Raman spectroscopy of calf lens γ-II crystallin: Direct evidence for the formation of mixed disulfide bonds with 2-mercaptoethanol and glutathione

This study presents Raman spectra of calf lens γ-II crystallin and its reaction products with reduced glutathione, 2-mercaptoethanol and p-hydroxymercuribenzoate. The absence of a disulfide vibration in γ-III crystallin (both in aqueous solution and in lyophilized state) indicates that the seven thiol groups in this protein are resistant to air oxidation, and are capable of maintaining their reduced state in the absence of added reducing agents during isolation. However, treatment of the protein with low molecular weight thiols such as glutathione and 2-mercaptoethanol results in mixed disulfide bonds. We have detected, for the first time, the S-S bond stretching vibration from the mixed disulfides at 510 cm^-1, which is very similar to the 508 cm^-1 reported for the inter/intramolecular disulfide bonds in intact mouse lenses (Yu, N.-T., DeNagel, D.C., Pruett, P.L. and Kuck, J. F. R., Jr. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 7965-8). Upon titration with five equivalents of p-hydroxymercuribenzoate, a strong Raman line was detected at 345 cm^-1, which is tentatively attributed to the Hg-S stretching vibration of the mercaptide complex. The S-H vibration region (2500-2700 cm^-1) exhibits two resolved peaks at 2562 and 2580 cm^-1 with an intensity ratio of 2:5. Both reactive surface thiol groups and buried cysteines give rise to the S-H vibration at 2580 cm^-1.