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Kinetics of sickle hemoglobin polymerization: I. Studies using temperature-jump and laser photolysis techniques
Journal article   Peer reviewed

Kinetics of sickle hemoglobin polymerization: I. Studies using temperature-jump and laser photolysis techniques

Frank A Ferrone, James Hofrichter and William A Eaton
Journal of molecular biology, v 183(4), pp 591-610
1985
DOI: https://doi.org/10.1016/0022-2836(85)90174-3
PMID: 4020872
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Abstract

HbCO Hb HbO 2
Using a combination of laser photolysis and temperature-jump techniques, the kinetics of hemoglobin S polymerization have been studied over a wide range of delay times (10 −3 to 10 5 s), concentrations (0.2 to 0.4 g/cm 3) and temperatures (5 to 50 °C). A slow temperaturejump technique was used to induce polymerization in samples with delay times between 10 2 seconds and 10 5 seconds by heating a solution of completely deoxygenated hemoglobin S. For samples with shorter delay times, polymerization was induced by photodissociating the carbon monoxide complex in small volumes (10 −9 cm 3) using a microspectrophotometer equipped with a cw argon ion laser. The photolysis technique is described in some detail because of its importance in studying hemoglobin S polymerization at physiological concentrations and temperatures. In order to establish conditions for complete photodissociation with minimal laser heating, a series of control experiments on normal human hemoglobin was performed and theoretically modeled. The concentration dependence of the tenth time is found to decrease with increasing hemoglobin S concentration. In the range 0.2 to 0.3 g/cm 3, the tenth time varies as the 36th power of the hemoglobin S concentration, while in the range 0.3 to 0.4 g/cm 3 it decreases to 16th power. As the tenth times become shorter, the progress curves broaden, with the onset of polymerization becoming less abrupt. For tenth times greater than about 30 seconds, measurements with the laser photolysis technique on small volumes yield highly irreproducible tenth times, but superimposable progress curves, indicating stochastic behavior. The initial part of the progress curves from both temperature-jump and laser photolysis experiments is well fit with an equation for the concentration of polymerized monomer, Δ( t) = A[cosh ( Bt)−1], which results from integration of the linearized rate equations for the double nucleation mechanism described in the accompanying paper (Ferrone et al., 1985). The dependence of the parameters A and B on temperature and concentration is obtained from fitting over 300 progress curves. The rate B has a large concentration dependence, varying at 25 °C from about 10 −4 s −1 at 0.2 g/cm 3 to about 100s −1 at 0.4 g/cm 3. The variation in A, which parametrizes the shape of the progress curves, is also large, increasing from about 10 −7 m m to about 10 −1 m m over the same concentration range. In the accompanying paper we show that the dependence on concentration of B and the combined parameter B 2 A is well fit with physically reasonable kinetic and thermodynamic parameters of the double nucleation mechanism, and that the stochastic behavior is also correctly predicted by the mechanism.

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Web of Science research areas
Biochemistry & Molecular Biology
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