PHOSPHATE AND PHOSPHATASE 227 



pound, 15% faster than phenyl phosphate, and 2-3 times more 

 rapidly than glycerophosphate, according to Bessey, Lowry, and 

 Brock. Either acid or alkaline phosphatase may be determined with 

 the substrate; it is only necessary to carry out the colorimetry in 

 alkaline solution, since the free nitrophenol, which would exist in 

 acid solution, is colorless. For other methods see pages 124, 208, 209, 

 and 280. 



Lundsteen and Verniehren Method for Inorganic Phosphate 

 and Phosphatase 



SPECIAL REAGENTS 



Substrate. Combine 8 ml. 1 A^ ammonium hydroxide, 12 ml. 1 N 

 ammonium chloride, 1 g. disodium-;S-glycerophosphate, 2 ml. 1 M 

 magnesium chloride, and make up to 100 ml. with water. 



10% Trichloroacetic Acid. 



Acid-Molybdate Solution. Combine 100 ml., 7.5% ammonium 

 molybdate, 45 ml. 10 A^ sulfuric acid, and 105 ml. water. 



Amidol Solution. Dissolve 15 g. sodium sulfite and 1.5 g. Amidol 

 (Agfa) in 100 ml, water. Store in the dark and cold, and dilute 

 five times before use. After about 2 weeks it turns red and can no 

 longer be used. 



PROCEDURE 



1. If blood is used, pipette 50 ^1. into 1 ml. of 0.9% sodium 

 chloride soln. and centrifuge out the cells. 



2. To 200 ix\. of the supernatant fluid or a tissue extract add 200 

 fj\. substrate soln. and place in thermostat for the digestion period 

 (for plasma 24 hr. at 37°). 



3. To another tube containing the same ingredients add 300 /*!. 

 10% trichloroacetic acid for a control experiment. 



4. Stop the reaction by adding 300 fA. 10% trichloroacetic acid, 

 and centrifuge out the precipitate from both the enzyme and control 

 tubes. 



5. To 400 /xl. of the supernatant in each case ^dd 100 /xl. acid- 

 molybdate reagent and 100 fA. Amidol soln. 



6. Measure the color intensity after the soln. has stood for 15 

 min., and obtain the quantity of free phosphate from a previously 

 determined calibration curve constructed from measurements with 

 known amounts of phosphate. 



