The physical characterization and antibacterial activity of herring protamines

Clarke, Annette June Morgan (1998) The physical characterization and antibacterial activity of herring protamines. Masters thesis, Memorial University of Newfoundland.

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Abstract

Protamines are a class of low molecular weight highly basic proteins found in the sperm nuclei of a variety of animals. These proteins are often heterogeneous with components that differ slightly in amino acid sequence. According to published data, herring protamine (or clupeine) can be fractionated into 3 components, namely clupeine Y-l, Y-ll and Z, via ion exchange chromatography. The amino acid compositions and sequences of these 3 components have also been published. -- In this study, protamines were isolated from the gonads of herring, Clupea harengus, and compared with commercially available herring protamine (Sigma clupeine). Protamines were first extracted from mature herring testes of uncertain stage in spermatozoan development. The protamine yield was low, therefore another extraction was performed on sexually mature, or ripe gonads and a much higher amount of protamine was obtained. Ion exchange chromatography suggested that the first protamine extract contained 9 components (labelled A through l)t while the extract from ripe gonads had 3 major components (labelled MA, MB and MC). The chromatogram of commercial clupeine also showed 3 components (labelled SA, SB, and SC). Despite the presence of multiple components in these protamine extracts, both the intact protamines and the components fractionated by chromatography each gave only a single protein band after polyacrylamide gel electrophoresis and subsequent protein staining. After SDS-PAGE and protein staining, there was no band observable for whole Sigma clupeine. However, salmon protamine (Sigma) and protamine extracted from ripe herring testes each had a single protein band. Protamines extracted from herring testes of uncertain stage in spermatozoan development gave 4 bands. The amino acid compositions of the fractionated components of isolated protamine, MA, MB and MC, and Sigma clupeine, SA, SB, and SC, correspond well with the published amino acid sequences of clupeine Y-ll, Y-l, and Z respectively. The molecular weights obtained by electrospray mass spectrometry of fractions MA, MB, MC, SA, SB and SC are higher than those predicted from the amino acid compositions and from the published molecular weights of clupeine Y-ll, Y-l and Z. This suggests that either the original sequences are incomplete or that microheterogeneity exists in these fractions. Whole protamine from ripe gonads and whole Sigma clupeine were also tested for their antimicrobial activity using a broth dilution assay. Both were found to inhibit the growth of the Gram-positive and Gram- negative organisms tested.

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