Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label.
Electron paramagnetic resonance was used to characterize the first use of a thio-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.
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Department of Physics, American University of Beirut, New York, NY 10022-6297, USA.
James
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Bhardwaj
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Butterfield
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D000110:Acetylcholinesterase / Q000097:blood; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry; D001205:Ascorbic Acid; D002794:Choline; D003497:Cyclic N-Oxides; D003579:Cytochrome Reductases / Q000097:blood; D042966:Cytochrome-B(5) Reductase; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005033:Ethylmaleimide; D006801:Humans; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000097:blood* / Q000737:chemistry*; D008698:Mesylates; D009994:Osmolar Concentration; D013049:Spectrin / Q000737:chemistry; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000032:analysis*
D001457:Anion Exchange Protein 1, Erythrocyte; D003497:Cyclic N-Oxides; D007202:Indicators and Reagents; D008565:Membrane Proteins; D008698:Mesylates; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D013049:Spectrin; C034248:(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D000110:Acetylcholinesterase; D010450:Endopeptidases; D002794:Choline; D005033:Ethylmaleimide; D001205:Ascorbic Acid