STUDIES ON INTERACTIONS BETWEEN METMYOGLOBIN AND HEPARIN

The complex formation between metmyoglobin and heparin was investigate d by absorbance and fluorescence spectroscopy as well as differential scanning microcalorimetry. In acidic pH region, three distinct complex es detected by absorbance measurements are formed depending on pH and time of equilibration. The kinetics of the conformational transition o f metmyoglobin-heparin complex equilibrated at neutral pH observed aft er pH change to acidic region comprises two steps. During the first st ep, characterized by rapid changes of the absorption spectra (approxim ately 5 minutes) as well as fluorescence intensities, reversible trans ition with pit = 6.5 +/- 0.1 occurs and the first type: of the complex forms. Below pH 0.2 the transition with pit = 5.7+/-0.1 is observed a nd the second type of the complex is formed. During the second slow st ep, the third type of the complex formed after 30 minutes of equilibra tion is characterized by a spectrum corresponding to low-spin form wit hout protein axial ligand bound. At neutral pH and 25 degrees C, the i nteraction between metMb and heparin only slightly alters absorption a nd fluorescence spectra. On the other hand, the formation of metMb-hep arin complex is established from the decrease of the transition temper ature 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 is oelectric point resulting in a considerable increase in the reversibil ity of thermal denaturation.