Kinetics of sickle hemoglobin polymerization. II. A double nucleation mechanism.

A double nucleation mechanism for the polymerization of sickle hemoglobin is described. The mechanism accounts for all of the major kinetic observations: the appearance of a delay, the high concentration dependence of the delay time, and the stochastic behavior of slowly polymerizing samples in small volumes. The mechanism postulates that there are two pathways for polymer formation: polymerization is initiated by homogeneous nucleation in the solution phase, followed by nucleation of additional polymers on the surface of existing ones. This second pathway is called heterogeneous nucleation. Since the surface of polymers is continuously increasing with time, heterogeneous nucleation provides a mechanism for the extreme autocatalysis that is manifested as an apparent delay in the kinetic progress curves. In this mechanism, each spherulitic domain of polymers is considered to be initiated by a single homogeneous nucleation event. The mechanism explains the irreproducibility of the delay time for single domain formation as arising from stochastic fluctuations in the time at which the homogeneous nucleus for the first polymer is formed. Integration of the linearized rate equations that describe this model results in a simple kinetic form: A[cosh(Bt)-1] (Bishop & Ferrone, 1984). In the accompanying paper (Ferrone et al., 1985) it was shown that the initial 10 to 15% of progress curves, with delay times varying from a few milliseconds to over 10(5) seconds, is well fit by this equation. In this paper, we present an approximate statistical thermodynamic treatment of the equilibrium nucleation processes that shows how the nucleus sizes and nucleation equilibrium constants depend on monomer concentration. The equilibrium model results in expressions for B and B2A as a function of monomer concentration in terms of five adjustable parameters: the bimolecular addition rate of a monomer to the growing aggregate, the fraction of polymerized monomers that serve as heterogeneous nucleation sites, the free energy of intermolecular bonding within the polymer, and two parameters that describe the free energy change as a function of size for the bonding of the heterogeneous nucleus to a polymer surface. This model provides an excellent fit to the data for B and B2A as a function of concentration using physically reasonable parameters. The model also correctly predicts the time regime in which stochastic behavior is observed for polymerization in small volumes.