The isolation and characterization of antibacterial factors from bovine seminal plasma and pancreas : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
An isolation procedure was developed for the extraction of antibacterial factor(s) from bovine seminal plasma (BSP). The procedure involved a batch separation on a cation exchange resin: adsorption onto cellulose phosphate, and elution of the active fraction with sodium citrate. For comparison, an antibacterial fraction was extracted from bovine pancreas by homogenization of the tissue in an acidic-citrate solution, followed by treatment with cellulose phosphate. Optimal conditions for the recovery of antibacterial material with high specific activity were determined. The antibacterial fractions were further purified by ethanol precipitation and the purity of various preparations was monitored by SDS gel electrophoresis. Purified bovine seminal plasma and pancreas preparations were used to immunize rabbits. The antisera obtained were run against homologous and heterologous bovine antigens in immunodiffusion agar plates. Whereas BSP preparations elicited a multicomponent antibody response in rabbits, pancreas preparations were either non-antigenic or only poorly antigenic. Cellulose phosphate column chromatography of BSP ethanol precipitated material confirmed the heterologous nature of "purified" preparations. Absorption studies on immunodiffusion agar using anti-BSP antiserum and "column-antigens" were used to determine relationships between the multicomponent BSP antibody system (anti-BSP antiserum) and the various column fractions. Antibacterial activity was associated with one peak,which elicited an indistinct antibody response, compared to other components,which gave sharp precipitin reactions in the presence of homologous antibody on immunodiffusion agar. Physico-chemical and biological properties of the seminal plasma and pancreas preparations were established and compared. Antibacterial activity was determined by the agar diffusion assay using Micrococcus lysodeikticus as the test organism. The active fractions from both seminal plasma and pancreas material contained basic polypeptides with molecular weights of approximately 15,000 dal tons. Both were dialyzable, heat stable, trypsin sensitive and were subject to little if any inactivation by anionic polymers such as deoxyribonucleic acid. Neither was associated with lysozyme or phospholipase activity. An amino acid analysis of a seminal plasma preparation with a high specific activity gave a lysine to arginine ratio of 1.27; this is similar but not identical to the various calf thymus histone fractions which have been reported in the literature. Further studies on BSP and pancreas material Involved comparisons of ethanol and ammonium sulphate precipitations, as well as ethanol reprecipitation of BSP material following acidification in the presence of citrate. Although the active fractions of both seminal plasma and pancreas possessed similar characteristics, the different behaviour of these fractions in precipitation studies suggested that these may be closely related proteins or modified forms of the same protein.