Journal of Thermal Analysis and Calorimetry, Vol. 61 (2000) 597–605 EFFECT OF OXYGEN FREE RADICALS ON MYOSIN IN MUSCLE FIBRES A DSC and EPR study D. Lõrinczy 1* , B. Gaszner 2 , F. Könczöl 3 and J. Belágyi 2 1 Biophysical Department, Faculty of Medicine, University of Pécs, Szigeti u. 12, Pécs 2 Central Research Laboratory, Faculty of Medicine, University of Pécs, Szigeti u. 12, Pécs 3 Institute of Forensic Medicine, Faculty of Medicine, University of Pécs, Szigeti u. 12, H-7624 Pécs, Hungary Abstract Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation transfer EPR) were used to study the motional dynamics and seg- mental flexibility of myosin in muscle fibres in the presence of free radical generating system. Muscle fibre bundles isolated from psoas muscle of rabbit were spin-labelled with maleimide- and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor do- main. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe mole- cules decreased with time following a single exponential curve. MgADP and MgATP plus ortho- vanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. The analysis of the EPR spectra of spin-labelled and oriented fibres showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the expense of the disordered population. This suggests a transition of myosin heads from weak- binding state into strong-binding state. DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves. Keywords: DSC-measurement, oxygen free radicals, skeletal myosin, spin-labelling Introduction Current models of muscle contraction assume that force is generated by actin-myosin interaction coupled to the ATPase cycle. In the absence of nucleotides the head re- gion of myosin (the catalytic domain) is rigidly attached to actin and forms a com- 1418–2874/2000/ $ 5.00 © 2000 Akadémiai Kiadó, Budapest Akadémiai Kiadó, Budapest Kluwer Academic Publishers, Dordrecht * Author for correspondence: Phone/fax: 36-72-314-017, e-mail: microcal@apacs.pote.hu