Investigation on the pH-dependent binding of vitamin B12 and lysozyme by fluorescence and absorbance

The binding reaction between vitamin B12 (B12, cyanocobalamin) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet–vis (UV) absorbance, and three-dimensional fluorescence. The intrinsic fluorescence of Lys was strongly quenched by the addition of B12 in different pH buffer solutions (pH 3.4, 7.4, and 9.0) and the spectroscopic observations are mainly rationalized in terms of a static quenching process at lower concentration of B12 (CB12/CLys < 5) and a combined quenching process at higher concentration of B12 (CB12/CLys > 5). The structural characteristics of B12 and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.4, 7.4, and 9.0). The effect of B12 on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of B12 to Lys causes apparent change in the secondary or tertiary structures of Lys. Furthermore, the effect of Zn2+ on the binding constant of B12 with Lys under various pH conditions (pH 3.4, 7.4, and 9.0) was also studied.

► A combined quenching process at higher concentration of B12 occurred. ► Binding affinities were determined at pH 3.4, 7.4, and 9.0. ► The effect of B12 on the conformation of Lys was analyzed. ► A new fluorescence emission band emerged around 380 nm.