A study of the aggregating cationic antibacterial proteins and peptides in bovine seminal plasma

Abstract

Several non-specific defence mechanisms are known to be involved in the protection of the mammalian host against invading microorganisms. Among them are well researched proteins like lysozyme and lactoferrin, systems like the myeloperoxidase- and lactoperoxidase-mediated system, and two complement systems. Also, a number of mammalian cationic proteins and peptides have been found to have antibacterial properties. The antibacterial activity in bovine seminal plasma has been reported to be due to proteins which appeared to be present in aggregated forms (Eschenbruch, 1980; Shannon et al., 1987). This investigation was designed to account for the aggregation and disaggregation of the antibacterial activity in bovine seminal plasma. The overall study demonstrated that most or all of the high molecular weight antibacterial proteins of bovine seminal plasma are aggregates. Three major antibacterial aggregates with molecular weights of around 500 kDa, 250 kDa and 20 kDa were identified and isolated using gel filtration chromatography at neutral pH. Acidic gel filtration chromatography, cation exchange chromatography using citrate buffer and anion exchange chromatography at pH 12.0 and pH 13.0 were found to be successful techniques for disaggregation of the antibacterial aggregates. FPLC of diluted samples using acetonitrile and trifluoroacetic acid was found to give complete disaggregation. The aggregates were found to be made up of two peptides, a basic antibacterial peptide with a molecular weight of about 1.2 kDa and an inactive acidic peptide with a molecular weight of about 1 kDa. However, the molecular weights estimated of the components and the aggregates could be larger than estimated in this study because of possible retardation on gel filtration chromatography. Both the peptides were shown to be necessary for the formation of the antibacterial aggregate. These peptides were found to form a stable antibacterial aggregate of about 20 kDa mainly through hydrophobic interactions. The aggregation beyond the 20 kDa aggregate, resulting in the formation of the 500 kDa and 250 kDa aggregates, was found to occur through ionic interactions. Furthermore, lysozyme was found to occur both in a free form and bound with the antibacterial aggregates. The possible significance of the aggregation on the antibacterial activity is discussed.