130 kDa acid phosphatase from the liver of labeo rohita:isolation, purification and some kinetic properties. | [Journal of Chemical Society of Pakistan • 2009]

Author(s):

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

2. Mehran Sherazi: Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan

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

4. Irshad Ali: Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan

5. Ahmad Saeed: Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan

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

7. Abdul Haleem Shah: Department of Biological Sciences, Gomal University, Dera Ismail Khan, Pakistan

8. Abdul Rahim Khan: Department of Biological Sciences, Gomal University, Dera Ismail Khan, Pakistan

9. Mushtaq Ahmad: Department of Biotechnology, University of Science and Technology, Bannu, Pakistan

10. Hidayatullah Khan: Department of Biotechnology, University of Science and Technology, Bannu, Pakistan

Abstract:

Summary: An isoenzyme of high molecular weight acid phosphatase (HM-ACP) from the liver of fish Rohu (Labeo Rohita) was isolated and purified to homogeneity. The enzyme had specific activity of 14.96 U/mg and a recovery of about 4 %. The purification procedure included ammonium sulphate precipitation and series of chromatographic separations on SP-Sephadex C-50, CM-Cellulose and Sephacryl HR-200 columns. Nearly 500-folds purification was achieved. The molecular weight was estimated to be 120-130 kDa by polyacrylamide gel electrophoresis (PAGE) of native enzyme and 130 kDa by gel filtration on calibrated Sephadex G-100 column. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced & non - reduced conditions showed a band corresponding to 66 kDa confirming the dimeric nature of enzyme. Para - nitrophenyl phosphate and flavin mononucleotide were hydrolyzed effectively by the enzyme and found to be good substrates. Optimum temperature for the enzyme was 50oC and temperature stability was 0o-50oC. Similarly optimum pH for the enzyme was 5.4 and pH stability was 4.8-6.0. The Km for the p-nitrophenyl phosphate was estimated to be 0.15 mM. The enzyme was competitively inhibited by the phosphate, vanadate, molybdate, tartrate, fluoride and pyridoxal-5 PO4 while pyridoxamine-5 PO4 showed poor inhibition. Metal ions such as Age, Cu2+, Zn2+ showed strong inhibition on the enzyme activity while other divalent ions like Mg2+, Mn2+ and Co2+ were found to be poor inhibitors. Modifiers like EDTA, methanol, ethanol, acetone and glycerol had no effect on the enzymes activity.

Page(s): 801-808

DOI: DOI not available

Published: Journal: Journal of Chemical Society of Pakistan, Volume: 31, Issue: 5, Year: 2009

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