Introduction Actin is one of the most abundant proteins, which can assemble into many different structures in cells [2–4]. The biological activity of actin is regulated by actin-binding proteins, which form a well-controlled regulatory network. During their interactions these bio- logical partners of actin affect its behaviour and conformational properties. In vitro model systems were often applied to elucidate the properties of actin fila- ments. Previous studies involved the characterisation of the effect of the polymerisation [5, 6], nucleotides [7, 8] and the interaction of actin with myosin [9–13]. Phalloidin, isolated from Amanita phalloides mushroom, and jasplakinolide from the marine sponge Jaspis johnstoni, are actin-binding toxins, and used ex- tensively for studying the conformational properties of actin filaments. These drugs competitively bind to fila- mentous actin with high affinity and causes the stabili- zation of the filaments [14–16]. Differential scanning calorimetry studies showed that phalloidin and jasplakinolide are also able to stabilize actin filaments at substochiometric concentration [17, 18]. It was in numerous cases that differential scan- ning calorimetry (DSC) is an effective method to study the thermal properties of actin [19–29]. In this work we used DSC to study the complexes of phalloidin and jasplakinolide with actin filaments (F-actin). Apart from the analyses of the heat absorp- tion vs. temperature curves we also took into account the kinetic nature of the irreversible denaturation pro- cesses. Using the model developed by Sanchez-Ruiz et al. [1] we performed the detailed analysis of the toxin-induced changes in the thermal stability of actin filaments. This method allowed us to determine the activation energies from the Arrhenius plots of the first-order rate constant of denaturation for both phalloidin and jasplakinolide stabilised actin fila- ments. The results were in agreement with previous observations that the stabilization of actin by these drugs is different. The differences appear not only in the extent of stabilization, but also in the kinetic prop- erties of the toxin modified processes. Materials and methods Materials KCl, MgCl 2 , CaCl 2 , EGTA, Tris and phalloidin were obtained from Sigma Chem Co. (St. Louis, MO, USA). Jasplakinolide was purchased from Molecular Probes (Eugene, OR, USA). ATP and 2-mercaptoethanol were obtained from Merk (Darmstadt, Germany). NaN 3 was purchased from Fluka (Switzerland). Protein preparation Aceton-dried powder from rabbit skeletal muscle was obtained as described previously [30]. Actin was pre- pared according to the method of Spudich and Watt [31] and stored in 2 mM Tris-HCl (pH 8.0), 0.2 mM ATP, 0.1 mM CaCl 2 , 0.5 mM MEA (buffer A). Before the measurements the actin monomer solution was clarified by ultracentrifugation at 100.000 g, 2 h, 4°C. The concentration of G-actin was determined spectro- 1388–6150/$20.00 Akadémiai Kiadó, Budapest, Hungary © 2005 Akadémiai Kiadó, Budapest Springer, Dordrecht, The Netherlands Journal of Thermal Analysis and Calorimetry, Vol. 82 (2005) 275–279 THE EFFECT OF TOXINS ON THE THERMAL STABILITY OF ACTIN FILAMENTS BY DIFFERENTIAL SCANNING CALORIMETRY Beáta Bugyi 1 , G. Papp 1 , Sz. Halasi 1 and B. Visegrády 2* 1 Department of Biophysics, Faculty of Medicine, University of Pécs, Pécs, Szigeti str. 12, 7624, Hungary 2 Research Group for Fluorescence Spectroscopy, Office for Academy Research Groups Attached to Universities and Other Institutions at the Department of Biophysics, Faculty of Medicine, University of Pécs, Pécs, Szigeti str. 12, 7624, Hungary In our present study we performed the detailed characterisation of jasplakinolide and phalloidin on the thermal stability of actin fila- ments. The heat absorption curves were analysed by using the model established by Sanchez-Ruiz et al. [1]. The analysis provided the activation energies attributed to the heat denaturation of actin filaments in the absence and in the presence of toxins. The results indicated that there are kinetic differences between the toxin-mediated stabilization of the Ca 2+ -and Mg 2+ -actin filaments. The effect of toxins appeared to be cation dependent. Keywords: actin, cation, differential scanning calorimetry, jasplakinolide, phalloidin, protein conformation and dynamics * Author for correspondence: balazs.visegrady@aok.pte.hu