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Biophys. J. BioFAST: First Published July 29, 2005. doi:10.1529/biophysj.105.067785
© 2005 by the Biophysical Society.

A more recent version of this article appeared on October 1, 2005.
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PROTEINS

Heterogeneous Nucleation in Sickle Hemoglobin: Experimental Validation of a Structural Mechanism

Maria A. Rotter 1, Suzanna Kwong 2, Robin W. Briehl 3 and Frank A. Ferrone 1*

1 Drexel University
2 Albert Einstein Col. of Medicine
3 Albert Einstein Col. of Med.

* To whom correspondence should be addressed. E-mail: fferrone{at}drexel.edu.

Submitted on June 1, 2005
Revised on July 11, 2005
Accepted on 14 July 2005


  Abstract
Sickle hemoglobin polymerizes by two types of nucleation: homogeneous nucleation of aggregates in solution, and heterogeneous nucleation on preexisting polymers. It has been proposed that the same contact that is made in the interior of the polymer between the mutant site {beta}6 and its receptor pocket on an adjacent molecule is the primary contact site for the heterogeneous nucleus. We have constructed cross-linked hybrid molecules in which one {beta} subunit is from HbA with Glu at {beta}6, and the other is from HbS with a Val at {beta}6. We measured solubility (using sedimentation) and polymerization kinetics (using laser photolysis) on cross-linked hybrids, and cross-linked HbS as controls. We find about 4000 times less heterogeneous nucleation in the cross-linked AS molecules than in cross-linked HbS, in strong confirmation of the proposal. In addition, changes in stability of the nucleus support a further proposal that more than one ?6 contact is involved in the homogeneous nucleus.

Key Words: hemoglobin, kinetics, nucleation, polymerization, protein structure, sickle-cell disease






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Copyright © 2005 by the Biophysical Society.