluted for use. When stock solutions are made. and in glass bottles (plastic bottles can leach UV-absorbing material) (Perrin and Dempsey. Blanchard Cu2þ or Fe2þ for hydroxylases). addition of protein should not vary more than  0. the buffer concentration should be as low as possible (Blanchard. with an appropriate pK ). and have been widely used in protein purification. Although buffers like Tris are inexpensive. particularly amine buffers. which makes cost a concern. 1976) will interfere with the Bradford dye-binding protein assay. or addition of salts. Before titrating a buffer solution. It may be important to use a buffer that does not absorb appreciably in the spectral region of interest. and dialysis. 1974). The effects of dilution of stock solutions. Spectroscopic measurement of enzyme rates is a commonly applied method. then the buffer concentration should be raised to 50 mM. If the pH changes too drastically (greater than  0. Buffer Preparation Once a suitable buffer has been chosen it must be dissolved and titrated to the desired pH. Tris and many inorganic buffers are widely used since they are relatively inexpensive. chromatographic separations. The pH prior to.1. Some of the Good buffers. Large amounts of buffer will be needed for centrifugation.06 at 25  C will have a pH of 8. Calibration should be done using commercially available pH . the pH meter must be calibrated.5 and its pK is temperature dependent (a solution made up to pH 8.

then titration with tetramethylammonium hydroxide can be done to avoid mineral cations.49 Buffers: Principles and Practice standards. Volatile buffers make it possible to remove components that may interfere in subsequent procedures.8 7.5 6.2 Types of systems for use as volatile buffersa a System pH range 87 ml glacial acetic acid þ 25 ml 88% HCOOH in 11 l 25 ml 88% HCOOH in 1 l Pyridine–formic acid Trimethylamine–formic acid Triethylamine–formic (or acetic) acid 5 ml pyridine þ 100 ml glacial acetic acid in 1 l 5 ml pyridine þ 50 ml glacial acetic acid in 1 l Trimethylamine–acetic acid 25 ml pyridine þ 25 ml glacial acetic acid in 1 l Collidine–acetic acid 100 ml pyridine þ 4 ml glacial acetic acid in 1 l Triethanolamine–HCl Ammonia–formic (or acetic) acid Trimethylamine–CO2 Triethylamine–CO2 24 g NH4HCO3 in 1 l Ammonium carbonate–ammonia Ethanolamine–HCl 20 g (NH4)2CO3 in 1 l 1. with other anions such as acetate.22 mm) to prevent bacterial or fungal growth. Volatile buffers are useful in electrophoresis. sulfate.5 8..8–8.3–3. especially with solutions in the pH 6–8 range.0 7–12 7–12 7. Most of the volatile buffers (Table 6.0 3–6 3. Stock solutions should be made with quality water (deionized and double-distilled.1 3. or are being investigated. Similarly. chloride. the substitution of the most commonly used counteranion.5 8. ion-exchange chromatography.9 8.5 4. Volatile Buffers In certain cases. To prevent heavy metals from interfering.5 3.5–7. 1987). bracketing the desired pH. 5.9 From Perrin and Dempsey (1974). . or glutamate.0–6.0 6.1 2.0–5.0 4.7 5.5–10. EDTA (10–100 mM) may be added to chelate any contaminating metals. If monovalent cations interfere.2) are transparent in the lower UV range except for the buffers Table 6. may have significant effects on enzyme activity or protein–DNA interactions (Leirmo et al.0–10.9 2. preferably) and filtered through a sterile ultrafiltration system (0.0–10. and digestion of proteins followed by separation of peptides or amino acids. it is necessary to remove a buffer quickly and completely.

