SENSITIZER‐INDUCED CONFORMATIONAL CHANGES IN LENS CRYSTALLIN—I. PHOTODYNAMIC ACTION OF METHYLENE BLUE AND N‐FORMYLKYNURENINE ON BOVINE α‐CRYSTALLIN

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Abstract
Abstract— Fluorescence and circular dichroic properties of bovine a‐crystallin have been monitored to detect changes in the structural integrity of the protein following photoreactions in the presence of sensitizer, either methylene blue or N‐formylkynurenine. Methylene blue‐sensitized photooxidation causes a change in the tertiary structure as manifested in the near‐UV CD; this is observed within 0.5 h of irradiation during which time tryptophan emission decreases rapidly. Using inhibitors specific for active species of oxygen, it has been shown that singlet oxygen predominantly causes this change but the sensitizer molecules also have some role in this process. Upon 6 h of irradiation in the presence of methylene blue under both aerobic and anaerobic conditions, the thiol groups that were in a non‐polar region of the protein are exposed to polar environments. In conformity with these fluorescence results. near‐UV CD (tertiary structure) suffers a drastic alteration whereas the far‐UV CD (secondary structure) remains virtually unchanged. The studies with inhibitors indicate that sensitizer molecule itself is primarily responsible for this process. This major change in the conformation has been explained by suggesting that a large portion of the protein unfolds in the photosensitized reaction, thereby altering microenviron‐ments, orientation, and intermolecular interactions of different amino acids. N‐formylkynurenine also shows some changes in the near‐UV CD, presumably, caused by H2O2 generated in the photosensitized reaction. But the major alteration in the microenvironments of thiol groups and in the near‐UV CD, as observed in the case of methylene blue, does not occur even when the protein is irradiated for 6 h in the presence of N‐formylkynurenine and air.