Zymographic Detection of Aspartic Proteinase Activities in Porcine Ovarian Extracts

Aims: Tightly regulated proteolytic activity is essential in the mammalian ovary to maintain follicular and luteal functions. Studies conducted on ovarian aspartic proteinases (APs) are limited. Previously it has been noted that the AP activity increases towards the latter part of luteal phase. The aim of this study was to isolate AP from porcine ovarian extract and to identify whether multiple AP activities are found in the extracts.

Place and Duration of Study: Department of Biochemistry, between December 2009 and February 2012.

Methodology: Porcine ovaries (n= 100) were collected and ovarian extracts were prepared. APs were fractionated, using anion exchange chromatography at pH 8.5, gel permeation chromatography and affinity chromatography. AP activity (U/ml) of the fractions were measured in the presence and absence of Pepstatin A. AP specific activities (U/mg) were calculated after measuring total protein concentrations (mg/ml) of the fractions. Fractions were analyzed using polyacrylamide gel electrophoresis conducted under denaturing (SDS-PAGE) and native (PAGE) conditions. PAGE was followed by zymography.

Results: With anion exchange chromatography, AP was recovered during column washing as an unbound fraction (~67%) and during elution as a bound fraction (~33%). Bound AP was recovered around 0.23 M NaCl with 0-1 M NaCl gradient used during elution. Both AP fractions had an apparent molecular mass of 40 kDa. AP activity was completely inhibited in the presence of 1 µM Pepstatin. Specific activity of AP increased with fractionation from 1 in the ovarian extract to 747 and 511 U/mg with unbound and bound AP respectively. SDS-PAGE showed elimination of impurities with the progress of fractionation. PAGE and zymography showed the presence of at least three AP activities in porcine ovaries.

Conclusion: Proteinases fractionated using three chromatography procedures were APs. Results showed the presence of multiple AP activities in porcine ovary.