g. diluted to a total of 100 ml: . Blanchard containing pyridine (Perrin and Dempsey.6 3.1 M solution of citric acid (21. 1909a.41 g C6H5O7Na3  2H2O in 1000 ml) x ml of A þ y ml of B.4 8. A detailed description of buffer mixtures which provide a wide range of buffering capacity with constant ionic strength is available (Ellis and Morrison.2 M HCl 50 ml of A þ x ml of B. 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. Most volatile buffers will not interfere with ninhydrin if the concentrations are not too high (e.50 Vincent S. One method would be a mixture of buffers that sufficiently covers the pH range of interest. An important consideration is interference in amino acid analysis (i.4 2. Stoll and John S. 1948) stock solutions  A: 0. Citrate buffer (Lillie.6 – 2. This may lead to nonspecific buffer interactions for which corrections must be made.e.4 3. 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).2 3.01 g in 1000 ml)  B: 0. Recipes for Buffer Stock Solutions 1. The Good buffers are ideal for this approach since they are structurally related and have relatively evenly spaced pK values. triethanolamine less than 0. As the pH passes the pK of one buffer it becomes nonparticipatory and therefore has no further function.2 16.1 M does not interfere). diluted to a total of 200 ml: x pH x pH 5.2 M solution of glycine (15. 1974).. this series). reactions with ninhydrin).8 2.1 M solution of sodium citrate (29. 6.0 6.01 g in 1000 ml)  B: 0.. Glycine–HCl buffer (Sorensen. These nonparticipating buffer components may show nonspecific buffer effects as well as raising the ionic strength with potential deleterious effects. 7.2 32.b) stock solutions  A: 0.8 24.

8 5.2 5.0 4.2 41.6 5.8 4.8 6.0 36.2 3. 1914) stock solutions  A: 0.0 31.0 37.5 14.4 g of C2H3O2Na or 27.7 6.2 3.0 18.5 7.5 18.0 9.5 6.2 39.0 5.1 M solution of citric acid (19.6 4.8 30.5 24.0 17.4 4.6 5. diluted to a total of 100 ml: .8 10.6 3. Acetate buffer (Walpole.65 g of Na2HPO4  7H2O or 71.5 28.2 g of C2H3O2Na  3H2O in 1000 ml) x ml of A þ y ml of B.4 4.5 27.6 3.5 8.51 Buffers: Principles and Practice x y pH 46.8 4.4 5.0 35.0 13.5 41.6 4.4 3.0 15.0 13.2 M solution of dibasic sodium phosphate (53.0 41.8 4.3 38.0 29.2 3. 1921) stock solutions  A: 0.3 44.2 M solution of acetic acid (11.0 36.0 5.5 25.7 11.5 23.3 10.0 33.0 24.0 13.7 40.0 34.0 16.7 g of Na2HPO4  12H2O in 1000 ml) x ml of A þ y ml of B.55 ml in 1000 ml)  B: 0.2 M solution of sodium acetate (16.5 22.2 3.2 4.0 20.2 45.0 25.5 35.0 3.5 42. diluted to a total of 100 ml: x y pH 46.0 4.8 9.2 4.0 6.8 3.2 5. Citrate–phosphate buffer (McIlvaine.5 43.8 3.2 19.21 g in 1000 ml)  B: 0.4 5.5 32.

9 43. Cacodylate buffer (Plumel.8 5.7 43.7 20.4 6.2 21.6 5.3 23.8 7.6 22.4 40. unpublished observation) stock solutions  A: 0.8 37.52 Vincent S.2 5.2 M NaOH 25 ml of A þ x ml of B. 1949) stock solutions  A: 0.5 5.8 26.1 33.3 24.1 17.2 4.3 22.2 3.9 15.0 30.0 19.0 13.7 30.8 4.0 23.6 2.1 16.7 17.3 30.5 10.0 5.4 5.2 M solution of sodium cacodylate (42.3 16.8 25.7 19.7 29.0 6.4 27.2 24.0 5.2 6.2 23.8 6.0 4.6 2.6 36.2 37.3 20.8 29.7 26.6 5.0 4.8 10.3 32.6 42. Stoll and John S.6 3.5 5.0 5. Succinate buffer (G.4 4.5 3. Blanchard x y pH 44.0 3.7 35.0 6.8 4.4 3.9 32.4 13.9 16.6 4.1 6.4 4.9 33.5 40.2 12.6 9.4 7.7 27.2 M solution of succinic acid (23. diluted to a total of 100 ml: x pH x pH 7.4 5.6 g in 1000 ml)  B: 0.7 25.2 M NaOH 50 ml of A þ x ml of B.8 6.8 26.6 4.2 39. Gomori.8 g of Na(CH3)2AsO2  3H2O in 1000 ml)  B: 0.8 3.1 34.3 34.3 14.6 6.2 5.2 4. diluted to a total of 200 ml: .

