Doctor of Philosophy
Department of Chemistry
Hood, Brian David, Seeing red but not blue: model studies on the ageing human lens, Doctor of Philosophy thesis, Department of Chemistry, University of Wollongong, 2002. http://ro.uow.edu.au/theses/1171
The lens is the primary medium by which light is transmitted onto the retina. The transparency of the lens to light in the visible region and its flexibility to focus this light are crucial in maintaining vision. Ageing of the human lens is characterised by the slow onset of a variety of physical changes that affect the way we visualise the world. As we age, the integrity of the lens deteriorates in several quantifiable ways. The protein in the lens becomes progressively more yellow, the effect of this filter being that the colours transmitted to the retina are diminished, especially the blue-green colours. In addition the lens proteins become more fluorescent and the crystallin protein, which makes up most of the dry weight of the lens, slowly becomes more insoluble. It is a widely held view that these physical manifestations of ageing in the lens are attributed to post-translational modification of the crystallin protein. Over time these modifications alter the integrity of the protein. Individual units aggregate and eventually cease to remain soluble. Increased insolubility of the protein is associated with the elderly suffering glare from bright lighting and the eventual loss of vision through cataract formation.
The human lens contains a number of low molecular weight fluorescent compounds derived from the metabolism of tryptophan, termed UV filter compounds. They absorb most of the UV light transmitted by the cornea between 295 and 400 nm. It has been speculated that the function of these compounds within the lens m a y be to protect the lens and the retina from harmful UV radiation and to increase visual acuity.
In this study tryptophan metabolites were examined for their ability to covalently modify crystallin protein of the lens. The primary objective was to find evidence for this modification on the protein. Secondary objectives of this study were to discover what physical effects these modifications had on parameters normally associated with the ageing lens, namely increases in protein 'colouration', fluorescence, aggregation and precipitation.
An important finding from research undertaken in our laboratory was that metabolites of tryptophan that contain the kynurenine side-chain, can spontaneously deaminate to form an a, B-unsaturated carbonyl compound. Model incubation studies were conducted with an analogue of this reactive intermediate in aqueous buffered solutions to determine if the intermediate can further react to form adducts with various nucleophilic amino acids. Evidence is given that not only shows that the intermediate is stable in aqueous buffered solution but that adducts are formed with thiol and s-amino groups of cysteine and lysine, respectively, in high yield.
Having established the feasibility of forming kynurenine-derived adducts with crystallin, we then established a test to quantify the amount of 3-hydroxykynurenine-O- (3-D-glucoside ( 30HKG - the most abundant kynurenine-derived U V filter) bound to protein from human lenses. By treating protein material with alcoholic hydroxide for 48 hours, a fluorescent compound was released that was identified as the cleavage product of 30HKG (2-amino-3-hydroxyacetophenone-0-P-D-glucoside, AHAG ) . AHAG was quantified from the protein extracts of over 100 individual human lenses spanning over eight decades of life. Quantification showed that there were negligible quantities of the bound fluorophore prior to approximately 40 years of age, whereafter an exponential increase could be observed. The amount of 30HKG bound to the crystallin protein also showed a good correlation to the observed 360 nm absorption and non-tryptophan fluorescence of the human lens protein.
The UV filter compounds were incubated with crystallin protein derived from young bovine lenses for up to five days with the aim of examining the parameters associated with the ageing lens. The crystallins obtained from the bovine lens have a close sequence homology to that of the analogous human lens proteins but the bovine species lack the UV filter metabolic pathway, hence the protein had not previously been in contact with these small molecules. The incubated protein material exhibited similar physical characteristics as protein from aged human lenses, namely increases were observed in 360 nm absorption, non-tryptophan fluorescence, protein aggregation and protein precipitation as incubation time and UV filter concentration were increased. The inclusion of glutathione, an anti-oxidant compound normally found in healthy human lens homogenates, delayed crystallin modification as measured by these parameters.
Finally, mass spectral evidence is provided for crystallin sequences that had been modified by the most abundant U V filter, 30HKG . B y incubating separate crystallin families with 30HKG for up to five weeks followed by digestion with trypsin, we found that cysteine residues from bovine oA and BB2 crystallin sequences were initial and major sites of covalent modification by this UV filter compound. Since 30HKG (and the other kynurenine-derived UV filters) are continually synthesised in the human lens over the lifetime of the individual, these findings m a y provide a biochemical link to the physical parameters associated with the ageing lens and cataract formation.