IDENTIFICATION OF THE SITE OF GLYCATION OF HUMAN INSULIN

This study evaluates the nature of glycated human insulin formed follo wing exposure to hyperglycemic conditions in vitro. Glycated insulin w as purified by RP-HPLC and its molecular mass (5971.3 Da) determined b y plasma desorption mass spectrometry (MS). The difference in mass (16 3.7 Da) from nonglycated insulin (5807.6 Da) corresponds to a single r educed glucose (glucitol) residue. Following reduction of insulin disu lfide bridges, MS confirmed that the B-chain was glycated. Enzymatic d igestions with trypsin, endoproteinase Glu-C, and thermolysin, followe d by RP-HPLC and identification of fragments by MS, localized glycatio n to the B-chain (1-5) region. Electrospray tandem MS identified the s ite of glycation as the B-chain NH2-terminal Phe(1) residue. This was confirmed by automated Edman degradation with glycated human insulin. Copyright (C) 1996 Elsevier Science Inc.