RESOLUTION OF THE ABSORBENCY 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 Oran ge (MO). The stoichiometries of the two HSA-MO complexes (1:1 (C-1) an d 1:2 (C-2)), their association constants (K-1,K-1 = 2.32 (0.18) x 10( 5) M(-1) and K-1,K-2 = 1.12 (0.15) x 10(11) M(-2)), and both absorbanc e and dichroic spectra have been determined by a computational approac h. Nearly 900 experimental points, consisting of absorbance and CD mea surements registered in the 340-550 nm interval and over a wide range of concentrations of protein and ligand, have been included in a uniqu e fitting procedure. The Scatchard plot indicates the existence of a u nique binding site which can accommodate up to two molecules of MO in a positive cooperative process. Calculation of the CD spectrum for the C-2 complex according to the DeVoe method reproduces the fitted dichr oic spectrum for the same complex. The shapes of the fitted absorbance and dichroic spectra, as well as the influence of concentrated NaCl o r ethylene glycol on the absorbances of both free MO and HSA-MO mixtur es are consistent with the presence of dominant electrostatic interact ions in C-1. The C-2 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 gen eral validity of this multitechnique, multiwavelength approach in the investigation of protein-ligand complexes is discussed.