Abstract
The drug-binding site subdomain IIA of human serum albumin (HSA) was characterized by absorption and fluorescence spectroscopy using 7-hydroxyquinoline (7-HQ) as a local reporter. The spectra of 7-HQ in solution indicate that a ztitterionic tautomer is stabilized by water in the ground state and produces a unique absorption peak at 400 nm and a fluorescence peak at 510 nm. By examining the spectral change in binary mixtures of water and 1,4-dioxane, three water molecules were estimated to stabilize this tautomer through direct interactions with the polar regions of the molecule. When 7-HQ is mixed with HSA, a reduction in the absorbance of the zwitterionic tautomer was observed which indicates a less polar environment around the molecule. The 7-HQ molecule is found to specifically bind in subdomain IIA of HSA and causes a reduction in the fluorescence intensity of the Trp-214 residue which is located in the same binding site. The reduction in the fluorescence of Trp-214 is due to energy transfer from the Trp-214 residue to the 7- HQ probe. The distance between Trp-214 and the probe was calculated using Förster theory for energy transfer to be 1.95 nm. This distance and the calculated quenching rate constant using a Stern-Valmer plot (kq = 3.04 x 1012 M-1s-1) both point to a static quenching mechanism. The binding constant and the number of binding sites of the complex were also estimated and the calculations show that the 7-HQ probe binds only in subdomain IIA. The change in the fluorescence intensity of HSA in the presence of the probe indicates that the 7-HQ molecule selectively interacts with the Trp-214 residue which results in partial unmasking of the fluorescence due to the Tyr-263 residue (located in the same site). A much stronger fluorescence from Tyr-263 is observed when HSA is chemically unfolded by 6.0 M GdnHCl. 7- HQ is found to still bind in subdomain IIA in the unfolded state of HSA and causes a reduction in the fluorescence intensities of both Trp-214 and Tyr-263. The present results propose 7-HQ as a useful photophysical probe in studying binding sites in proteins and exploring their hydrophobic environment.
Original language | English |
---|---|
Title of host publication | Progress in Biomedical Optics and Imaging - Proceedings of SPIE |
Volume | 7576 |
DOIs | |
Publication status | Published - 2010 |
Event | Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications II - San Francisco, CA, United States Duration: Jan 25 2010 → Jan 27 2010 |
Other
Other | Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications II |
---|---|
Country | United States |
City | San Francisco, CA |
Period | 1/25/10 → 1/27/10 |
Keywords
- 7-Hydroxyquinoline
- Biological probes
- Chemical unfolding
- Drug binding sites
- Fluorescence
- Human serum albumin
- Resonance energy transfer
- Tautomerization
ASJC Scopus subject areas
- Biomaterials
- Electronic, Optical and Magnetic Materials
- Radiology Nuclear Medicine and imaging
- Atomic and Molecular Physics, and Optics