Acid phosphatase surface inactivation

Two major difficulties must be considered in any assay for acid phosphatase. The enzyme is subject to surface inactivation (23, 24). Accordingly, reproducible initial hydrolytic rates are not always obtained, and the kinetic behavior should be checked in any new assay developed. Discrepancies between the amount of inorganic phosphate produced and phenol liberated from phenolic phosphates may be substantial if extensive phosphotransferase activity occurs because of phosphoryl acceptor action on the part of hydroxylic buffers or other constituents of the incubation mixture (25, 26). Fluorogenic assays have been developed with very high sensitivity (27). Reference will be made to particular assays in the discussion of the specific enzymes. 

Solutions of acid phosphatase are particularly sensitive to surface inactivation. Figure 3 (88) shows the inactivation rate of the enzyme in the presence and absence of surface-active detergents. The inactivation process is temperature sensitive and the protection by detergent is total. Most of the enzyme inactivation proceeds with first-order kinetics. A variety of agents—gelatin, bovine serum albumin, egg albumin, and Tween-80—protect the enzyme against inactivation. 

Fig. 3. Surface inactivation rate of prostatic acid phosphatase by shaking and protection by added surface-active agent. Shaking mixtures (20 ml) contained purified enzyme (056 /ug of protein/ml) in 0.05 M acetate buffer at pH 5.5. The solutions were shaken in 50 ml volumetric flasks using a mechanical shaker (Burrell, model CC). Temperatures were maintained by immersion of the flasks in an appropriately set water bath. After specified intervals of shaking, duplicate 0.1 ml ahquots were removed into tubes containing Triton X-100. All tubes were assayed simultaneously, following the shaking procedure, with 0.05 M phenyl phosphate as substrate. Curve 1 Enzyme + Triton X-100 at 0°C and 29°C. Curve 2 Enzyme alone at 0°C. Curve 3 Enzyme alone at 29°C. From Tsuboi and Hudson (88).

Prostatic acid phosphatase is reversibly inactivated by p-mercuri-benzoate and by Cu2+ and Fe3+ (59). In contrast to red cell acid phosphatase, prostatic acid phosphatase is only partially inactivated even after prolonged periods of incubation at high concentrations of p-mercuri-benzoate. Addition of cysteine to the p-mercuribenzoate-treated enzyme produces complete reactivation. Binding of SH groups by p-mercuri-benzoate renders the enzyme more labile to thermal denaturation, but no difference is obtained with surface inactivation (23). Similar partial inactivation with Cu2+ is also subject to reactivation. 

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