Luminescent/paramagnetic probes of order and orientation in biological assemblies: the transformation of luminescent probes into pi-radicals by photochemical reduction within the contractile apparatus.
Several unsubstituted xanthene dyes (eosin, erythrosin, and fluorescein) were irradiated by laser light at their absorption maximum in the presence of different reducing agents. Due to photochemical reduction the quinoidal structure of the xanthene ring is transformed into a semiquinone and a pi-radical is formed having a characteristic electron paramagnetic resonance (EPR) signal of an unpaired electron spin with proton hyperfine interactions. A strong EPR signal is observed from the dye in solution or when specifically attached to myosin following irradiation in the presence of dithiothreitol or cysteine. The spectroscopic methods of fluorescence polarization and EPR are useful in the study of ordered biological assemblies. These methods generate complementary information about the order of the system but a consistent quantitative interpretation of the related data is complicated because the signals arise from different donors. Our method allows us to detect both signals from the same donor. We applied our new technique to the study of skeletal muscle fibers. The fluorescent dye iodoacetamidofluorescein was covalently attached to the reactive thiol of the myosin molecule in muscle fibers. Fluorescence polarization and EPR spectroscopy were performed on the labeled fibers in rigor. Both signals indicate a highly ordered system characteristic of cross-bridges bound to actin.
Burghardt
T P
TP
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905.
Ajtai
K
K
D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D004801:Eosine Yellowish-(YS) / Q000737:chemistry; D004923:Erythrosine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008163:Luminescent Measurements; D015335:Molecular Probes / Q000737:chemistry*; D009119:Muscle Contraction; D009132:Muscles / Q000737:chemistry* / Q000166:cytology; D010084:Oxidation-Reduction; D010777:Photochemistry; D013113:Spin Labels; D014966:Xanthenes
D005456:Fluorescent Dyes; D015335:Molecular Probes; D013113:Spin Labels; D014966:Xanthenes; D004923:Erythrosine; D004801:Eosine Yellowish-(YS)