Resolution of the Absorbance and CD Spectra and Formation Constants of the Complexes between Human Serum Albumin and Methyl Orange

Difference absorbance and circular dichroism techniques show that two complexes are formed between human serum albumin (HSA) and Methyl Orange (MO). The stoichiometries of the two HSA‐MO complexes (1:1 (C1) and 1:2 (C2)), their association constants (K1, 1 = 2.32 (0.18) × 105 M−1 and K1, 2 = 1.12 (0.15) × 1011 M−2), and both absorbance and dichroic spectra have been determined by a computational approach. Nearly 900 experimental points, consisting of absorbance and CD measurements registered in the 340–550 nm interval and over a wide range of concentrations of protein and ligand, have been included in a unique fitting procedure. The Scatchard plot indicates the existence of a unique binding site which can accommodate up to two molecules of MO in a positive co‐operative process. Calculation of the CD spectrum for the C2 complex according to the DeVoe method reproduces the fitted dichroic spectrum for the same complex. The shapes of the fitted absorbance and dichroic spectra, as well as the influence of concentrated NaCl or ethylene glycol on the absorbances of both free MO and HSA‐MO mixtures are consistent with the presence of dominant electrostatic interactions in C1. The C2 complex can be envisaged as a unique chromophore, consisting of two MO units associated in a stacking process into the same binding site of HSA, leading to a well‐defined chirality. The general validity of this multitechnique, multiwavelength approach in the investigation of protein‐ligand complexes is discussed. How does albumin bind ligands? Two complexes between human serum albumin and Methyl Orange (1:1 and 1:2) have been characterized by fitting a large set of UV/Vis and CD data. The computed formation constants and the shapes of the resolved electronic spectra show that the 1:1 complex results from electrostatic interactions, while the 1:2 complex arises from stacking processes with strong positive cooperativity (see Figure).