title: Structure of the methyl orange-binding site on human serum albumin and its color-change mechanism.
abstract: The goal in this study was to clarify the color-change mechanisms of methyl orange (MO) bound to human serum albumin (HSA) and the structure of the binding site. The absorbance of the MOHSA complex was measured at 560 nm in solutions of varying pH (pH 2.4-6.6). The obtained pH-dependent experimental data were consistent with the data calculated using the Henderson-Hasselbalch equation and pKa values (3.8, MO; 1.4, carboxyl group). The extent of the binding of MO to an HSA molecule was determined to be 1-4 by performing surface plasmon resonance analysis. Furthermore, the binding of MO to HSA was inhibited by warfarin. A fitting model of MO to HSA was created to evaluate these results based on PDB data (warfarin-HSA complex: 2BXD) and protein-structure analysis. The color-change mechanism of the MO-HSA complex appears to be as follows: the dissociated sulfo group of MO binds to Arg218/Lys444 sidechains through electrostatic interaction in the warfarin-binding site, and, subsequently, the color change occurs through a proton exchange between the diazenyl group and the γ-carboxyl group of Glu292. The color-changed MO is fixed in the warfarin-binding site. These results could support the development of a reliable dye-binding method and of a new method for staining diverse tissues that is based on a validated mechanism.
authors: Department of Physiological Chemistry, Osaka Medical College.
authors: Shigenori
authors: S
authors: Ito
authors: Daisuke
authors: D
authors: Yamamoto
mesh_terms: D001391:Azo Compounds / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions
chemical_list: D001391:Azo Compounds; D012709:Serum Albumin; D012996:Solutions; C100258:methyl orange