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CHARACTERIZATION OF SKELETAL MUSCLE ACTIN LABELED WITH THE TRIPLET PROBE ERYTHROSIN‐5‐IODOACETAMIDE
Author(s) -
Ludescher Richard D.,
Liu Zane
Publication year - 1993
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1993.tb04984.x
Subject(s) - iodoacetamide , skeletal muscle , actin , chemistry , biophysics , photochemistry , biochemistry , biology , anatomy , cysteine , enzyme
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 μs while in F‐actin the decay was approximately monoexponential with a lifetime of 278 μs. 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°C and the anisotropy increased to ≈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.