Purification and Characterization of Alkaline Phosphatase from Water Buffalo Liver (Bubalus bubalis)

Two forms of alkaline phosphatases (EC 3.1.3.1) designated P1 and P2 isoenzymes were purified to homogeneity from the liver of water buffalo after anion exchange chromatography on DEAE-cellulose column and gel filtration on Sephacryl S-300 column. The native molecular weights of the two isoenzymes P1 and P2 are 188 +/- 4 kDa and 194 +/- 3 kDa and both of them exhibited homotrimeric structure. The purified isoenzymes displayed their maximum activity at pH 10. Both P1 and P2 isoenzymes showed isoelectric point (pI) value at pH 5.6-5.8. MnCl2 increased the AP activity of P1and P2 isoenzymes while ZnCl+2 was potent inhibitor of both P1 and P2 isoenzymes. The enzyme was sensitive to EDTA and the thiol compounds in the following order: DTT > GSH > cysteine > beta mercaptoethanol. The P2 isoenzyme was more sensitive to beta-mercaptoethanol and phenylalanine than P1.