13C-n.m.r. of the cyanylated β-lactoglobulins: Evidence that Cys-121 provides the thiol group of β-lactoglobulins A and B

Abstract

The thiol groups of β-lactoglobulins A and B have been cyanylated using [13C]KCN. The samples of [cyanato-13C]cyanylated-β-lactoglobulins A and B which we prepared had signals at 109.7 p.p.m and 114.4 p.p.m. We conclude that the thiocyanate carbon having a chemical shift of 109.7 p.p.m. is in an apolar environment similar to a cyclohexane solvent, whereas the thiocyanate carbon having a chemical shift of 114.4 p.p.m. is in a polar environment similar to water. The signals with chemical shifts of 109.7 p.p.m. are assigned to the thiocyanate carbons of the native [cyanato-13C]cyanylated-β- lactoglobulins A and B. We deduce that the signal at 114.4 p.p.m. is due to an irreversibly denatured/unfolded species produced by alkaline denaturation, which is caused by intramolecular thiol/disulphide exchange occurring during our cyanylation procedure. We propose that Cys-119 is cyanylated in the irreversibly denatured species and Cys-121 is cyanylated in the native[cyanato-13C] cyanylated-β-lactoglobulins A and B. We suggest that the same intramolecular thiol-disulphide exchange reactions occurred when McKenzie and co-workers alkylated β-lactoglobulins with iodoacetamide. Therefore the one mol of thiol/mol of monomer in the native β-lactoglobulins is due to the thiol of Cys-121 and is not due to an equimolar mixture of Cys-119 and Cys-121 as they suggested.