Year

2001

Degree Name

Doctor of Philosophy

Department

University of Wollongong. Dept. of Chemistry

Abstract

Kynurenine (Kyn) and 3-hydroxykynurenine (3-OHKyn) are present in the human lens where they play a protective role. With age, human lens proteins become increasingly yellow. A marked increase in protein colouration is also observed with the increasing severity of age related nuclear cataract. The molecular basis for the increase in colour with age is not well understood, however, it has been proposed, at least in result from modification by UV filter compounds such as 3-OHKyn. Until recently, however, no direct evidence for these modifications has been found. To explore the of these UV-filters in the lens, model studies have been used to develop and verify analytical methods (based on mass spectrometry in combination with HPLC). These methods enabled the direct analysis of amino acid modifications in human lens proteins.

Since UV filter compounds can often only be isolated in small quantities from the this study used mass spectrometry as the basis for the identification and characterization of novel kynurenine-derived compounds. A combination of accurate mass measurement and tandem mass spectrometry was used to characterise the major fragment ions observed in the ESI mass spectrum of kynurenine. Three major fragmentation pathways were evident, resulting from the initial elimination either of ammonia, H20 and CO the imine form of glycine.

The major autoxidation products of 3-OHKyn as well as their relative rates of formation and the role that H2O2 played in mediating the autoxidation process was examined. Oxidation of 3-OHKyn generated a number of compounds, namely: xanthommatin (Xan), p-quinone, 4,6-dihydroxyquin-olinequinonecarboxylic acid (DHQCA) and a compound that has a structure consistent with that of hydroxyxanthommatin (OHXan). Xan was the major product formed initially, however, it was found to be unstable, particularly in the presence of H2O2, and rapidly degraded to yield DHQCA. Hydrogen peroxide was formed rapidly upon oxidation of 3-OHKyn, and significantly influenced the rate of autoxidation.

Using an acid hydrolysis procedure in combination with HPLC and LC-MS we were able to detect covalent adducts of kynurenine bound to histidine and lysine residues. 3-OHKyn adducts of His and Lys residues, however, were not sufficiently stable under the acid hydrolysis conditions to enable them to be analysed by this method. Kyn modifications to Cys residues were found at very low levels due to their poor stability under physiological pH conditions.

Proteins isolated from human lenses were shown to contain significant levels of the coloured UV-filter, kynurenine, covalently bound to histidine and lysine residues at concentrations up to 3.6 and 0.2 nmol/mg of lens protein respectively. Analysis of human lens tissue over a wide range of ages indicated that the level of bound kynurenine increased with the age of the individual. The analysis of human cataract tissue, however, revealed that the level of Kyn modification to lens tissue decreased with the increasing severity of cataract. This indicated that Kyn once bound to lens protein may play a role in the progression of cataract.

Overall the work described in this thesis has provided strong evidence that posttranslational modification of lens proteins by tryptophan metabolites appears responsible, at least in part, for the age-dependent colouration of the human lens and that covalently-bound Kyn plays a significant role in the progression of cataract.

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