Zinc dependent acid phosphatases from camel liver: Purificaion and chracterization. | [Journal of Chemical Society of Pakistan • 2013]

Author(s):

1. Mehrin Sherazi: Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan

2. Asma Saeed: Department of Biological Sciences, Gomal University, Dera Ismail Khan, Pakistan

3. Rubina Naz: Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan

4. Shakil Abbas: Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan

5. Aisha Siddiqua: Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan

6. Shazia Ameen: Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan

7. Amber Zaman: Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan

8. Ahmad Saeed: Department of Chemistry, University of Science & Technology, Bannu, Pakistan

Abstract:

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 Km against p.nitrophenyl phosphate was estimated to be 0.5 mM and Vmax was 9.7 µmol of substrate hydrolysed /min/mg of protein. The optimal pH for this enzyme was between5-6 and optimum temperature was 50° 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 Ki 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 α-naphthyl phosphate and β-glycero phosphate were hydrolysed at reasonable rates. Other substrates like phospho-amino acid, sugar phosphate and nucleoside phosphate were hydrolysed poorly.

Page(s): 829-834

DOI: DOI not available

Published: Journal: Journal of Chemical Society of Pakistan, Volume: 35, Issue: 3, Year: 2013

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