The binding of C.I. Acid Red 2 to human serum albumin: Determination of binding mechanism and binding site using fluorescence spectroscopy

The mechanism of interaction between C.I. Acid Red 2 and human serum albumin was studied using different spectroscopic methods. The binding constants for the formation of a complex between the dye and albumin were 2.557, 2.461 and 2.383 × 105 M−1 at 298, 304 and 310 K, respectively. The associated changes in enthalpy and entropy were −4.512 kJ mol−1 and 88.38 J mol−1 K−1, indicating that hydrophobic interactions as well as H-bonding were the dominant intermolecular forces stabilizing the complex. Site marker competitive experiments revealed that the binding of the dye to albumin occurred in subdomain IIA; the distance between dye and albumin was 3.91 nm according to fluorescence resonance energy transfer theory. Changes in the albumin secondary structure imparted by the dye were confirmed using synchronous fluorescence, electronic absorption, circular dichroism and three-dimensional fluorescence spectroscopy.