The stoichiometry of the reaction of the spin labeling of F-actin and the effect of orientation of spin-labeled F-actin filaments

Abstract

The electron spin resonance (ESR) spectra of actin from rabbit skeletal muscle spin labeled with nitroxide analogs of N-ethylmaleimide were studied. When N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide was added to F-actin there was a preferential reaction of 1 mole of label per 46,000 g of actin. The ESR spectrum of oriented films of spin labeled F-actin varied depending on the orientation with respect to the applied magnetic field, suggesting that the label had a preferred orientation on the actin monomers. The 2pπ orbital on the N-atom that holds the unpaired electron of the spin label was more nearly parallel than perpendicular to the helical axis of F-actin. It was confirmed that spin labeling did not inhibit the G-F transformation and that during polymerization of G-actin spin labels became more strongly immobilized. On reaction of maleimide spin labels having varying chain lengths with F-actin both strongly and weakly immobilized labels were observed and the proportion of weakly immobilized labels increased as the chain length was increased.