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Studies on the delta haemolysin of Staphylococcus aureus.
thesisposted on 2015-11-19, 09:07 authored by Gillian Margaret. Smith
The production of haemolysins by several strains of Staphylococcus aureus was investigated and strain RN25 chosen for further study. Three methods for the purification of delta haemolysin were compared for efficiency and purity of the products. The products were characterised using polyacrylamide gel electrophoresis, amino acid analysis and K-terminal sequence analysis. The method of Heatley (1971 and 1976) was found to be the most efficient and yielded the product of highest purity and specific activity. Prom polyacrylamide gel electrophoresis in the presence of SDS the miolecular weight of delta haemiolysin was estimated to be less than 5 000. N-terminal sequence analysis of the products of the purification procedures led to the observation that the preparations of delta haemiolysin contained a proportion of N-terminally blocked protein. Thin layer chromatography of the products revealed the three preparations of delta haemolysin to be heterogeneous. The toxin was further purified using hydrophobic affinity chromatography. The molecular weight of native delta haemolysin was estimated from gel filtration to be 150 000. The effect of chemical modification of delta haemiolysin on its haemolytic activity indicated that the N-terminus of the toxin is involved in its mechanismi of action and that the amino groups of the molecule are very important for its activity. The results were closely comparable to those observed for melittin of bee venom. Inhibitors of extracellular protein production, procaine and cerulenin, were found to inhibit the production of delta haemolysin and total extracellular protein. Using cerulenin and harvesting cultures of strain RN25 in the mid-exponential phase of growth, the possibility of the existence of a precursor of delta haemolysin was investigated using techniques of immunoprecipitation.
Date of award1982-01-01
Awarding institutionUniversity of Leicester