Preparation Of Buffers For Use In Enzyme St Udies

PreParaTion of buffers for use in enzyme sTudies*
G. Gomori
The buffers described in this section are suitable for use either in enzymatic or histochemical studies. The accuracy of the tables is within ± 0.05 pH at 23°. In most cases the pH values will not be off by more than ± 0.12 pH even at 37° and at molarities slightly different from those given (usually 0.05 M). The methods of preparationdescribed are not necessarily identical with those of the original authors. The titration curves of the majority of the buffers recommended have been redetermined by the writer. The buffers are arranged in the order of ascending pH range. For more complete data on phosphate and acetate buffers over a wide range of concentrations, see Vol. I [10].* *From Gomori, in Methods in Enzymology, Vol. 1,Colowick and Kaplan, Eds., Academic Press, New York, 1955, 138. With permission. TAbLe 1 hydrochloric AcidPotassium Chloride buffer*
x 97.0 78.0 64.5 51.0 41.5 33.3 26.3 20.6 16.6 13.2 10.6 8.4 6.7
* Stock solutions

TAbLe 3
x 46.7 39.6 33.0 26.4 20.3
*

Phthalate-hydrochloric Acid buffer*
pH 2.2 2.4 2.6 2.8 3.0 x 14.7 9.9 6.0 2.63 pH 3.2 3.4 3.6 3.8

Stock solutions

A: 0.2 M solutionof potassium acid phthalate (40.84 g in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml reference
1. Clark and Lubs, J. Bacteriol., 2, 1 (1917).

TAbLe 4
x 15.0 21.0 28.0 36.0 44.0 52.0 60.0 68.0 76.0
* Stock solutions

Aconitate buffer*
x 83.0 90.0 97.0 103.0 108.0 113.0 119.0 126.0 pH 4.3 4.5 4.7 4.9 5.1 5.3 5.5 5.7

pH 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.81.9 2.0 2.1 2.2

pH 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1

A: 0.5 M solution of aconitic acid (87.05 g in 1,000 ml) B: 0.2 M NaOH 20 ml of A + x ml of B, diluted to a total of 200 ml reference
1. Gomori, unpublished data.

A: 0.2 M solution of KCl (14.91 g in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml reference
1. Clark and Lubs, J. Bacteriol., 2, 1 (1917).TAbLe 5 Citrate buffer*
x 46.5 43.7 40.0 37.0 35.0 33.0 31.5 28.0 25.5 23.0 20.5 18.0 16.0 13.7 11.8 9.5 7.2
* Stock solutions

y 3.5 6.3 10.0 13.0 15.0 17.0 18.5 22.0 24.5 27.0 29.5 32.0 34.0 36.3 38.2 41.5 42.8

pH 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

TAbLe 2
x 5.0 6.4 8.2 11.4
* Stock solutions

Glycine-hCL buffer*
x 16.8 24.2 32.4 44.0 pH 2.8 2.62.4 2.2

pH 3.6 3.4 3.2 3.0

A: 0.2 M solution of glycine (15.01 g in 1,000 ml) B: 0.2 M HCl 50 ml of A + x ml of B, diluted to a total of 200 ml reference
1. Sørensen, Biochem. Z., 21, 131 (1909); 22, 352 (1909).

721

722
A: 0.1 M solution of citric acid (21.01 g in 1,000 ml) B: 0.1 M solution of sodium citrate (29.41 g C6H5O7Na3 · 2H2O in 1,000 ml; the use of the salt with 5½ H2O isnot recommended). x ml of A + y ml of B, diluted to a total of 100 ml reference
1. Lillie, Histopathologic Technique, Blakiston, Philadelphia and Toronto, 1948.

handbook of biochemistry and Molecular biology
B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HPO4 · 7H2O or 71.7 g of Na2HPO4 · 12H2O in 1,000 ml) x ml of A + y ml of B, diluted to a total of 100 ml reference
1.McIlvaine, J. Biol. Chem., 49, 183 (1921).

TAbLe 8
x 7.5 10.0 13.3 16.7 20.0 23.5
* Stock solutions

Succinate buffer*
x 26.7 30.3 34.2 37.5 40.7 43.5 pH 5.0 5.2 5.4 5.6 5.8 6.0

TAbLe 6
x 46.3 44.0 41.0 36.8 30.5 25.5 20.0 14.8 10.5 8.8 4.8
* Stock solutions

Acetate buffer*
y 3.7 6.0 9.0 13.2 19.5 24.5 30.0 35.2 39.5 41.2 45.2 ph 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6

pH 3.8 4.04.2 4.4 4.6 4.8

A: 0.2 M solution of succinic acid (23.6 g in 1,000 ml) B: 0.2 M NaOH 25 ml of A + x ml of B, diluted to a total of 100 ml reference
1. Gomori, unpublished, data.

A: 0.2 M solution of acetic acid (11.55 ml in 1,000 ml) B: 0.2 M solution of sodium acetate (16.4 g of C2H3O2Na or 27.2  g of C2H3O2Na · 3H2O in 1,000 ml) x ml of A + y ml of B, diluted to a total of 100 ml...