Phosphatase Activity of Human Erythrocytes

Abstract

Evidence has accumulated which indicates that phosphatases occur in the erythrocytes of dog, beef, horse, rabbit, rat, and man. To provide further information about phosphomonoesterase occurring in the normal human red blood cells the enzymatic activity of red cell hemolysates was studied as to (1) the presence of alkaline and acid pliosphatase, (2) the effect of fluoride and magnesium upon the enzymatic activity, (3) the relation of erythrocyte phosphatase to plasma phosphatase.

Procedure. The erythrocytes were washed 3 times with normal saline. The washed cells obtained from x cc of oxalated or heparinized blood were hemolyzed by adding distilled water to bring the total volume of the hemolysate up to 1.5 times x cc. If the hematocrit value is known the phosphatase activity of the erythrocytes can be calculated from the activity of the hemolysates. The phosphatase activity of the hemolysate as well as of the plasm was tested by detertnining the amount of phosphorus (according to Fiske and Subbarowl) liberated from a sodiuni glycerophosphate substrate, or by measuring the amount of phenol freed from a discd' Jium phenylphosphate substrate, according to King and Armstrong. 2 Michaelis' 3 veronal-acetate buffer was used preferably, since certain buffer salts interfere with the colorimetric determination of phenol. In a few of the experiments with glycerophosphate as substrate, citrate and veronal buffers were used. The results are expressed in mg of phosphate or phenol liberated per 100 cc of hemolysate or plasma during one hour of incubation at 37°C.

Results. (1) Phosphatase activity of red cell hemolysates was found to be constantly present (Fig. 1). Strong activity was observed between pH 4.8 and pH 6.1. Below and above this zone of acidity hydrolysis drops abruptly. The optimum pH of the phosphatase of human erythrocytes was found around pH 5.3. Its action upon P-glycerophosphate is slow compared to the strong action upon disodium monophenylphosphate. A second zone of optimal activity at alkaline reaction as reported by Roche and Bullinger 4 for horse blood cells, was missing. If confirmed, this result would preclude the occurrence of an “alkaline phosphatase” in human erythrocytes.