Effects of temperature and pH on prothrombin fragment 1 conformation as determined by nuclear magnetic resonance.

Abstract

The effects of temperature and pH on the solution conformation of native prothrombin fragment 1 were examined with 1H NMR spectroscopy. A calcium-dependent quenching of the intrinsic protein fluorescence was used to monitor calcium binding to fragment 1 as an indicator of functional protein. The native fragment 1 NMR spectrum contained several features indicative of a folded protein: (a) nonequivalent histidyl C-2 resonances at 7.9 and 8.1 ppm, (b) two resonances of nearly equal intensity at 7.26 and 7.32 ppm, and (c) a resonance at -1.04 ppm. Temperature studies showed that thermal unfolding of fragment 1 (even at 80 degrees C) was reversible; however, there was an irreversible inactivation step which occurred subsequent to the unfolding. The basis for this inactivation appeared to include disulfide exchange reactions. On the basis of NMR spectra, fragment 1 retained its conformation from pH 7.0 to pH 11.5. From pH 7.0 to pH 5.0, the protein showed a reversible conformational change, and below pH 5, the protein self-associated. The pH dependence of the chemical shift of the tyrosyl resonances indicated a pKa of approximately 10 for the tyrosyl residues. These data suggest that the tyrosyl residues are accessible to solvent in the native protein.