Fig. 2

Cystine quantification method utilized herein: A N-ethylmaleimide (NEM) blocked non-cystine thiols before cell lysis; cystine lysed from lysosome was reduced by NaBH₄ to cysteine. B The inactive disulfide derivative of papain was activated in the presence of cysteine. C Papain cleaves the substrate N-benzoyl-L-Arginine-4-nitroanilide hydrochloride (L-BAPNA) releasing the p-nitroaniline chromophore detectable at 410 nm. Values were quantified by comparison to a cysteine standard. Quantity of cysteine was therefore stoichiometrically equal to p-nitroaniline thus serving as a surrogate for lysosomal cystine concentration