C-13 NMR OF THE CYANYLATED BETA-LACTOGLOBULINS - EVIDENCE THAT CYS-121 PROVIDES THE THIOL-GROUP OF BETA-LACTOGLOBULIN-A AND BETA-LACTOGLOBULIN-B

The thiol groups of a-lactoglobulins A and B have been cyanylated usin g [C-13]KCN. The samples of [cyanato-C-13]cyanylated-beta-lactoglobuli ns 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 solv ent, 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 che mical shifts of 109.7 p.p.m. are assigned to the thiocyanate carbons o f the native [cyanato-C-13]cyanylated-beta-lactoglobulin A and B. We d educe that the signal at 114.4 p.p.m. is due to an irreversibly denatu red/unfolded species produced by alkaline denaturation, which is cause d by intramolecular thiol/disulphide exchange occurring during our cya nylation procedure. We propose that Cys-119 is cyanylated in the irrev ersibly denatured species and Cys-121 is cyanylated in the native [cya nato-C-13]cyanylated-beta-lactoglobulins A and B. We suggest that the same intramolecular thiol-disulphide exchange reactions occurred when McKenzie and co-workers [McKenzie, Ralston and Shaw (1972) Biochemistr y 11, 4539-4547] alkylated beta-lactoglobulins with iodoacetamide. The refore the one mol of thiol/mol of monomer in the native beta-lactoglo bulins is due to the thiol of Cys-121 and is not due to an equimolar m ixture of Cys-119 and Cys-121 as they suggested.