Isolation, Characterization, and Biological Activity of Phospholipase A2 (PLA2) and Hyaluronidase from Iranian Honey Bee Venom (Apis Mellifera meda)

Abstract

ObjectiveBee venom contains various enzymes such as hyaluronidase and phospholipase A2, which have medical and pharmaceutical applications. In several studies, the activity of phospholipase A2 and hyaluronidase has been reported in different bee species, but there is no report about these enzymes and their activities in Iranian honey bee venom. Therefore, the purpose of this research was to isolate, identify phospholipase A2 and hyaluronidase enzymes and measure their activity in crude venom and its fractions of Iranian honey bee (Apis mellifera meda).Materials and methodsOne hundred mg of the crude venom was purified using gel filtration chromatography on sephadex G-50, equilibrated with 0.05mM ammonium acetate buffer. SDS-PAGE was used to determine the protein profile of BV and its fractions. Protein concentration, phospholipase A2 and hyaluronidase enzyme activity of Apis mellifera crude venom and its fractions were measured. Protein concentration of Apis mellifera crude venom and its fractions was determined using Bradford method. Phospholipase A2 (PLA2) activity was determined using suspension of egg yolk as substrate. The activity of hyaluronidase was determined turbidimetrically.ResultsThe preliminary results showed that the 56% of crude venom was protein. Crude venom produced three fractions. Phospholipase A2 activity was observed in the crude venom as well as in the first and second fractions. The second fraction showed the highest phospholipase activity. Hyaluronidase activity was observed in crude venom and only in the first fraction. The optimal activity of bee venome phospholipase A2 enzyme was obtained at pH 7 and 37 °C. In this study, it was found that the activity of hyaluronidase in Iranian honey bee venom has the highest activity at 37 to 39 °C and gradually loses its activity as the temperature increases. Also, the results of this study indicate that the optimum pH for hyaluronidase enzyme activity is 5.5.ConclusionsIranian honey bee venom has both phospholipase and hyaluronidase activities, which can be separated using gel filtration chromatography. This study can be introduced as a simple method to isolate, purify and measure the activity of hyaluronidase and phospholipase A2 enzymes of Iranian honey bee venom. Purified hyaluronidase and phospholipase A2 enzymes can be used in industry and research.

Keywords

• apis mellifera meda
• phospholipase
• hyaluronidase