1H-NMR studies of bovine platelet factor 4: histidine assignments and interactions with heparin

Abstract

1H-NMR spectroscopy has been used to assign and to characterize the two histidine C2H resonances of the heparin binding protein, bovine platelet factor 4. One histidine has a pKa value of 6.51 at 27 degrees C; the second histidine exhibits 2 pKa values of 5.52 and 5.66 at 27 degrees C. The two histidine resonances have been assigned by an analysis of their deuterium exchange kinetics. Both resonances are solvent accessible with half-times of exchange at pH 8.8 of 3.3 and 4.0 days. These two resonances have been assigned by digesting partially deuterated protein with Staphylococcus aureus V-8 proteinase, separating and purifying the resulting peptides, and determining their relative and residual hydrogen content by NMR. The results indicate that His-38 has the lower pKa value and the slower deuterium exchange rate, whereas His-50 has the higher pKa value and the faster deuterium exchange rate at pH 8.8. The 1H-NMR resonance of His-38 of bovine platelet factor 4 is preferentially perturbed by the introduction of heparin. This observation and the presence of His-38 within the belt of positively charged residues around the platelet factor 4 tetramer supports the model of the platelet factor 4-heparin complex in which the polysaccharide crosses over each of the 2 alpha-helices of the 2 dimers at right angles.