PHOSPHATE AND PHOSPHATASE 229 



Furthermore, acceleration in color development may be accomplished by 

 increasing the molybdate to 1.5% in 0.05 A'' acid, and the ascorbic acid 

 concentration to values that do not exceed a final concentration of 0.2%. 



Bessey, Lowry, and Brock Method for Phosphatase 



SPECIAL REAGENTS 



Buffer-Substrate Solution. (pH 10.3 to 10.4). Prepare soln. A by 

 dissolving 7.50 g. (0.1 mole) glycine and 95 mg. (0.001 mole) 

 magnesium chloride in 700 to 800 ml. water; then add 85 ml. 1 N 

 sodium hydroxide and dilute to 1 1. 



Prepare soln. B, which is 0.4% disodium p-nitrophenyl phos- 

 phate in 0.001 A^ hydrochloric acid. (The authors reported that the 

 Eastman Kodak Co. product contained about 50% inert material; 

 hence twice the quantity of this preparation should be used. 

 Purification may be carried out by recrystallization from hot S7% 

 alcohol.) Adjust the pH of soln. B to 6.5 to 8.0 with acid or base, 

 if necessary. Test for free nitrophenol by diluting 1 ml. with 10 

 ml. 0.02 A^ sodium hydroxide and measuring the absorption at 

 415 m^. If the extinction is greater than 0.08 {i.e., transmission 

 less than 83% for 1 cm. liquid, or 70% for 2 cm.) remove the 

 free phenol by extracting two to three times with equal vol. water- 

 saturated butyl alcohol followed by three extractions with water- 

 saturated ether. (All butyl alcohol must be removed since it in- 

 hibits phosphatase activity.) Aerate off the traces of ether and 

 store in the cold. 



Mix equal vol. of solns. A and B; adjust the pH to 10.3 to 10.4 

 if necessary with strong sodium hydroxide or hydrochloric acid, 

 and store in the cold, or better, in the frozen state. When 2 ml. 

 diluted with 10 ml. 0.02 A^ sodium hydroxide has an extinction 

 greater than 0.1 for 1 cm., either discard or extract it with butyl 

 alcohol, as above, and readjust the pH. 



Standard Solutions. Prepare solns. containing 1, 2, 4, and 6 milf 

 p-nitrophenol (molecular weight 139.1) per liter. 



PROCEDURE 



1. Place 5 ^1. serum in the bottom of a 6 X 50 mm. serological 

 tube, immerse in ice water, and rapidly add 50 jxl. of the ice-cold 

 buffer-substrate soln. with a constriction pipette. Alix by tapping 

 with the finger. 



