Spin labeling of protein sulfhydryl groups by spin trapping a sulfur radical: application to bovine serum albumin and myosin.
Reaction of sulfhydryl-containing compounds, RSH, with Ce4+ in the presence of the spin trap phenyl-N-t-butylnitrone results in the appearance of a nitroxide ESR spectrum, which is greatly diminished if the sulfhydryl group is blocked prior to reaction. The spectra have short lifetimes which can be increased two- to fivefold to half-lives of 5-60 min by prior flushing of the solutions with nitrogen. For small molecules, such as cysteine, N-acetylcysteine, glutathione, and 2-mercaptoethanol, the spectrum is that of a freely rotating nitroxide while for the proteins, bovine serum albumin and myosin, the spectrum is characteristic of a strongly immobilized nitroxide spin label rigidly attached to the protein. Since Ce4+ is reported to oxidize the sulfhydryl group via the thiyl radical, RS, the following reactions are proposed to account for the formation of the nitroxide: (formula; see text) These reactions permit the spin labeling of sulfhydryl proteins such that the nitroxide is much closer to the point of attachment than when using conventional spin-labeling methods.
D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D009132:Muscles; D009218:Myosins; D011487:Protein Conformation; D011817:Rabbits; D012710:Serum Albumin, Bovine; D013113:Spin Labels; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds
D013113:Spin Labels; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; D009218:Myosins