Analytical techniques involving the determination of acid and alkaline phosphatases are based on the analysis of the products released by the enzymatic hydrolysis ofa monoester of orthophosphoric acid, i.e., phosphate released or the liberated phenolic moiety. Various studies have been undertaken with a wide variety of monoesters of phosphoric acid; in practically all determinations a spectrophotometric technique is employed to analyze the released products. The most frequently used substrates include fl-glycerophosphate 1, phenyl phosphate 2, p-nitrophenyl phosphate 3, ~-naphthyl phosphate 4, and phenolphthalein phosphate 5. These procedures have been applied in manual, automated, and kinetic determinations of acid and alkaline phosphatases.
In the present study the enzymatically released phenols are titrated coulometrically with generated bromine to a biamperometric end-point. This titration is applied to the determination of phenols in standard solutions and to phenols enzymatically released by wheat germ acid phosphatase and purified alkaline phosphatase.
EXPERIMENTAL
Apparatus
Constant-current coulometric titrations were carried out with a coulometric apparatus constructed in this laboratory. Details of the construction may be found elsewhere 6. The equipment could deliver any generating current ranging from 0.03 to 5.00 mA and the titration time could be read accurately to 0.1 sec. The titration cell (a 10-ml beaker) contained a platinum-wire generating anode, 20 gauge, and an isolated Nichrome-wire cathode. The isolation was accomplished by means of a 6-mm pyrex tube with a 7-mm piece of"thirsty glass" (porous Vycor, Corning Glass Works, Coming, N.Y.) sealed at the end with a Manolok Teflon sleeve (Manostat Corp., 20 N. Moore St., New York, N.Y.). The isolation tube was filled with 0.5 M potassium bromide.
The end-point was detected biamperometrically, with 135 mV applied across the two platinum-wire indicator electrodes. This applied potential was chosen from preliminary experiments of the bromine-bromide couple. The two wire indicating * Taken in part from the Ph.D.