Acta Biochimica et Biophysica 10. (1975)
1975 / 3. szám - Belágyi, J.: Orientation Dependence in the Epr Spectra of Spin Labels in Glycerinated Muscle Fibres
234 Belágyi: Orientation Dependence of Spin Labels in Muscle Fibres Materials and methods The preparation followed was essentially that of Huxley (1963). The fibre bundles prepared from psoas muscle of the rabbit were glycerinated for three weeks or longer in refrigerator at — 16°C. After washing the fibre bundles in 0.1 M KC1, 0.001 M MgCI2 and 0.07 M phosphate buffer (pH 7.0) at room temperature to remove the glycerol, the preparations were kept in the same solution containing the spin label. The reaction of spin label with glycerinated muscle fibres was carried out on ice. The following spin labels were used: (i) 3-maleimido-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl as label I, (ii) 3-(maleimidomethyl)-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl as label II, (iii) 3-(3-maleimidopropyl)carbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl as label III, (iv) 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl as label IV, (v) 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinoxyl as label V. All the spin labels were purchased from Synvar Associates. The glycerinated muscle fibres have reacted with 0.3—0.5 or 1.5 —1.6 M of spin label per 10° g of protein for 60 or 180 min in the case of maleimide spin labels or for 24 hours with iodoacetamide spin label. The unreacted label was removed by washing the fibre bundles in a large amount of salt solution for 16 hours at 4°C. Thermal dénaturation was carried out in thermostat at 50+1 °C for 20 min. The shortening of the muscle fibre bundles was elicited in a medium containing 4 mM ATP and 4 mM MgCl2. All chemicals used in the experiments were analytical grade and obtained from Reanal (Budapest). The electron paramagnetic resonance spectra were taken at room temperature using a Zeiss Model ER 9 spectrometer operating at 9 kMc/s. The temperature within the cell was 23°C. The orientation of the glycerinated muscle fibres with respect to the applied field was obtained by appropriate alignment of short segments of the fibres in a flat cell. The epr spectra were characterized by the ratio of the two down-field peaks [denoted by I+1/(A)/I + ,(B)] where A means the spectral component due to a strong immobilization of the attached label, while В the spectral component due to a label with greater degree of rotational freedom, the weakly immobilized spin labels and the hyperfine coupling parameter 2AZZ measured between the outermost hyperfine extrema. It should be noted that the I + 1(B) peak is strongly influenced by the orientational anisotropy of the strongly immobilized spin labels; therefore, the ratio of the two down-field peaks can reflect the change in the degree of orientation of spin labels. This can cause some difficulty in the interpretation of epr spectra, especially after MgATP-induced modification. Spectra were recordced twice and were found to be very well reproducible. Field calibration was done by using peroxylamine disulphonate ion radicals in low concentration. Acta Biochimica et Biophysica Academiae Scientiarum Hungaricae 10, 1975