Human Platelet Factor 4 Monomer–Dimer–Tetramer Equilibria Investigated by 1HNMR Spectroscopy

As a function of protein concentration, proton NMR spectra of human platelet factor 4 (PF4) differ. Correlation with low-angle laser light scattering data has allowed identification of concentration-dependent NMR spectral changes to PF4 aggregation, with tetramers being the largest aggregates formed. Well-resolved aromatic ring proton NMR resonances were assigned to Tyr-60, His-I, and His-II in monomer, dimer, and tetramer states. Since Tyr-60 3,5 ring proton resonances are well resolved from state to state, estimation of fractional populations in each state was possible. By varying the PF4 concentration, changes in these populations when plotted according to the Hill equation show a bimolecular mechanism of aggregation which proceeds from monomers to tetramers through a dimer intermediate. Equilibrium constants for dimer association (K_D) and tetramer association (K_T) have been estimated as a function of pH and ionic strength. At pH 4, where K_D and K_T approach the same value, resonances associated with all three aggregate states are observed. Lowering the pH shifts the equilibrium to the monomer state, while raising the pH shifts the equilibrium to dimer and tetramer states. Analysis of the pH dependence of K_D and K_T suggests that electrostatic interactions, probably arising from Glu/Asp and Lys/Arg side chains, play a role in the binding process. Increasing the solvent ionic strength stabilizes the tetramer state especially at low pH, suggesting that intersubunit, repulsive electrostatic interactions probably between/among cationic side chains (Lys/Arg) attenuate the aggregation process. Information based primarily on histidine pK_a values and photo-CIDNP ¹H NMR data suggests that Tyr-60 and His-I, but not His-II, are significantly affected by the aggregation process.