Interaction of brilliant red X-3B with bovine serum albumin and application to protein assay.
The interaction of brilliant red X-3B (BRX) with bovine serum albumin (BSA) in three pH media has been characterized by the spectral correction technique. The binding number maximum of BRX was determined to be 102 at pH 2.03, 82 at pH 3.25 and 38 at pH 4.35 and the binding mechanism was analyzed in detail. The effects of ionic strength from 0 to 1 mol L(-1) and temperature from 20 to 70 degrees C on the binding were investigated. The results showed that the interaction of BRX with BSA responded to the Langmuir adsorption isothermal model and the binding constant was determined. From the correlation between the binding number and the number of basic amino acid residues, the ion-pair attraction induced the union of non-covalent bonds including H-bond, van der Waals force and hydrophobic bond and the binding model was illustrated. The binding of BRX to BSA has resulted in change of the BSA conformation confirmed by means of circular dichroism. Using this interaction at pH 2.03, a sensitive method named the absorbance ratio difference spectrometry was established and applied to the protein assay and the limit of detection of protein was only 6 microg L(-1). Two samples were determined and the results were in agreement with those obtained by the classical coomassie brilliant blue colorimetry.
Chen
Fang-Fang
FF
State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China.
Wang
Shi-Long
SL
Liu
Xiang-Hu
XH
Xu
Ran
R
Gao
Hong-Wen
HW
D000818:Animals; D001391:Azo Compounds / Q000737:chemistry*; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D009282:Naphthalenesulfonates / Q000737:chemistry*; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods*; D013696:Temperature
D001391:Azo Compounds; D009282:Naphthalenesulfonates; D011506:Proteins; C520101:Reactive brilliant red X-3B; D012710:Serum Albumin, Bovine