Studies on protein photo-oxidation.
thesisposted on 19.11.2015, 09:08 authored by Nicholas Maurice Charles. Kaye
Photo-oxidation is a light-dependent reaction, catalysed by photo-sensitive dyes, which can result in the oxidation of a large variety of substances. The photo-oxidation of enzymes with the anionic dye rose bengal was shown to follow one of two types of pH dependence; with an increase in pH the rates of photo-inactivation either increased (conventional photo-oxidation pH profiles), or decreased (unconventional photo-oxidation pH profiles). Photo-oxidation of the same enzymes with the cationic dye methylene blue gave rise to conventional pH profiles, irrespective of the type of pH dependence obtained with rose bengal. The production of unconventional photo-oxidation pH profiles was found to be dependent on the anionic nature of the photo-sensitiser. Using visible absorption spectroscopy and enzyme kinetic studies, rose bengal, but not methylene blue, was found to be capable of binding to and inhibiting the activity of enzymes. However, amino acid analysis of pig heart citrate synthase demonstrated that the occurrence of unconventional photo-oxidation pH profiles and dye-enzyme interactions was not accompanied by increased specificity of residue destruction. Some restriction of dye-protein interactions was achieved by the immobilisation of rose bengal on Sepharose. The photo-oxidation of various enzymes with this immobilised form of the dye was found to result in the formation of unconventional pH profiles only, irrespective of the type of photo-oxidation pH profile originally observed with the free form of the dye. The total prevention of all contact between rose bengal and enzymes was attained by compartmentalising the immobilised dye within a dialysis sac, the protein remaining on the outside. Photo-oxidation of enzymes with this compartmentalised dye again resulted only in the formation of unconventional pH profiles. Both the free and immobilised states of rose bengal were shown to photo-oxidise the five known susceptible amino acids---cysteine, histidine, methionine, tryptophan and tyrosine. The cysteine-containing peptide glutathione, and a glutathione-Sepharose complex, were also shown to be sensitive to photo-oxidation by free rose bengal, and the formation of conventional pH profiles only was observed. Although glutathione was shown to be similarly sensitive to photo-oxidation by the immobilised dye, no loss of cysteine occurred when the glutathione-Sepharose complex was irradiated in the presence of either immobilised or compartmentalised dye. Furthermore, the cysteine residues of mercaptalbumin proved insensitive to photo-oxidation by these two states of the dye. This demonstrated the necessity of direct interactions between rose bengal and cysteine residues as a prerequisite for their photo-oxidation. In the case of photo-oxidation of enzymes with free or immobilised rose bengal, the involvement of singlet oxygen was indicated by the enhanced rates of photo-inactivation obtained when H2O was replaced by D2O as solvent and also by the protective effects of sodium azide. It is therefore suggested that the pH profile obtained on photo-oxidation of enzymes by rose bengal consists of two components; a component at high pH which involves the photo-oxidation of cysteine residues by a direct contact mechanism and a component at low pH which involves the photo-oxidation of other susceptible residues by a singlet oxygen mechanism. This suggestion is supported by the demonstration of a correlation between the type of pH profile obtained on the photo-oxidation of various enzymes with free rose bengal and the presence of cysteine residues in their active sites, as indicated by inactivation experiments with the thiol-specific reagents DTNB and pHMB. The combined use of all the three forms of rose bengal may therefore both increase the specificity for residue destruction and provide information on the mechanisms involved in the photo-oxidation of biological macromolecules to a greater extent than is possible with the free dye alone. Immobilised rose bengal was found capable of photo-oxidising the active site of Acinetobacter citrate synthase yet, in contrast to free rose bengal, was incapable of desensitising the enzyme to NADH inhibition. The advantages and potential offered by the use of immobilised and compartmentalised rose bengal are discussed in this thesis and attention is drawn to the difficulties encountered and the caution required in the interpretation of photo-oxidation pH profiles.