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

• Published in: Biochemical journal 1994-09, Vol.302 (2), p.511-516
• Main Authors: Phelan, P, Malthouse, J.P.G
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; beta-lactoglobulin; Biological and medical sciences; Cattle; Chromatography, Gel; composition; cysteine; Cysteine - chemistry; Disulfides - chemistry; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Holoproteins; Hydrogen-Ion Concentration; Lactoglobulins - chemistry; Lactoglobulins - metabolism; Magnetic Resonance Spectroscopy; milk; Models, Molecular; Other proteins; Polymers; Potassium Cyanide - chemistry; Protein Denaturation; Proteins; spectral analysis; Sulfhydryl Compounds - chemistry; sulfhydryl groups; Thiocyanates - chemistry
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3020511

The thiol groups of beta-lactoglobulins A and B have been cyanylated using [(13)C]KCN. The samples of [cyanato-(13)C]-cyany lated-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-(13)C]cyany lated-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-(13)C]cyany lated-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.