Figure 2. Hydrolysis of the oxidized ß-chain of insulin by ZapA. A solution of oxidized ß-chain (20 µM) was incubated with 2.8 pM purified native ZapA in 50 mM Tris-HCl and 2 mM CaCl₂, pH 8.0, at 37ºC for 3 h. The hydrolysis products were analyzed by HPLC as described in Material and Methods. The chromatogram in panel B shows the profile of the intact polypeptide, and panel C shows the production of the fragments generated by the incubation with ZapA. The bonds cleaved by ZapA, indicated by arrows in the sequence of the ß-chain of insulin (panel A), were determined by mass spectrometry as described in Material and Methods. ¯ = known cleavage sites; ß = newly detected cleavage site.