Premium
Resolution of the Absorbance and CD Spectra and Formation Constants of the Complexes between Human Serum Albumin and Methyl Orange
Author(s) -
Ambrosetti R.,
Bianchini R.,
Fisichella S.,
Fichera M.,
Zandomeneghi M.
Publication year - 1996
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.19960020206
Subject(s) - absorbance , chromophore , chemistry , human serum albumin , circular dichroism , methyl orange , analytical chemistry (journal) , molecule , crystallography , photochemistry , chromatography , organic chemistry , catalysis , photocatalysis
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 (C 1 ) and 1:2 (C 2 )), their association constants ( K 1, 1 = 2.32 (0.18) × 10 5 M −1 and K 1, 2 = 1.12 (0.15) × 10 11 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 C 2 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 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 general validity of this multitechnique, multiwavelength approach in the investigation of protein‐ligand complexes is discussed.