The binding of caffeine to human serum albumin (HSA) under physiological conditions has been stud-ied by the methods of fluorescence,UV-vis absorbance and circular dichroism (CD) spectroscopy. The mechanism of quenching of HSA fluorescence by caffeine was shown to involve a dynamic quenching procedure. The number of binding sites n and apparent binding constant Kb were measured by the fluorescence quenching method and the thermodynamic parameters △H,△G,△S were calculated. The results indicate that the binding is mainly enthalpy-driven,with van der Waals interactions and hydrogen bonding playing major roles in the reaction. The distance r between donor (HSA) and acceptor (caffeine) was obtained according to the Frster theory of non-radiative energy transfer. Synchronous fluorescence,CD and three-dimensional fluorescence spectroscopy showed that the microenvironment and conformation of HSA were altered during the reaction.
