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J Mol Biol. 1986 Jun 5;189(3):553-71.

Kinetics of sickle hemoglobin polymerization. III. Nucleation rates determined from stochastic fluctuations in polymerization progress curves.

Abstract

The polymerization kinetics of sickle cell hemoglobin are found to exhibit stochastic variations when observed in very small volumes (approximately 10(-10) cm3). The distribution of progress curves has been measured at several temperatures for a 4.50 mM-hemoglobin S sample using a laser-photolysis, light-scattering technique. The progress curves at a given temperature are superimposable when translated along the time axis, showing that the variability of the kinetic progress curves results primarily from fluctuations in the time at which polymerization is initiated. The shapes of the initial part of the progress curves are well-fitted using the functional form I(t) = Io + As exp (Bt), derived from a dual nucleation model. When the distribution of the measured tenth times is broad, the rate of homogeneous nucleation can be obtained by fitting the exponential tail of the distribution. As the distribution sharpen, the rate of homogeneous nucleation can be estimated by modelling the width of the distribution function using a simple Monte-Carlo simulation of the polymerization kinetics. Using the rates of homogeneous nucleation obtained from the distributions, the rates of heterogeneous nucleation and polymer growth can be obtained from the experimental parameters As and B. The resulting nucleation rates are roughly 1000 times greater than those obtained from an analysis of bulk kinetic data. The results provide strong support for the dual-nucleation mechanism and show that the distribution of progress curves provides a powerful independent method for measuring the rate of homogeneous nucleation and thereby obtaining values for the other principal rates of the mechanism.

PMID:
3783684
DOI:
10.1016/0022-2836(86)90324-4
[Indexed for MEDLINE]

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