Proteolysis of native proteins - Trapping of a reaction intermediate

When limited proteolysis of the mouse major urinary proteins by trypsin was stopped by rapid denaturation of the proteinase, a covalent adduct of the two proteins was observed. The formation of this complex required active tr ypsin, was favored at low pH, and could be reversed by the addition of cova lent or non-covalent trypsin inhibitors. Electrospray mass spectrometry of the complex demonstrated that it was an acyl-enzyme complex, formed after a n unusual exopeptidase attack on the C-terminal-Arg-Glu-OH sequence by tryp sin. The complex could sequester over 50% of the trypsin in a digestion mix ture, and as anticipated, the protein was an effective trypsin inhibitor.