Analysis of drug binding sites on human serum albumin using multidimensional fluorescence measurements

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Stern-Volmer fluorescence quenching, fluorescence lifetime measurements and fluorescence detected circular dichroism quenching (FDCDQ) are used to investigate the properties of the site I and site II binding areas on human serum albumin (HSA). The binding sites are probed using the site I specific fluorescent markers warfarin, dansylamide, and the site II markers salicyclic acid and naproxen. Two nonionic quenching probes, acrylamide and 2,2,2-trichloroethanol, are used to measure the relative degree of exposure and hydrophobicity of the binding sites. Quenching measurements confirm that the microenvironment of the site I binding area is more hydrophobic than site II. The fluorescence lifetimes for the site I probes increase by an order of magnitude upon binding to HSA. The change in lifetime upon binding and lack of dynamic quenching of the bound fluorophores indicates that the site I and site II binding areas are located in pockets on the protein which are formed as the probes bind. Lifetime measurements in the presence of both quenchers provide evidence that the site II probes bind to two regions of different microenvironment. One of these sites is quenched by acrylamide and does not contribute to the FDCDQ signal. The salicylic acid bound to the second and predominant binding area on HSA which produces the optical activity for the complex is not quenched by acrylamide. © 1988.

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Spectrochimica Acta Part B: Atomic Spectroscopy

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