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Proceedings Paper

Multidimensional Fluorescence Detected Circular Dichroism Quenching Of Bilirubin Albumin Complexes
Author(s): Mark P Thomas; Gabor Patonay; Isiah M Warner
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Paper Abstract

The chiral and fluorescent complexes that are formed when bilirubin binds to three mammalian serum albumins [Human (HSA); Bovine, (BSA); and Rabbit, (RSA)] were studied using conventional fluorescence quenching, circular dichroism, fluorescence detected circular dichroism (FDCD), and FDCD quenching (FDCDQ). The fluorescence of the bilirubin complex with HSA at pH = 7.3 was quenched by the hydrophobic quencher 2, 2, 2-trichloroethanol yielding Stern-Volmer value of 2.89 M for traditional fluorescence quenching and 3.06 tel for FDCDQ. A Stern-Volmer value of 3.10 M was obtained using trichloroethanol as the quencher for the bilirubin-HSA complex at pH = 4.2. These results suggest that neither the addition of quencher to the system, nor the changes which occur upon acidification of the bilirubin-HSA complex change the microenvironment of the bound bilirubin fluorophore. The bilirubin complexes with BSA and RSA showed an increase in the fluroescence intensity upon the addition of trichloroethanol. Differences in the quenching of the mammalian serum albumin complexes by 2,2,2-trichloroethanol indicate a difference in the hydrophobicity of the microenvironment surrounding the bilirubin binding sites. Traditional fluorescence quenching measurements using triethylamine led to nonlinear Stern-Volmer plots for the bilirubin complexes with BSA and HSA. In addition, FDCDQ experiments detected large conformational changes in the bilirubin complexes with HSA and BSA when triethylamine was added. The fluorescence quenching and FDCDQ results with triethylamine may be interpreted to confirm previous bilirubin albumin studies which suggested that the bilirubin bound to one site on albumin produces a large Cotton effect with a small contribution to the fluorescence while the bilirubin bound to the second site on albumin produces intense fluorescence with a smaller contribution to the optical activity of the complex.

Paper Details

Date Published: 27 April 1988
PDF: 7 pages
Proc. SPIE 0910, Fluorescence Detection II, (27 April 1988); doi: 10.1117/12.945439
Show Author Affiliations
Mark P Thomas, Emory University (United States)
Gabor Patonay, Emory University (United States)
Isiah M Warner, Emory University (United States)

Published in SPIE Proceedings Vol. 0910:
Fluorescence Detection II
E. Roland Menzel, Editor(s)

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