Mechanism of dye binding in the protein assay using eosin dyes.
Eosin B and eosin Y have been used to estimate micro- and submicrogram quantities of proteins respectively as shown in our previous reports. In the present study we describe the mechanism of eosin binding to proteins. At pH lower than 3.0 the absorbance of unbound dye is greatly reduced. After the dye binds to protein, the absorption maximum of the dye changes from 514 to 530 +/- 5 nm. The absorbance and bathochromatic shift in absorption maximum of the protein-dye complex are proportional to the concentration of protein. The pH of the assay solution does not change due to protein. Arginine, histidine, and lysine (at both acidic and neutral pH) and tryptophan (at acidic pH) residues of a protein bind electrostatically to carboxylic and phenolic groups of the dye to produce a stable water-soluble protein-dye complex. The binding constants of eosin B with poly-L-arginine, poly-L-histidine, poly-L-lysine, and poly-L-tryptophan at pH 1.96 are 0.37, 0.32, 0.33 and 0.33 nmol/nmol of amino acid, respectively. The binding constants of eosin B and eosin Y with bovine serum albumin (BSA) at pH 1.96 are essentially the same, i.e., 0.82 nmol/nmol of reactive amino acid of BSA. The binding constant varies with solution pH so that a wide range of protein concentrations can be estimated. The reason for the higher absorbance of protein-eosin Y complex compared to that of protein-eosin B complex is discussed.
Waheed
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AA
Department of Protein Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo, 173-0015, Japan. awaheed@tmig.or.jp
Rao
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KS
Gupta
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PD
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004355:Drug Stability; D010697:Eosine I Bluish; D004801:Eosine Yellowish-(YS) / Q000737:chemistry / Q000378:metabolism*; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis
D005452:Fluoresceins; D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine; D010697:Eosine I Bluish; D004801:Eosine Yellowish-(YS)