Demonstration of the Feasibility of Observing Nuclear Magnetic Resonance Signals of super(77)Se Covalently Attached to Proteins

• Published in: The Journal of biological chemistry Vol. 257; no. 3; pp. 1142 - 1144
• Main Authors: Odom, J D, Luthra, N P, Costello, R C, Dunlap, R B
• Format: Journal Article
• Language: English
• Published: 01.01.1982
• Subjects: 6,6'-diselenobis(3-nitrobenzoic acid); chemical modification; lysozyme; N.M.R; ribonuclease A; sulfhydryl groups
• ISSN: 0021-9258

Previous super(77)Se NMR relaxation time studies established the utility of super(77)Se NMR spectroscopy in studying low molecular weight (< 500) selenium-containing molecules. Since the spin rotation and chemical shift anisotrophy mechanisms contributed significantly to the super(77)Se spin-lattice relaxation in these compounds, it was questionable as to whether the latter mechanism would be efficient enough to enable super(77)Se resonances to be observed in a reasonable period in high molecular weight selenobiomolecules. Thus, to address this problem, disulfide bonds of ribonuclease-A and lysozyme were reductively cleaved under denaturing conditions, and the resulting 7-8 sulfhydryl groups were treated with a new sulfhydryl group reagent containing selenium, 6,6'-diselenobis(3-nitrobenzoic acid), to give proteins containing covalently attached selenium in the form of selenenyl sulfides. The observation of high resolution super(77)Se NMR spectra of these proteins under denaturing conditions was accomplished. Five to six super(77)Se NMR resonances, which fell in a chemical shift range of 14-15 ppm, were observed for each protein and are selenenyl sulfides derived from cysteine.