Electrospray mass spectrometry studies show that the binding of Pd(II) complexes and the site of cleavage are highly dependent on the secondary structure and local environment of oxidized insulin B chain (B). The hydrolytic cleavage of denatured B by Pd(II) complexes is monitored by HPLC, which follows first order kinetics with half-life of 4.8 days at 40 degrees C. Two amide bonds at Leu6-Cys7 and Gly8-Ser9 are selectively cleaved.