Electrophoretic separation of alkaline and acid phosphatase isoenzymes from the pulp of monkey teeth

Abstract

Franzén, A. Hasselgren, C. Electrophoretic separation of alkaline and acid phosphatase isoenzymes from the pulp of monkey teeth.

Monkey pulps were homogenized in a Triton tris solution. After three centrifugation steps (800, 20000, and 105000 g) the supernatant was applied on acryl amide columns at pH 7.5 in a trisdiethyl barbituric acid buffer. Electrophoresis was performed at a constant current of 2.5 mA per gel column at 18–20°C.

Incubations for alkaline phosphatase (E.C. 3.1.3.1) were carried out at pH 8.3 using naphthol-AS-MX-phosphate as substrate and Fast Red Violet LB salt as coupler. Incubations for acid phosphatase (E.C. 3.1.3.2) were undertaken at pH 5.0 using α-naphtyl phosphate as substrate and hexazotized pararosanilin as coupling agent. After the incubations for alkaline phosphatase as well as acid phosphatase two bands showing enzyme activity were demonstrated. By means of treatment with heat (56° C) prior to incubation or addition of vanadate or pyrophosphate to the muation medium it was shown that the main part of the fast moving alkaline phosphatase band was sensitive to these procedures. The alkaline phosphatase of the slow moving band appeared to be resistant to heat or the addition of inhibitors.