Luminescent/paramagnetic probes for detecting order in biological assemblies: transformation of luminescent probes into pi-radicals by photochemical reduction.
Luminescent/paramagnetic probes for detecting order in biological assemblies: transformation of luminescent probes into pi-radicals by photochemical reduction.
复制标题
用于检测生物组装中的顺序的发光/顺磁性探针：通过光化学还原将发光探针转化为π自由基。
DOI：
10.1021/bi00132a018
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Burghardt,TP
中科院分区：
文献类型：
--
作者：
Ajtai,K;Burghardt,TP
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905 Received September 12, 1991; Revised Manuscript Received February 10, 1992 abstract: The spectroscopic methods of fluorescence polarization and electron paramagnetic resonance (EPR) are used to study order and orientation of extrinsically labeled protein elements of ordered biological systems. These methods generatecomplementary 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. We introduce a new method that allows us to detect both signals from the same donor. 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 Tr-radical is formed having a characteristic 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 a protein following irradiation in the presence of dithiothreitol or cysteine. We applied this technique to the study of skeletal muscle fibers. The fluorescent dye (iodoacetamido) fluorescein 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 orderedsystem characteristic of cross-bridges bound to actin. Our use of the same signal donor for fluorescence and EPR studies of probe order is a promising new technique for the study of order in protein elements of biological assemblies. e techniques of luminescence polarization and electron paramagnetic resonance (EPR) spectroscopy employing ex-trinsic probes are useful in quantitating orientation and order tThis work was supported by the National Science Foundation (DMB 8819755), the National Institutes of Health (R01 AR 39288-01A2), the American Heart Association (Grant-in-Aid 900644), and the Mayo Foundation. TPB is an Established Investigator of the American Heart Association. in biological assemblies because of their highsensitivity to differences or changes in probe orientation (Griffith & Jost, 1976; Arata & Shimizu, 1981; Morales et al., 1982; Gergely & Seidel, 1983; Vanderkooi & Berger, 1989). In the highly ordered skeletal muscle fiber system, twospatially separated points on the surface of the myosin cross-bridge, the reactive thiols SHI and SH2, are routinely specifically modified by covalent probes of this type (Morales et al., 1982; Gergely & Seidel, 1983; Ajtai & Burghardt, 1989; Burghardt & Ajtai,
