A number of alphaA-crystallin mutants are associated with hereditary cataract including cysteine substitution at arginine 49. We report the formation of affinity-driven disulfide bonds in the interaction of alphaA-R49C with betaB1-crystallin. To mimic cysteine thiolation in the lens, betaB1-crystallin was modified by a bimane probe through a disulfide linkage. Our data suggest a mechanism whereby a transient disulfide bond occurs between alphaA- and betaB1-crystallin followed by a disulfide exchange with cysteine 49 of a neighboring alphaA-crystallin subunit. This is the first investigation of disulfide bonds in the confine of the chaperone/substrate complex where reaction rates are favored by orders of magnitude. Covalent protein cross-links are a hallmark of age-related cataract and may be a factor in its inherited form.