Characterization of skeletal muscle actin labeled with the triplet probe erythrosin-5-iodoacetamide.
We have labeled rabbit skeletal muscle actin with the triplet probe erythrosin-5-iodoacetamide and characterized the labeled protein. Labeling decreased the critical concentration and lowered the intrinsic viscosity of F-actin filaments; labeled filaments were motile in an in vitro motility assay but were less effective than unlabeled F-actin in activating myosin S1 ATPase activity. In unpolymerized globular actin (G-actin), both the prompt and delayed luminescence were red-shifted from the spectra of the free dye in solution and the fluorescence anisotropy of the label was high (0.356); filament formation red shifted all excitation and emission spectra and increased the fluorescence anisotropy to 0.370. The erythrosin phosphorescence decay was at least biexponential in G-actin with an average lifetime of 99 microseconds while in F-actin the decay was approximately monoexponential with a lifetime of 278 microseconds. These results suggest that the erythrosin dye was bound at the interface between two actin monomers along the two-start helix. The steady-state phosphorescence anisotropy of F-actin was 0.087 at 20 degrees C and the anisotropy increased to approximately 0.16 in immobilized filaments. The phosphorescence anisotropy was also sensitive to binding 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.
Ludescher
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RD
Department of Food Science, Rutgers, State University, New Brunswick, NJ 08903-0231.
Liu
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Z
D000199:Actins / Q000737:chemistry*; D000818:Animals; D004355:Drug Stability; D004923:Erythrosine / Q000737:chemistry*; D005260:Female; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D007460:Iodoacetamide / Q000737:chemistry*; D049449:Luminescence; D008297:Male; D009132:Muscles / Q000737:chemistry; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D005456:Fluorescent Dyes; D004923:Erythrosine; D007460:Iodoacetamide