Melittin, a major component of bee venom: Determination by capillary electrophoretic methods

In recent times, bee venom from honey bees (Apis Mellifera) have become subject of excessive pharmacological and biological investigation because of its form of alternative and preventive medication for treatment of variety of ailments. Numerous studies have been showed its capability in treating arthritis, rheumatism, pain, angiocardiopathy, cancerous tumors, sclerosis and skin disease. Bee venom has been used since pheristoric times and in the ancient civilization of China, India, Egypt, Babylon, and Greece for apitherapy, but its composition was established few years ago. Bee venom is natural toxin that produced by the venom gland located in the abdominal cavity of a bee and contains considerable biologically active peptides, including melittin, apamin, adolapin, mast cell degranulation peptide, and enzymes such as phospholipase A2 and hyaluronidase, and also nonpeptides components such as histamine, dopamine and epinephrine. Melittin is the main component and the major pain producing substance of bee venom. It was first identified as a haemolytic factor by Habermann in 1954. Melittin comprises approximately 50% of dry weight of bee venom. It is a small linear peptide composed of 26 amino acids residues. The first twenty amino acids of this peptide are mostly apolar whereas the C-terminal hexapeptide is polar and highly basic. Capillary electrophoresis (CE) is a modern analytical method that offers advantages of short analysis time and minimum consumption of both reagents and samples. It is well suited for the separation of aminoacids, peptides, proteins and has been used for this purpose. In CE, peptides function differently in terms of separation efficiency than small molecules. They differ in electric charge, relative molecular mass, conformation, hydrophobicity, and specific binding. Attributed to these properties, electrophoretic separation can be achived by differences in electrophoretic mobility, size, charge, hydrophobicity, specific interactions with other biomolecules. The aim of this study is determination of mellitin content in bee venom via CE. It was observed that separation of basic peptide melittin has loss of efficiency because of adsorption on the internall surface on the capillary wall. The adsorption occurred due to the electrostatic interactions between positively charged residues of the basic peptide and negatively charged fused silica surface. Two strategies have been tried to prevent wall adsorption. The first is to supresss the negative surface charge of the silica capillary by lowering the pH of the separation buffer, and the other is coating the inner wall of capillary with a positively charged polymer to eliminate the wall negative charge. This study deals with the determination of predominant melittin component using bare fused silica capillary and polymeric coated capillary electrophoresis, and critically compares these two approaches.