1. Reaction of free thiols with 5,5'-dithiobis(2-nitrobenzoate) (DTNB). The resulting modified thiols absorb strongly at 410-412 nm (see G. Allen, "Sequencing of proteins and peptides", Laboratory tecniques in biochemistry and molecular biology, vol. 9, Elsevier, where the reference to the original work by Zahler & Cleland and Walsh, et al. are given). Pierce Chemical Co. (and I am sure others) sell the required reagents.
2. Reaction of free thiols with any of the "standard" cysteine alkylation reagents, e.g. iodoacetic acid, N-ethylmaleimide, 4-vinylpyridine, etc. Depending on the size of the protein and the mass resolution/accuarcy of the mass spectrometer used one can cound how many cysteines were alkylated.
Many proteins may have to be denatured (e.g. 6 M Guanidine-HCl) in order to expose the free thiols to the alkylating agent. Also, one can alkylate any free thiols with one alkylating agents and then reduce disulfides and alkylate with another in order to identify which cysteines were originally reduced and which were oxidized. Care should be taken (e.g. by keeping he pH low) to avoid disulfide scrambling.
Ioannis Papayannopoulos
Astra Arcus USA, Inc.
Worcester, MA
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