Conformational changes of β_H-crystallin in riboflavin-sensitized photooxidation

The effect of riboflavin-sensitized photooxidation on calf lens β_H-crystallin has been investigated by using fluorescence and circular dichroism techniques. β_H-Crystallin showed a pronounced change in its tertiary structure (conformation) as manifested in the near-u.v. circular dichroism spectra and fluorescence yield of tryptophan residues. The rate of tryptophan photolysis was significantly diminished under anaerobic conditions, but was not affected appreciably when D₂O was used in the reaction mixture instead of H₂O. Ferricyanide and ferricytochrome c added to the solution prior to irradiation inhibited the rate of photolysis of tryptophan, suggesting the involvement of O₂^- anion in the photoreactions. Quantitative assays of O₂^- and H₂O₂ in the irradiated protein solution strongly suggest that the Type I photosensitization pathway is involved in the RF-sensitized photooxidation of β_H crystallin. The effect of photolysis on the cysteine residues of the protein was also studied. The sulfhydryl specific fluorophore N-iodoacetyl-N′-(5-sulfonaphthyl) ethylenediamine (1,5-IAEDANS) was used to study the change in the microenvironment of the cysteine (sulfhydryl) residues of the protein by photolysis. The results indicate that there is a quantitative loss of IAEDANS labeling sites due to photooxidation as well as structural changes of the protein. Fluorescence lifetime measurements indicate that the probe is bound in two environments-the major one (95%) is exposed and the minor one (5%) hydrophobic. A decrease in the lifetimes of the bound label occurs after photooxidation. However, the relative proportion of the hydrophobic IAEDANS-labeling sites increases in the photooxidized β_H crystallin, probably due to the formation of supra-aggregated protein by photolysis.