Determination of arginine in the reactive site of proteinase inhibitors by selective and reversible derivatization of the arginine side chain.
Reversible and selective modification of the guanidino group by 1,2-cyclohexanedione or 2,3-butanedione in borate buffer was applied to identify arginine residues in the reactive sites of proteinase inhibitors. The ability of the method to distinguish between arginine and lysine residues was examined. Trypsin inhibitors with arginine at the reactive site were almost completely inactivated within 2 h, e.g. the low-molecular weight inhibitors from soybeans (Kunitz), peanuts and porcine seminal plasma. Inhibitors of the lysine-type were not inactivated by 1,2-cyclohexanedione/borate, e.g. the bovine trypsin/kallikrein inhibitor, the trypsin inhibitors from cow colostrum, porcine pancreas, or snail epidermis (isoinhibitor K from Helix pomatia), and the human alpha1-proteinase inhibitor (alpha1-antitrypsin). However, 2,3-butanedione/borate does not show this high specificity. The method was applied to four high-molecular weight proteinase inhibitors from the albumin gland of the snail (Helix pomatia). All four inhibitors were subject to inactivation by the reagent, indicating an arginine residue at the reactive site. Evidence is given by NMR, UV, and titration data that a complex is formed from 1,2-cyclohexanedione and borate. Due to this complex formation, the amount of free carbonyl component is drastically decreased. Since 1,2-cyclohexanedione without borate lacks the high specificity for arginine residues, the borate complex of the reagent is presumed to be responsible for the high specificity. The new procedure for identification of reactive sites is based on a reversible modification of the guanidium group of the arginine residues.
D001120:Arginine / Q000032:analysis*; D001665:Binding Sites; D001881:Borates / Q000494:pharmacology; D003512:Cyclohexanones; D003931:Diacetyl; D008239:Lysine / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000032:analysis* / Q000494:pharmacology
D001881:Borates; D003512:Cyclohexanones; D014361:Trypsin Inhibitors; D001120:Arginine; D014357:Trypsin; D003931:Diacetyl; D008239:Lysine