Glycation of Proteinous Inhibitors: Loss in Trypsin Inhibitory Activity by the Blocking of Arginine and Lysine Residues at Their Reactive Sites

Abstract

Lysine-type trypsin inhibitors [Japanese quail ovomucoid (QOM) and Bowman−Birk inhibitor (BBI)], and arginine-type trypsin inhibitors [chicken ovomucoid (COM) and Kunitz soybean trypsin inhibitor (KSTI)] were modified with glucose by amino−carbonyl reaction at 50 °C and 65% relative humidity for 15 days. Free amino groups of inhibitors were blocked rapidly and decreased to <20% within 5 days. On the other hand, their free guanidino groups began to decrease after a time lag for a few days, and 6−40% of the free guanidino groups remained after the 15-day incubation. Both lysine- and arginine-type trypsin inhibitors were inactivated by the reaction. A linear relationship between the loss in inhibitory activities for the lysine-type inhibitors but not for the arginine-type inhibitors and the decrease of their free amino groups was observed. The soybean trypsin inhibitors were almost completely inactivated by the 15-day incubation with glucose without severe structural or chemical damage. Denaturation of COM and KSTI incubated with glucose was scarcely observed with immunoassay, though protein polymerization of COM−, QOM−, and BBI−glucose complexes was detected by SDS−PAGE. Keywords: Chicken ovomucoid; Japanese quail ovomucoid; Kunitz soybean trypsin inhibitor; Bowman−Birk inhibitor; amino−carbonyl reaction; Maillard reaction; protein modification; inactivation; trypsin inhibitory activity