PHOSPHATE AND PHOSPHATASE 209 



4. Centrifuge all the tubes at one time with the stannous chloride 

 soln. up. 



5. Compare the colors on a milk glass background illuminated by- 

 two lamps arranged to avoid shadows. The colors fade by about 

 10% during the first few min. but this change occurs equally in all 

 of the tubes and hence need not interfere with the comparisons. 



PHOSPHATASE 



By an adaptation of the method of King (1932) to capillary tube 

 colorimetry, Weil and Russell (1940) worked out a procedure for 

 the determination of phosphatase which could be applied to less 

 than 1 ix\. plasma with an average deviation of ±3.0%. Their 

 technique employs capillary tube procedures for the determination 

 of the inorganic phosphate, but the enzymatic digestion procedure 

 is based on the use of pipettes, reaction tubes, and other apparatus 

 common to the titrimetric techniques. As in the method of Siwe 

 (1935b) (page 226), aminonaphtholsulfonic acid is used to reduce 

 the phosphomolybdate. For other methods see page 226, 



Weil and Russell Method for Phosphatase 



SPECIAL REAGENTS 



Standard Phosphate Solutions. Prepare standards in the range of 

 0.02-1.00 /xg. phosphorus/15 /A. differing from one another by 

 0.02 or 0.04 ixg. 



Molybdic-Sulfuric Acid Reagent. 5% ammonium molybdate con- 

 taining 15% by vol. cone, sulfuric acid. 



Ajninonaphtholsulfonic Acid Solution. Dissolve 0.5 g. of the 1,2,4 

 acid, 30 g. sodium bisulfite, and 6 g. crystalline sodium sulfite in 

 water by shaking, and make up to 250 ml. Filter and, if filtrate 

 is not clear, leave overnight and again filter. Prepare fresh 

 every 2 weeks. 



Veronal Buffer, pH 9.0, with magnesium. 0.0015 M magnesium 

 chloride in buffer consisting of 9.36 ml. 0.1 M sodium diethyl 

 barbiturate + 0.64 ml. 0.1 N hydrochloric acid. 



Substrate Solution. 0.1 M sodium-/3-glycerophosphate. 



10% Trichloroacetic Acid. 



