The central goal of this work remains a complete understanding of the gelation process of sickle hemoglobin, including homogeneous and heterogeneous nucleation, polymer alignment, and degelation, both as a paradigm for biological assembly and as a fundamental component of a debilitating disease. The overall strategy we have adopted is to experimentally separate the complex process of gelation into its constituents. Their separate study not only allows a more precise understanding of the particular process, but also is valuable for insights into similar processes which occur in other assembly systems. Nucleation rates will be extracted by decomposition of the bulk progress curves and by observation of stochastic fluctuations of the formation rate. By measurements on samples gelled in high phosphate buffers, the effects of non-ideality can be removed. Finally, the use of wavelength and angle dependence of the light scattering will further characterize the initial nucleation process. We will map the concentration and temperature dependence of domain formation, which is strongly dependent on the heterogeneous process. Monomer diffusion into the domain will be measured. Angular dependence of the scattering will be used to construct a structure factor for the domain. The temperature dependence of the alignment transition will be studied. The theory we have advanced makes specific predictions which can be tested. These include the prediction that alignment should parallel a free edge of surface. Since the melting process will be influenced by the structure of the domain through which ligands must diffuse, depolymerization studies will also provide insights into the internal structure of domains. By examining how partially melted domains regrow as a function of domain incubation we can gain complimentary information to that from depolymerization studies. Studies are to be primarily carried out on a unique instrument constructed at Drexel. Continuous laser photolysis of the CO derivative or rapid temperature jump of the deoxy derivative is used to induce gelation of HbS. The spatial and temporal growth of the resulting polymerization is monitored by light scattering (of the photolysis beam) birefringence intensity (transmitted between crossed polarizers), or absorption measurements. Any two of the three signals can be alternately collected by a Silicon Intensified Target Vidicon to provide the spatial resolution.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL028102-07A1
Application #
3339492
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1988-07-01
Project End
1991-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Drexel University
Department
Type
Schools of Arts and Sciences
DUNS #
061197161
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Cho, M R; Ferrone, F A (1990) Monomer diffusion into polymer domains in sickle hemoglobin. Biophys J 58:1067-73
Zhou, H X; Ferrone, F A (1990) Theoretical description of the spatial dependence of sickle hemoglobin polymerization. Biophys J 58:695-703
Ferrone, F A (1989) Kinetic models and the pathophysiology of sickle cell disease. Ann N Y Acad Sci 565:63-74
Basak, S; Ferrone, F A; Wang, J T (1988) Kinetics of domain formation by sickle hemoglobin polymers. Biophys J 54:829-43
Ferrone, F A; Basak, S; Martino, A J et al. (1987) Polymer domains, gelation models and sickle cell crises. Prog Clin Biol Res 240:47-58
Ferrone, F A; Hofrichter, J; Eaton, W A (1985) Kinetics of sickle hemoglobin polymerization. II. A double nucleation mechanism. J Mol Biol 183:611-31