A Comparative Study of the Effect of Exogenous and Endogenous Photostabilizers on Lens Crystallin Photodegradation

Abstract

The purpose of the present study was to determine in vitro the effect of sodium azide, ethanol, trans-β-carotene, and the reduced form of glutathione on phototransformations in the lens crystallin. These photostabilizers show a specific affinity for different kinds of free radicals. The water-soluble protein from the cortical part of the eye was irradiated with doses of UV C ranging from 0 to 4.07 J/cm2. Changes in the structure of the crystallins have been monitored by steady-state absorption and fluorescence spectroscopy. Irradiation of dialyzed samples of these proteins at a wavelength of 254 nm (1.13 ± 0.02 mW/cm2) caused photooxidation of aromatic residues; the crystallin solutions became opaque and turbid. The samples displayed increasing attenuance at a wavelength of 280 nm as photodamage proceeded. The fluorescence of tryptophan at 333 nm systematically decreased and a new band between 400 and 500 nm appeared during the UV C irradiation. Our results show that the antioxidants can protect proteins from UV C-induced photodegradation and the protective effect is significantly dependent on their concentration in the protein solution. There are no dramatic differences in the rates of exogenous and endogenous scavenging of generated free radicals for all concentrations used, with rate constants varying by a factor no greater than 2. The mechanism and the rate of scavenging or quenching are dependent on the nature of the radical species and the photostabilizer structure. Although this study provides evidence for free radical scavenging and protein protection, extrapolations to possible antioxidant effects in vivo must be made cautiously.