Physicochemical studies on the binding of chemicals with proteins. 3. The binding of methyl orange with lens proteins

Abstract

The binding of methyl orange with bovine lens protein, α-crystallin and β-crystallin, was studied in various pH values by equilibrium dialysis method in 0.1 M phosphate buffer of pH 4.7∼8.9. At equilibrium, the relationship between the amount of bound methyl orange and the concentration of methyl orange followed the Langmuir-type equation for each protein. The maximum possible number of methyl orange bound to each protein remained constant independent of the pH values examined and the number with α-crystallin was 10-4 (mole methyl orange/g. α-crystallin) and that with β-crystallin was 10-5. Binding constant (k) with each protein decreased markedly with increasing pH. The binding affinity (Δμ) of methyl orange with each protein was calculated, variation of Δμ with pH values was examined, and the effect of protein charge at the binding was discussed. It was concluded that in the binding of methyl orange with these proteins, the electrostatic force was the most important factor and that the binding with α-crystallin was greatly affected by electrostatic force than that with β-crystallin.