CHARACTERIZATION OF SKELETAL-MUSCLE ACTIN LABELED WITH THE TRIPLET PROBE ERYTHROSIN-5-IODOACETAMIDE

We have labeled rabbit skeletal muscle actin with the triplet probe er ythrosin-5-iodoacetamide and characterized the labeled protein. Labeli ng decreased the critical concentration and lowered the intrinsic visc osity of F-actin filaments; labeled filaments were motile in an in vit ro motility assay but were less effective than unlabeled F-actin in ac tivating myosin S1 ATPase activity. In unpolymerized globular actin (G -actin), both the prompt and delayed luminescence were red-shifted fro m the spectra of the free dye in solution and the fluorescence anisotr opy of the label was high (0.356); filament formation red shifted all excitation and emission spectra and increased the fluorescence anisotr opy to 0.370. The erythrosin phosphorescence decay was at least biexpo nential in G-actin with an average lifetime of 99 mu S while in F-acti n the decay was approximately monoexponential with a lifetime of 278 m u s. These results suggest that the erythrosin dye was bound at the in terface between two actin monomers along the two-start helix. The stea dy-state phosphorescence anisotropy of F-actin was 0.087 at 20 degrees C and the anisotropy increased to approximate to 0.16 in immobilized filaments. The phosphorescence anisotropy was also sensitive to bindin g the physiological ligands phalloidin, cytochalasin B and tropomyosin . This study lays a firm foundation for the use of this triplet probe to study the large-scale molecular dynamics of F-actin.