Human Platelet Factor 4 Subunit Association/Dissociation Thermodynamics and Kinetics

Mu Jung Chen, Kevin H. Mayo

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24 Scopus citations

Abstract

Platelet factor 4 (PF4) monomers (7800 daltons) form dimers and tetramers in varying molar ratios under certain solution conditions [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469]. The presence of a simplified aromatic region (one Tyr and two His) and resolved monomer, dimer, and tetramer Y60 3,5 ring proton resonances makes study of PF4 aggregate association/dissociation thermodynamics and kinetics possible. PF4 protein subunit association/dissociation equilibrium thermodynamic parameters have been derived by 1H NMR (500 MHz) resonance line-fitting analysis of steady-state Y60 3,5 ring proton resonance monomer-dimer-tetramer populations as a function of temperature from 10 to 40 °C. Below 10 °C and above 40 °C, resonance broadening and overlap severely impaired analysis. Enthalpic and entropic contributions to dimer association Gibb’s free energy [-5.1 kcal/mol (30 °C)] are +2.5 ± 1 kcal/mol and +26 ± 7 eu, respectively, and for tetramer association Gibb’s free energy [-5.7 kcal/mol (30 °C)], they are -7.5 ± 1 kcal/mol and -7 ± 3 eu, respectively. These thermodynamic parameters are consistent with low dielectric medium electrostatic/hydrophobic interactions governing dimer formation and hydrogen bonding governing tetramer formation. Association/dissociation kinetic parameters, i.e., steady-state jump rates, have been derived from exchange-induced line-width increases and from 1H NMR (500 MHz) saturation-transfer and spin-lattice (T1) relaxation experiments. From dissociation jump rates and equilibrium constants, association rate constants were estimated. For dimer and tetramer equilibria at 30 °C, unimolecular dissociation rate constants are 35 ± 10 s-1 for dimer dissociation and 6 ± 2 s-1 for tetramer dissociation. The association rate constants at 30 °C are (13 ± 4) × 104 and (8 ± 2) × 104 M-1 s-1, respectively. Activation enthalpies and entropies have been derived from the temperature dependence of forward/reverse rate constants. Kinetically, tetramers are preferred over dimers due primarily to a slower dissociation rate, and thermodynamically, tetramer formation is enthalpy-driven, while dimerization is entropy-driven.

Original languageEnglish (US)
Pages (from-to)6402-6411
Number of pages10
JournalBiochemistry
Volume30
Issue number26
DOIs
StatePublished - Jul 1 1991

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