Isolation, Purification And Characterization Of Acid Phosphatase From APIS CERANA

Acid phosphatase (ACPase, E.G.3.1.3.2) was isolated andpurified from Apis cerana, and properties of the enzyme had been studied. The ACPase activities of the fresh bee pupa, honey, royal jelly and Apis cerana were compared, then the Acid phosphatase was partially obtained from Apis cerana by homogenate, ammunium sulfate fractionation, chromatography with DEAE-sepharose and gel filtration with Sephadex G-200.The purified enzyme moved as a single electrophoretic band in PAGE. The purification multiple was 77.24, and the specific activity was 16.22 unit/mg.Pr with pNPP as its substrate. The optimum pH value for the enzymes was pH 4.1. The optomum temperature was 52@. The Michaelis-Menten constant (Km) was 1.25×10-3 mol/L on the pNPP. ACPase was stable for its relative activity was above 60% under 50@. The molecular weight of ACPase was 135kDa determined with gel filtration and its subunit weight was 63.1kDa determined with SDS-PAGE. Isoelectric focusing study showed that pI of the enzyme were 4.46 and 4.79. NR/R single and two dimensions SDS-PAGE found that the enzyme contained intrachain disulfate link. CD spectrum was investigated and it was found that a-helix, p-sheet and random coil were 13.84%, 25.68% and 56.34% respectively. Amino acid composition analysis showed that there were about 507 amino acids in the ACPase, with plenty of Asp.The enzyme was activated by Zn2+, Mg2+, K+, while inhibited by ions of Cu2+, Fe3+, Cr3+. Pb2+ in low concentration activated the enzyme and in high concentration inhibited it. The effect of Cd2+ was lower than that of Pb2+ on the activity of the enzyme. ACPase is inactivated by urea. The inhibition of Cu2+, Cr3+ and Fe3+was demonstrated to be noncompetitive inhibition by using Lineweaver-Burk method and those Ki values were 3.58×10-3mol/L, 3.25×10-4mol/L and 6.15×10-4mol/L determined by Dixon method. Study on the UV spectrum found: Zn2+ led to the increase of the maximal ultraviolet absorption peak; Mg2+and K+ led to the decrease of it; Cd2+ led to the increase of the maximal ultraviolet absorption peak and the maximal wavelength; Pb2+ made the maximal ultraviolet absorption peak extinct gradually. The effect of Cu2+, Cr3+ and Fe3+onthe UV spectrum of the ACPase was significant. Low contents of them could led the enzyme lose its UV spectrum characters. Urea could increase the maximal ultraviolet absorption peak of the enzyme. The effects of different modification reagents on the ACPase catalytic activity and its fluorescence spectra were determined. The result indicated that when the tryptophane residue of ACPase, sulfhydryl group and disulfide link of cysteine residue were modified, the related activity of the enzyme was decreased and its fluorescence emission peak intensity was reduced; when the amino acids with hydroxyl group were modified, the related activity of the enzyme was inhibited and the fluorescence emission peak intensity was increased. Modification of histidine residue did not change the activity of the enzyme and its fluorescence emission peak intensity.