7 7.0 72.7 7.5 56.4 6.0 6.8 5.9 6.0 45.0 84.2 M solution of dibasic sodium phosphate (53.0 13.0 8.3 6.5 7.8 g in 1000 ml)  B: 0.0 90.7 5.8 6. Barbital buffer (Michaelis.8 43.5 77.0 9.0 7.0 22.0 90.0 6.3 15.0 28.0 19.2 M HCl 50 ml of A þ x ml of B. 1909a.5 43.2 7.3 55.6 7.2 7.8 8.7 17. diluted to a total of 200 ml: x pH x pH 1.8 45.5 6.2 7.0 6.2 9.6 9.0 2.5 62.5 91.6 45.2 g in 1000 ml)  B: 0.0 10.0 39.3 6.1 7.4 47.5 9.8 .b) stock solutions  A: 0.4 13.0 18.8 7.5 51.5 37.0 6.0 5.5 73.0 61.2 7.0 94.6 6.5 7.0 5.7 85.0 7.7 g of Na2HPO4  12H2O in 1000 ml) x ml of A þ y ml of B.2 M solution of sodium barbital (veronal) (41.5 2.0 39.8 6.0 12.0 16.0 5.2 8.5 8.5 32.4 7.0 77.5 92.2 34.5 68.7 6.0 87.8 7. diluted to a total of 200 ml: x y pH x y pH 93.2 18. 1930) stock solutions  A: 0.4 8.0 13.5 27.9 8.4 7.5 8.0 5. Phosphate buffer (Sorensen.5 31.9 7.0 23.65 g of Na2HPO4  7H2O or 71.5 4.53 Buffers: Principles and Practice x pH x pH 2.6 8.5 26.3 6.0 8.0 87.2 M solution of monobasic sodium phosphate (27.5 22.0 33.0 81.3 7.7 6.0 10.2 6.4 29.6 6.5 49.0 67.1 6.5 93.6 7.0 43.5 39.3 7.8 5.0 5.0 81.0 4.2 5.3 6.

9 7. especially in the cold.8 8.54 Vincent S.2 10.4 21. diluted to a total of 200 ml: .03 g in 1000 ml)  B: 0.5 59.2 g in 1000 ml)  B: 0.6 16.2 8.2 7.3-propanediol (Ammediol) buffer (Gomori.1 4. Blanchard Solutions more concentrated than 0.6 41.8 12.05 M may crystallize on standing.5 30.2 8. diluted to a total of 200 ml: x pH 5.2 M solution of 2-amino-2-methyl-1. this series) stock solutions  A: 0.0 83.8 8.4 g in 1000 ml)  B: 0.2 M HCl 50 ml of A þ x ml of B.4 26. diluted to a total of 200 ml: x pH x pH 2.0 8.0 8.7 8.2 M HCl 50 ml of A þ x ml of B.0 32.05 g in 1000 ml.6 22.5 17. 1943) stock solutions  A: 0.2 M solution of tris(hydroxymethyl)aminomethane (24.0 3.0 9.2 11.4 7.3-propanediol (21.0 115. 0. 9.3 11.4 44.1 9.0 42.8 38.5 7.4 8.5 8.9 9. 2-Amino-2-methyl-1.6 7.9 8. Tris(hydroxymethyl)aminomethane (Tris) buffer (Hayaishi. Boric acid–borax buffer (Holmes.1 8.05 M solution of borax (19. 1946) stock solutions  A: 0.2 M solution of boric acid (12.5 7.0 8.8 8.2 M in terms of sodium borate) 50 ml of A þ x ml of B.0 9.4 7. Stoll and John S.

