Zinc Dependent Acid Phosphatases from Camel Liver: Purificaion and Chracterization

Zn++-dependent acid phosphatase from camel liver was partially purified 333-fold by ammonium sulfate precipitation, ion exchange chromatography on DEAE- Cellulose and SP Trisacryl columns, gel filtration on Sephadex G-100 and by affinity chromatography on Sepharose 4B-L-Tartramic amide column. The specific activity of 10 U/mg of protein was obtained with recovery of 1% with respect to starting material. The molecular weight of native enzyme was 102 kDa as determined by gel filtration on calibrated Ultrogel AcA 44 column. The subunit molecular mass of purified enzyme, as determined by SDS-Polyacrylamide gel eletrophoresis, was 50 kDa suggesting a dimeric nature of protein consisting of two similar subunits. The K-m against p. nitrophenyl phosphate was estimated to be 0.5 mM and V-max was 9.7 mu mol of substrate hydrolysed /min/mg of protein. The optimal pH for this enzyme was between 5-6 and optimum temperature was 50 degrees C. The enzyme was activated by Zn++. It was also activated by Mn++ and Co++ but to lesser extent. Other cations such as Hg++, Pb++, Al+++ and Fe+++ showed inhibition on Zn++-dependent acid phosphatase activity. The tartrate and phosphate were found competitive inhibitors with K-i values 3mM and 0.5mM, respectively while fluoride was found insensitive to inhibition. Substrates specificity study showed that p.nitrophenyl phosphate and phenyl phosphate were found good substrates while alpha-naphthyl phosphate and beta-glycero phosphate were hydrolysed at reasonable rates. Other substrates like phospho-amino acid, sugar phosphate and nucleoside phosphate were hydrolysed poorly.