Influence of pH on radical reactions between kynurenic acid and amino acids tryptophan and tyrosine. Part II. Amino acids within the protein globule of lysozyme.

An acidosis, a decrease of pH within a living tissue, may alter yields of radical reactions if participating radicals undergo partial or complete protonation. One of photosensitizers found in the human eye lens, kynurenic acid (KNA-), possesses pKa 5.5 for its radical form that is close to physiological pH 6.89 for a healthy lens. In this work we studied the influence of pH on mechanisms and products of photoinduced radical reactions between KNA- and amino acids tryptophan (Trp) and tyrosine (Tyr) within a globule of model protein, Hen White Egg Lysozyme (HEWL). Our results show that the rate constant of back electron transfer from kynurenyl to HEWL• radicals with the restoration of initial reagents - the major decay pathway for these radicals - does not change in the pH 3-7. The quantum yield of HEWL degradation is also pH independent, however a shift of pH from 7 to 5 completely changes the outcome of photoinduced damage to HEWL from intermolecular cross-linking to oxygenation. HPLC-MS analysis has shown that four of six Trp and all Tyr residues of HEWL are modified in different extents at all pH, but the lowering of pH from 7 to 5 significantly changes the direction of main photodamage from Trp62 to Trp108 located at the entrance and bottom of enzymatic center, respectively. A decrease of intermolecular cross-links via Trp62 is followed by an increase in quantities of intramolecular cross-links Tyr20-Tyr23 and Tyr23-Tyr53. The obtained results point out the competence of cross-linking and oxygenation reactions for Trp and Tyr radicals within a protein globule and significant increase of oxygenation to the total damage of protein in the case of cross-linking deceleration by coulombic repulsion of positively charged protein globules.

Influence of pH on radical reactions between kynurenic acid and amino acids tryptophan and tyrosine. Part I. Amino acids in free state.

In the human eye lens the endogenous chromophores of UV-A light (315-400 nm) are able to sensitize radical reactions leading to protein modifications during normal aging and the cataract progression. Kynurenic acid (KNA-) is the most photochemically active dye of the human eye lens reported to date with pKa(KNAH2•) 5.5 for its radical form. Cataract is thought to develop under oxidative stress which could be accompanied by acidosis, an acidification of the intracellular environment. Protonation of kynurenyl radicals at mildly acidic conditions may change the outcome of radical reactions leading to additional damage to proteins. In this work we investigated the influence of pH on the degradation of initial reagents and the formation of products in photoinduced radical reactions between KNA- and amino acids tryptophan (Trp) and tyrosine (Tyr) in free states. Our results have shown that pH variation has minor influence on kinetics of reagent decay and accumulation of products in reactions between tyrosyl and kynurenic acid radicals. However in the case of Trp a two-fold decrease of the reagent degradation without visible changes in the composition of formed products was observed with pH decrease from 7 to 3. Time-resolved measurements have shown similar acidification-induced two-fold acceleration of decay of kynurenyl and tryptophanyl radicals via Back Electron Transfer (BET) with the restoration of initial reagents. Experiments with tryptophan derivatives with different pKa values for their radical forms point out the protonation of tryptophanyl radical as the driving force for BET acceleration at low pH. Our results demonstrate that the protonation of kynurenyl radical does not change its reactivity towards amino acids radicals but the total yield of radical photodamage decreases with the protonation of tryptophanyl radicals. It could be expected that radical induced damage to proteins will depend on the pKa of tryptophanyl radicals within a protein globule.