7 5.01 g in 1000 ml)  B: 0.0 17.0 22.5 16.8 9.0 9.05 g in 1000 ml.0 8.4 9.35 9.0 3.6 45.2 9.6 9.b) stock solutions  A: 0.4 27.6 9.0 10. 1945) stock solutions  A: 0.02 M in terms of sodium borate)  B: 0.5 9.5 34.0 38. Glycine–NaOH buffer (Sorensen.0 37. 1909a.4 22.8 12.6 8.1 14.6 13.0 9.05 M solution of borax (19. 1917) stock solutions  A: 0. diluted to a total of 200 ml: x pH x pH 4.8 8. Carbonate–bicarbonate buffer (Delory and King.2 M solution of glycine (15.6 8.2 M NaOH 50 ml of A þ x ml of B.7 8. 0.2 9.8 9.0 16.55 Buffers: Principles and Practice x pH x pH 2.9 10.0 11.7 10.6 23.0 9.5 9.7 9.5 8.2 8.0 38. Borax–NaOH buffer (Clark and Lubs.6 43.0 29.8 g in 1000 ml) x ml of A þ y ml of B.2 32.8 8.28 9. diluted to a total of 200 ml: x pH 0.2 M NaOH 50 ml of A þ x ml of B.0 29.4 9. diluted to a total of 200 ml: .6 9.8 12.0 7.7 41.8 10.2 M solution of sodium bicarbonate (16.0 10.0 46.4 8.0 34.5 12.2 M solution of anhydrous sodium carbonate (21.0 43.8 9.0 7.4 10.0 6.2 g in 1000 ml)  B: 0.

(this series).. 22.0 30. Bradford. 2. Johnson. Vol.0 9. J.0 25. 404. 1. 87.3 10. PA. C. and Singh. (1921). and Morrison. S. Buffers for pH and Metal Ion Control.. p. 144. Harrison. M. Biochem. S. (this series). Perrin. 87.0 27.0 17... Biol.0 10. (1966). Proc. (1976). Good. Connolly.5 20. M.. 163. Anat. Bacteriol. Bull. (1917).5 28.5 9.. M. W. Histopathologic Technique. (this series). 183. Winter. 24. Chem. W. Rec. D.. p. S.2 9. 352. W. M. and Lubs.5 42. (1987). Burgess. Unpublished observations. and Izawa.. Walpole. Delory. 245. 1. 21. (1943). Gomori. 104. P. D. 105. N.. 248.5 40. Chim. 2501. W. (this series). 22. 39. Soc. J. J. Exp.. Soc. (1948).5 9. Chapman & Hall.5 38. Anal. E. 62.0 22. Clark.5 9. Biochemistry 5. A.0 46. Michaelis. 30. E. 104. Anal. G.1 10.0 16. London. (1914). . S. 86.5 5.0 7. Plumel. (1980). J.7 REFERENCES Blanchard. M.2 10. M. R.4 9. C. L. M. Vol.3 9. Biol.5 11.0 14. N.9 10. 33.8 9. Holmes.56 Vincent S. 53.5 7. Vol.5 37. 300. Z. Biochemistry 26. 131. (1909b). S. Gomori. J. Med.. R. Stoll and John S.7 9.6 10. (1946). L. and Metzler. L.0 33. (1949).. K. (this series). G. Biochem. D. R. 467.0 19. J.0 34. 33. G. and Dempsey. (1930). 22. and King. Biochem. S. Biol.. G.. Biol.. J.4 10. Philadelphia. R. H. P.0 35. et al. E. Biochem. p.0 42. Sorensen. Chem. 2095. D. (1945). Sorensen. Vol. D. Blanchard x y pH 4. Chem. p. Izawa. Lillie.5 45. Good. Ferguson. R.0 25. G. Leirmo. Vol. T. Biochem. p. 3. Ellis. 405. Hayaishi. T. J. 129. J.. McIlvaine. S. (1974). O.5 13. E. Soc.5 22. T. J. D. S. F. Cayley.5 40. J.5 10. (1909a). and Record. 49. Blakiston. Winget. N.6 9. E. Z. B.5 30.