13C-n.m.r. of the cyanylated beta-lactoglobulins: evidence that Cys-121 provides the thiol group of beta-lactoglobulins A and B.
The thiol groups of beta-lactoglobulins A and B have been cyanylated using [13C]KCN. The samples of [cyanato-13C]-cyanylated-beta-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-beta-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-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) Biochemistry 11, 4539-4547] alkylated beta-lactoglobulins with iodoacetamide. Therefore the one mol of thiol/mol of monomer in the native beta-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.
Phelan
P
P
Department of Biochemistry, University College Dublin, Ireland.
Malthouse
J P
JP
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011108:Polymers; D011190:Potassium Cyanide / Q000737:chemistry; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013861:Thiocyanates / Q000737:chemistry*
D004220:Disulfides; D007782:Lactoglobulins; D011108:Polymers; D013438:Sulfhydryl Compounds; D013861:Thiocyanates; D003545:Cysteine; D011190:Potassium Cyanide