Biophysical Study on the Interaction of Ropivacaine with Human Serum Albumin Using Spectroscopic Methods

In this study, the interaction of human serum albumin (HSA) with ropivacaine was investigated by UV-vis, fluorescence, synchronous fluorescence and circular dichroism (CD) spectroscopic methods. Experimental results confirmed the complex formation between HSA and ropivacaine molecules under physiological conditions. Ropivacaine quenched the intrinsic fluorescence spectrum of HSA by static quenching mechanism. The binding constant of this system was calculated as 5.49x104 M-1 at 298K. The stability of HSA-ropivacaine complex illustrated a decrease within increasing temperature. The number of binding sites was found to be 1. Thermodynamic parameter values were calculated by using van't Hoff equation. According to sign and magnitude of thermodynamic parameters (Delta H = -735 J mol(-1) and Delta S = 10.3 J mol(-1) K-1), hydrogen bonding and hydrophobic forces were found as the effective interaction forces between HSA and ropivacaine molecules. Synchronous fluorescence and circular dichroism spectroscopic methods proved the alteration of secondary structure of HSA in the presence of ropivacaine. Fluorescent resonant energy transfer study proved the existence of energy transfer with a distance of 2.03 nm. Binding site study revealed that ropivacaine locates in the site I of HSA.