Multidimensional fluorescence detected circular dichroism quenching of bilirubin albumin complexes
© 1988 SPIE. All rights reserved. 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 a Stern-Volmer value of 2.89 M-1 for traditional fluorescence quenching and 3.06 M-1 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 f luroescence 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.
Publication Source (Journal or Book title)
Proceedings of SPIE - The International Society for Optical Engineering
Thomas, M., Patonay, G., & Warner, I. (1988). Multidimensional fluorescence detected circular dichroism quenching of bilirubin albumin complexes. Proceedings of SPIE - The International Society for Optical Engineering, 910, 54-60. https://doi.org/10.1117/12.945439