Combined fluorescence and electrochemical investigation on the binding interaction between organic acid and human serum albumin

Human serum albumin (HSA) is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants, Such binding plays a crucial role in determining the ADME (absorption, distribution, metabolism, and excretion) and bioavailability of the pollutants. The binding interaction between HSA and acetic acid (C2), octanoic acid (C8) and dodecanoic acid (C 12) has been investigated by the combination of site-specific fluorescent probe, tryptophan intrinsic fluorescence and tyrosine electrochemistry. For the study of the fatty acid interaction with the two drug-binding sites on HSA, two fluorescent probes, dansylamide and dansyl-L-proline were employed in the displacement measurements. Intrinsic fluorescence of tryptophan in HSA was monitored upon addition of the fatty acids into HSA. Electrocattalyzed response of the tyrosine residues in HSA by a redox mediator was used to investigate the binding interaction. Qualitatively, observations from these three approaches were very similar. HSA did not show any change in the fluorescence and electrochemical experiments after mixing with C2, suggesting there is no significant interaction with the short-chain fatty acid. For C8, the measured signal dropped in a single-exponential mode, indicating an independent and non-cooperative binding. The calculated association constant and binding ratio were 3.1 x 10(6) L/mol and I with drug binding Site I, 1.1 X 10(7) L/mol and I with Site II, and 7.0x 10(4) L/mol and 4 with the tryptophin site, respectively. The measurements with C12 displayed multiple phases of fluorescence change, suggesting cooperativity and allosteric effect of the C12 binding. These results correlate well with those obtained by the established methods, and validate the new approach as a viable tool to study the interactions of environmental pollutants with biological molecules.