The cysteine thiol in E3CaG-1 is at position 974.

(A) E3CaG-1 in pH 8.0 buffer containing either 0.1 mM (lanes 2 and 3) or 2 mM Ca⁺⁺ (lanes 4 and 5) was untreated (lanes 2 and 4) or reacted with NTCB (lanes 3 and 5). The peptide bond N-terminal of the cyanylated cysteine was then cleaved at pH 9. The protein was reduced and alkylated and resolved by SDS-PAGE. The M_r markers are shown in lane 1. (B-D) The 3 Coomassie-stained fragments were cut from the gel, digested with trypsin, and the resulting peptides analyzed by MALDI-rTOFMS. (B) Profile of band i in panel A, lane 5; (C), profile of band ii in panel A, lane 5; (D), profile of band iii in panel A, lane 5. Band i is undigested E3CaG-1, and bands ii and iii are the result of a single cleavage of band i. The M_r's of the major peptides were matched to the E3CaG-1 amino acid sequence. The amino acid residues of selected peptides are shown in the tables at the right of the profiles.