Purification and Biochemical Properties of Acid Phosphatase Enzyme from Seedlings of Rumex dentatus (Curly dock)

Abstract

The study was designed to purify enzyme from weed plant in association with wheat crop to assess and compare the purification and biochemical properties with acid phosphatases of seedlings of various plants. Acid phosphatase from seedlings of Rumex dentatus (Curly dock) was purified by different chromatography and chromatofocusing techniques with specific activity of 63U/mg of protein. The yield was about 3%. Single band was detected on SDS-polyacrylamide gel electrophoresis confirming the enzyme was homogeneous. Apparent molecular weight of 48 kDa was obtained. Gel filtration experiment indicated that native enzyme had a approximately molecular weight of 96 kDa, suggesting that this enzyme is homodimer. The enzyme showed Km value of 0.5 mM and Vmax of 60 and#181;M of p-nitrophenyl phosphate hydrolysed/min/mg of protein under experimental conditions. The optimum pH of 5.0 and temperature of 50and#176;C were obtained. Divalent cations such as copper and zinc ions caused acid phosphatase inhibition but the presence of 20 mM EDTA in the enzyme-metal ions incubation mixtures reversed the enzyme inhibition to some extent. The activity of 54 % and 63% were recovered back, respectively. Ca2+ and Mg2+ had very small activating effect on activity in the absence or presence of EDTA. The reaction of enzyme with iodoacetic acid or N-ethyl-maleimide had no inhibitory effect, pointing to a non-involvement of cysteine residues in enzyme action. Further, β-mercaptoethanol or dithiothreitol at low concentrations had very little activating effect revealing that SH-group containing amino acid in the enzyme may not be significant for its catalytic activity. The pH dependent variation of Km study showed that histidine may constitute a part of the active site. Acid phosphatase was competitively inhibited by phosphate and vanadate. Fluoride and Zn2+ acted as non-competitive inhibitors while molybdate showed mixed type of inhibition.