Influence of NaCl and sorbitol on the stability of conformations of cytochrome c.

Influence of ionic (NaCl) and non-ionic (sorbitol) additives on structural transitions of cytochrome c was investigated by circular dichroism, optical and EPR spectroscopy. Transformations of cytochrome c, induced by the acidification of solution and temperature perturbation, were monitored in the heme pocket together with changes in the secondary structure. NaCl and sorbitol exhibited antagonistic effect on the acid-induced transition of the protein. Sorbitol enhanced the stability of native conformation while NaCl destabilized this state. The midpoints of acid-induced transitions in the axial coordination of heme as well as in the secondary structure occurred nearly at the same pH values. However, temperature-induced transitions in the unfolding of the secondary structure were almost coincidental with the cleavage of Met80-Fe bond only in the sorbitol solutions. In the salt solution the Met80-Fe bond was markedly more labile than the secondary structure.

Studies on interactions between metmyoglobin and heparin.

The complex formation between metmyoglobin and heparin was investigated by absorbance and fluorescence spectroscopy as well as differential scanning microcalorimetry. In acidic pH region, three distinct complexes detected by absorbance measurements are formed depending on pH and time of equilibration. The kinetics of the conformational transition of metmyoglobin-heparin complex equilibrated at neutral pH observed after pH change to acidic region comprises two steps. During the first step, characterized by rapid changes of the absorption spectra (approximately 5 minutes) as well as fluorescence intensities, reversible transition with pK = 6.5 +/- 0.1 occurs and the first type of the complex forms. Below pH 6.2 the transition with pK = 5.7 +/- 0.1 is observed and the second type of the complex is formed. During the second slow step, the third type of the complex formed after 30 minutes of equilibration is characterized by a spectrum corresponding to low-spin form without protein axial ligand bound. At neutral pH and 25 degrees C, the interaction between metMb and heparin only slightly alters absorption and fluorescence spectra. On the other hand, the formation of metMb-heparin complex is established from the decrease of the transition temperature from 80.4 +/- 0.5 degrees C to 74.7 +/- 0.5 degrees C. Moreover, the binding of heparin prevents the aggregation of the protein at isoelectric point resulting in a considerable increase in the reversibility of thermal denaturation.