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

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet–vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC (CBZC/CLys < 9) and a combined quenching process at higher concentration of BZC (CBZC/CLys > 9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC 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 BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn2+ on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied.

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet–vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium.Figure optionsDownload as PowerPoint slideHighlights
► The intrinsic fluorescence of Lys was quenched by the addition of benzocaine.
► A combined quenching process at higher concentration of benzocaine occurred.
► Binding affinities were determined at pH 3.5, 7.4, and 8.4.
► The effect of benzocaine on the conformation of Lys was analyzed.