Phenolphthalein diphosphate

Appleyard (64) noted that addition of ethanol to incubation mixtures of sodium phenolphthalein diphosphate with prostatic extract increased the rate of free phenolphthalein formation. Phosphate ion failed to show a comparable increase, and this discrepancy was attributed to transphosphorylation. Phosphoryl transfer may be effected by prostatic phosphatase to acceptors other than solvent (65-67). Nigam and Fishman (25) studied phosphoryl transfer under conditions of 60-80% transfer to an acceptor. In the case of 1,4-butanediol, the optimal concentration was 0.8 M. In this experiment, water molecules outnumbered acceptor molecules by 55/0.8 or 70-fold. In spite of this, transfer far exceeded hydrolysis. Phosphoryl transfer to aliphatic alcohols can be easily measured when phosphates are used as donor compounds. The difference between alcohol formation from the substrate and phosphate ion production is a measure of the transfer reaction. Table IX (25) shows that four different substrates can transfer phosphoryl to butanediol with high efficiency. Table X (25) shows that aliphatic alcohols are good acceptors... 

Phenolphthalein diphosphate followed by alkali Reduction of the tetrazolium salt of MTT P-Naphthyl phosphate and diazo salt of fast blue RR... 

A number of substrates can be used for this enzyme. Two common ones are />-nitrophenyl phosphate and phenolphthalein diphosphate (Figure 6.8). In the case of />-nitrophenyl phosphate the />-nitrophenol released is bright yellow in alkaline solution and can be assayed spectrophotometrically. The test is used for the differentiation of streptococci. A variation on this test determines the phosphatase present in cow s milk as a measure of the efficiency of pasteurisation. 

We have already discussed the properties of human erythrocytic acid phosphatase (Section 3.3), and we pointed out that, like acid phosphate in other tissues, it may exist in several isoenzymatic forms. In 1963, Hopkinson et al. (H13) subjected hemolysates of human red cells from an English population to horizontal starch-gel electrophoresis for 17 hours at 5°C. The gels were then sliced horizontally, covered with 0.05 M phenolphthalein sodium diphosphate at pH 6.0, and allowed to incubate for 3 hours at 37°C. Five different electrophoretic patterns of acid phosphatase activity could be distinguished in different individuals. Shortly thereafter Lai and his associates (L2) confirmed these findings and discovered an additional sixth pattern which had been predicted by Hopkinson et al. (H13). The distribution of these patterns in various types of population was assiduously pursued within the next several years, and several new ones were discovered in Negro populations (G3, K2). 

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