<b>Structure of the methyl orange-binding site on human serum albumin and its color-change </b><b>mechanism </b>

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 MO-HSA complex was measured at 560 nm in solutions of varying pH (pH 2.4-6.6). The obtained pHdependent experimental data were consistent with the data calculated using the HendersonHasselbalch 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.Metachromasy is the characteristic color change of an acidic or basic dye used for staining biological tissues or cells (1, 7). The mechanism underlying metachromasy is considered to involve hydrophobic and hydrophilic interactions between basic (cationic) dyes, such as toluidine blue and methylene blue, and polyanions, such as nucleic acids and mucopolysaccharides containing phosphate and sulfate groups, respectively, and the resulting color change occurs through dye aggregation. In the case of acidic (anionic) dyes, the dyes interact with the amino groups of proteins (14, 16). Many pH indicators such as bromocresol green (BCG), bromocresol purple (BCP), and phenolphthalein exhibit metachromasy, and when albumin, which can adsorb various organic compounds (2), is added to a pH indicator in a buffered solution, the resulting color change in the indicator is equivalent to that which occurs in a solution of high or low pH. In clinical environments, dye-binding methods involving the use of BCG and BCP that are employed for quantifying human serum albumin (HSA) are based on this property of albumin (3). Because the color change occurs in a buffered solution whose pH is constant, a proton-exchange reaction occurs between the molecules. In the case of pH indicators, the amount of pH-dependent color change is measured using a photometric method after the binding of the indicator to HSA, and the amount is calculated using the Henderson-Hasselbalch equation. In our previous work, a comparison between experimental and calculated pH-dependent data indicated that color change

Formulation and Characterization of Bovine Serum Albumin-Loaded Niosome

“…6). Very recently, Ito and Yamamoto have investigated absorbance of the MO-human serum albumin (HSA) complex at 560 nm in the solutions with various pH values (pH 2.4-6.6) (25). Also, they have used surface plasmon resonance (SPR) analysis for confirming MO-HSA binding and reported that one or two MO molecules bind to a HSA molecule at pH 3.6-7.2 (25).…”

“…In this method, the protein and dye molecules, MO, form a complex. Color change of the MO-protein complex is caused by a proton-exchange reaction, and its intensity depends on the pH of the reaction solution (25). Easy handling is another advantage of this method.…”

Selective detection of human serum albumin by near infrared emissive fluorophores: Insights into structure-property relationship

“…Therefore, it is likely that a basic residue deprotonates the phenolic -OH, and hence a large red shift (~ 35 nm) of absorption maxima was observed when HSA was added into the solution of 2 at physiological buffer (figure 4). 49…”

Screening of Detergents for Stabilization of Functional Membrane Proteins

“…It may also interact with hydrophobic patches on proteins or other macromolecules containing such patches. For example, interactions with human serum albumin have been reported (Ito & Yamamoto, 2015). To our knowledge, methyl orange alone does not interact with DNA; however, when used in Lenoir et al…”