Journal
of Molecular Biology
Volume
336, Issue 1 , 6 February 2004, Pages 43-59
Mechanisms of
Homogeneous Nucleation of Polymers of Sickle Cell Anemia Hemoglobin in Deoxy
State
Oleg Galkin and Peter G. Vekilov
Department of Chemical Engineering, University of Houston, Houston, TX
77204-4004, USA
Received 14 August 2003; revised 25 November 2003; accepted 2 December
2003; Edited by K. Nagai Available online 19 December 2003.
The primary pathogenic event of
sickle cell anemia is the polymerization of the mutant hemoglobin (Hb) S within
the red blood cells, occurring when HbS is in deoxy state in the venous
circulation. Polymerization is known to start with nucleation of individual
polymer fibers, followed by growth and branching via secondary
nucleation, yet the mechanisms of nucleation of the primary fibers have never
been subjected to dedicated tests. We implement a technique for direct
determination of rates and induction times of primary nucleation of HbS fibers,
based on detection of emerging HbS polymers using optical differential
interference contrast microscopy after laser photolysis of CO-HbS. We show
that: (i) nucleation throughout these determinations occurs homogeneously and
not on foreign substrates; (ii) individual nucleation events are independent of
each other; (iii) the nucleation rates are of the order of 106–108 cm−3 s−1;
(iv) nucleation induction times agree with an a priori prediction based
on Zeldovich's theory; (v) in the probed parameter space, the nucleus contains
11 or 12 molecules. The nucleation rate values are comparable to those leading
to erythrocyte sickling in vivo and suggest that the mechanisms deduced
from in vitro experiments might provide physiologically relevant
insights. While the statistics and dynamics of nucleation suggest mechanisms
akin to those for small-molecule and protein crystals, the nucleation rate values
are nine to ten orders of magnitude higher than those known for protein
crystals. These high values cannot be rationalized within the current
understanding of the nucleation processes.
Author Keywords: sickle
cell anemia; hemoglobin S polymerization; fiber nucleation; homogeneous
nucleation rate; nucleus size
Abbreviations: Hb,
hemoglobin; DIC, differential interference contrast