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Title: Studies on the amino acid sequence of cytochrome c
Author: McLaren, William B.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1964
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The purpose of this work was to obtain information about the sequence of amino acid residues in the peptide chain of cytochrome c, and at the same time to study methods for obtaining ouch information. Cytochrome c Was isolated from horse heart d purified by chromatography on ion exchange resin (Amberlite IRC 50) and on sephadex. Attempts to identify the N-terminal amino acid residue yielded negative results, and it was concluded that the protein may not have a free terminal amino group. This was subsequently confirmed by Kreil and Tuppy who showed that the N-terminal group is acetylglycine. To investigate the internal sequence of the protein, cytochrome c was hydrolysed with trypsin to yield a mixture of peptides. Chromatography on carboxymethyl cellulose, brought about partial resolution of this mixture, and the application of high voltage paper electrophoresis permitted the isolation of some pure homogeneous peptides from the material thus obtained. These methods, however, seemed inadequate for the exhaustive analysis of the peptide mixture. An effort was made, therefore, to reduce the severity of the problem of peptide fractionation, by reducing the complexity of the mixture to be separated. This was achieved by acylating the lysine amino groups of cytochrome c, and thus restricting the action of trypsin to linkages involving the carbonyl groups of the two arginine residues in the protein. Methoxy-carboxyl chloride was firs tried as an acylating agent, but proved unsuitable. Ethyl thiotrifluoroacetate was satisfactory and, at pH 9, this ester reacted with all the amino groups of the protein. It was found also that the substituent trifluoroacetyl groups could subsequently be removed by means of 0.5 M ammonia, without affecting the peptide structure. The action of trypsin on the trifluoroacetyl protein yielded a mixture of peptides which was rather more complex the had been anticipated. On the assumption that this was due to the action of traces of chymotrypsin in the trypsin preparation the time of exposure to the protease was reduced, and it was eventually possible by means of chromatography on sephadex to isolate a small number of large peptides. The amino acid compositions of these peptides, and the nature of their N-terminal residues were determined. Also, one of them was hydrolysed with chymotrypsin, and the resulting mixture of small peptides fractionated on a column of ZeoKarb 225 x 2. The peptides thus separated were analysed and their sequence in the original peptide determined. The results of the analysis carried out during the course of this work were internally consistent, and in general found agreement with the recently published no acid sequence for cytochrome c.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available