Covalent attachment of sulfhydryl-specific, electron spin resonance spin-labels to Fab' fragments of murine monoclonal antibodies that recognize human platelet membrane glycoproteins. Development of membrane protein specific spin probes.
A general method for the production of high-affinity, nitroxide-labeled, protein-specific spin probes is described in this paper. Fab' fragments are generated from protein-specific, murine monoclonal antibodies by pepsin digestion and mild reduction with cysteine. The free sulfhydryl group located in the carboxy-terminal region of these molecules and produced de novo by this manipulation is then alkylated by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), thereby generating spin-labeled Fab' fragments of these monoclonal antibodies. Two prototypic monoclonal antibodies were tested, each specific for a different integral membrane glycoprotein of human blood platelets. The results indicate that Fab' spin probes generated by this method retain the ability to bind to these glycoproteins within the membrane of intact platelets. These reagents thus represent probes that can be generally used to monitor integral membrane protein mobility on the surface of the intact cell.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D001792:Blood Platelets / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000302:isolation & purification; D008565:Membrane Proteins / Q000097:blood*; D051379:Mice; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000032:analysis
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003497:Cyclic N-Oxides; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008565:Membrane Proteins; D013113:Spin Labels; D013438:Sulfhydryl Compounds; C003959:TEMPO