1. Detail Modes of Binding Assessed by Bulk and Single Molecular Level Fluorescence, MD Simulation, and Its Temperature Dependence: Coumarin 152 with Human Serum Albumin Revisited

This study describes the effect of temperature on binding parameters as well as mode of binding between coumarin 152 (C152) and human serum albumin (HSA). Site marker competitive experiment, Förster resonance energy transfer (FRET), and molecular docking study show that C152 binds to the digitoxin binding site in domain III of HSA. The binding constant calculated at the single molecular level experiment matches well with the ensemble average measurement, which indicate that at even low dye concentration the binding reaction proceeds with equal probability. Further, FRET and molecular dynamic simulation confirm that the binding location of C152 is independent on temperature (278 K to 323 K). It has been revealed that the binding affinity of C152 to HSA was almost unaffected until 298 K; afterward, it decreases continuously on increasing temperature forming two distinct regions. Thermodynamic parameters for association indicate that strong electrostatic and hydrophobic interactions are operational at lower temperature region, whereas hydrogen bonding predominates at higher temperate region.