Study On Purification And Quality Standard Of Melittin

In this paper, we have established a new technique to extract and purify melittin from bee venom, in which only gel filtration chromatography was used. The sample was tested by SDS-PAGE and HPLC,and quality standard of melittin was established.1. Extraction and purification of melittin from bee venom by gel filtrationThe condition of column chromatography: All chromatographic procedures were carried out at room temperature using an fraction collector, and the absorption was monitored at 280 nm . Melittin was isolated and purified from bee venom by gel filtration chromatography on columns of Sephadex G-25 and Sephadex G-50 in 0.1M formate buffer (pH4.5) and on columns of Sephadex G-75 in 0.02M formate buffer (pH3.0). The sample was eluted at a flow rate of 30mlh-1 and 22 mlh-1. Well-separated peaks were obtained, and the target sample was found to be consistent with melittin in Rf and retention time on SDS-PAGE and HPLC .2. Study on the quality standard of melittinSDS-PAGE and HPLC were applied to establish the determination of melittin. Homogenicity and molecular weight of melittin were determined by SDS-PAGE and HPLC. The conditions of HPLC were selected as follows: protein-pak 125 column, water-methanol-trifluoroacdtic acid (98:1.8:0.2) as mobile phase, flow rate at 1.0ml/min, detecting wavelength at 280 nm. The sample was a single peptide-chain with a molecular mass of about 2800 dalton as revealed by SDS-PAGE. The average recovery of HPLC was 99.21% and RSD was 1.10%. Melittin prepared by a combination of gel filtration chromatography had high purity. The method for the determination of milittin applying SDS-PAGE and HPLC was accurate, reliable and simple.