Previous work of Hansson and Rasmusson
(1) has shown that low concentrations (lO-' M) of ascorbic acid enhance the rate of purified milk acid phosphatase catalyzed hydrolysis of phenyl phosphate by 500/o, and that higher concentrations of ascorbic acid (lo-'M) are completely inhibitory.
It was also reported that lo-'M ascorbic acid without the enzyme promotes substrate hydrolysis with an optimum pH of 4.3, which is similar to the pH optimum of the acid phosphatase. Substrate hydrolysis in these studies was followed by measuring the amount of liberated phenol with 2,6-dibromoquinone 4-chlorimide. The latter is known to react in alkaline solution with phenol in water to form an intensely colored phenolindophenol
(2). Hansson and Rasmusson (1) indicated that this color test for phenol was influenced by the presence of ascorbic acid and attempted to correct for its action.
In view of the possible analytical and biological interest of these observations, it was decided to reinvestigate the influence of ascorbic acid on nonenzymic phosphate ester hydrolysis. Consideration was given to the selection of an .analytical method which would not give interference by ascorbic acid. The quantitative aspects of the effect were also studied since only relative data were presented in the original report by Hansson and Rasmusson (1). Finally, the influence of various experimental variables on the reaction were determined. METHODS Materials. All chemicals other than the phosphate esters were reagentgrade materials and were used without purification. The phosphate esters were obtained from Sigma Chemical Co. and were stored according to the instructions on the labels. Sigma type I wheat germ and type 11 potato acid phosphatase were stored at 0' after being received and were used