The main pepsinogen from the mucosa of the shark, Centroscymnus coelolepis, has been purified and characterized. This zymogen, the most abundant protein in terms of quantity and activity (yield 72%), is a homogeneous monomer of molecular weight 42+/-0.7 kDa, as determined by electrophoresis. The aspartyl proteinase nature of this enzyme was confirmed by the considerable inhibition by pepstatin. Its specificity as to the oxidized B-chain of bovine insulin was determined using electrospray ionization mass spectrometry (ESI-MS) coupled with reversed phase high pressure liquid chromatography (RP-HPLC). The 15-16 Leu-Tyr bond was rapidly cleaved in this substrate, followed by the 24-25 Phe-Phe, 25-26 Phe-Tyr, and 11-12 Leu-Val bonds.