The proteomic maps of lenses of experimental animals (Wistar and OXYS rat strains) of different age and cataract progression have been obtained. The determination of the proteomic content in the lens, depending on the age and the stage of cataract progression, has been performed by the quantitative analysis of water-soluble and water-insoluble protein fractions. Mass-spectrometric analysis revealed post-translational modifications of proteins from the eye lens with a mature cataract.
Proteomic characterization of O. felineus excretory-secretory (ES) products and parasite surface (tegument) resulted in 31 unique protein identifications. The most abundant proteins of ES product and tegument are five isoforms of glutathione transferase (GST), thioredoxin peroxidase, enolase, ferritin, superoxide dismythase and cathepsins. GSTs and thioredoxin peroxidase play an important role in the detoxification of organism, being responsible for the parasite survival. The presence of cathepsin proteases is accounted to their role in the digestion of host tissue for nutritional purposes. Other enzymes in ES and tegument, such as ferritin, superoxide dismythase and enolase, are key players in the protection of O. felineus from the host immune response.
The content of kynurenine and other molecular UV filters was determined in the human lenses with various degrees of the cataract. For the first time, the products of kynurenine decomposition were found in the human cataractous lenses. It has been shown that the low level of antioxidant glutathione is responsible for the presence of these molecules. The obtained results support a key role of chemical reactions with participation of kynurenine and its derivatives in the lens protein modification and the cataract development.
Our experiments revealed the detailed mechanism of spontaneous decomposition of kynurenine and its derivatives in aqueous solutions under physiological conditions. It has been shown that decomposition products, carboxyketoalkenes, can covalently attach to the amino acid residues of lens proteins; the rate constants of the covalent attachment reactions were measured. Our results demonstrate that glutathione can bind reactive molecules of carboxyalkenes and, thus, this antioxidant could be regarded as the main protecting agent, preventing undesired modifications of lens proteins.
Detailed investigation of photophysical processes, occurring in kynurenine molecule under UV irradiation, revealed that the ultrafast internal conversion is the main channel of the excited S1 state decay. Our results speak in favor of intermolecular hydrogen bonding interactions to be responsible for very fast radiationless transition S1->S0. In aprotic solvents, in absence of intermolecular hydrogen bonds, the lifetime of S1 state tremendously increases, which results in the augmentation of the yield of reactive triplet state.
The mechanisms of photochemical reactions of kynurenine and its derivatives were studied within last several years. For the first time, it has been found that the triplet state of kynurenine could be populated via the direct photolysis. This triplet state is photochemically active and it can react with amino acid residues (tryptophan and tyrosine) via electron transfer mechanism. It has been shown that ascorbate is the most effective quencher of kynurenine triplet state among all antioxidants present in the human eye lens. The covalent attachment of molecular UV filters to proteins markedly augments their photochemical activity.