| Autor: |
Frank A. Ferrone, Alexey Aprelev, Antonio Emanuele, Suzanna Kwong, Yihua Wang, Robin W. Briehl |
| Rok vydání: |
2010 |
| Předmět: |
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| Zdroj: |
Biophysical Journal. 98(3) |
| ISSN: |
0006-3495 |
| DOI: |
10.1016/j.bpj.2009.12.1377 |
| Popis: |
We have developed a novel method for measuring light scattering to observe critical fluctuations in hemoglobin (Hb) solutions. A small rectangular cell (0.2 x 4.0 x 30 mm) is filled with 24 μL of Hb solution. An optical fiber with outer diameter of 125 μm (62.5 μm core) is sealed into the cell in contact with the solution, and light scattering is measured at 90°. The flat faces of the cell permit measuring absorbance spectra to ensure sample integrity. The scattering source is a 785 nm laser diode that delivers 1.5 mW to the sample. Scattered light is detected by a Hamamatsu GaAs(Cs) PMT via a LWD microscope objective. Measured scattered light intensity agrees (±10%) with scattered intensity expected from Hb fluctuations. Sample temperature is controlled by a thermoelectric stage and raised in a series of user-controlled steps. Temperature may be conveniently returned to prior values to insure reversibility. Assuming that the divergence in scattering that is observed signifies a spinodal, all our measured experiments agree with published spinodals for deoxyHbS, and deoxyHbA. In addition we have obtained data for COHbA, and cross-linked deoxyHbA. Spinodal lines for COHbA and deoxyHbA are essentially indistinguishable, despite differences in quaternary structure. Spinodal lines for cross-linked deoxyHbA and regular deoxyHbA coincide at high c values, but differ significantly at lower ones. In conclusion, changes of quaternary structure cause alterations to spinodal lines when polymerization is possible. This kind of data can be used to explore free energy landscapes having features inaccessible to techniques based on equilibrium thermodynamic properties. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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