Human serum albumin–amlodipine binding studied by multi-spectroscopic, zeta-potential, and molecular modeling techniques

Abstract

The fluorogenic property of amlodipine (AML) was for the first time exploited at various pH conditions to analyze the interaction with human serum albumin (HSA) as a probe. The techniques employed included fluorescence spectroscopy, zeta-potential measurements, polydispersion index (PDI) determination, three-dimensional fluorescence spectroscopy, circular dichroism (CD), and molecular modeling. The fluorescence of HSA was remarkably quenched by AML, and the quenching mechanism was considered as static through formation of a complex in the pH range of 5.4–9.4. The association constants and the number of binding sites were calculated at varying pH which implies that there was two set of binding site for AML upon HSA molecule. The obtained results display that there were more binding affinity between AML with HSA at pH = 7.4 and 8.4, whereas the least affinity was observed at pH = 9.4. The HSA–AML binding distances were determined to be shorter than 8 nm at the different pH, suggesting that energy transfer from HSA to AML may occur and HSA–AML complex was formed. Zeta-potential and resonance light scattering spectra results showed that the interaction between AML with HSA demonstrated different behaviors at the various pH such that with an increasing pH, the CCIAC (critical induced aggregation concentration) was decreased. In addition, the PDI values illustrated that pH effects caused differences in heterogeneity. According to CD data, the secondary structure contents of HSA upon interaction with AML were found to depend on pH, and an HSA–AML complex was clearly seen. Furthermore, the molecular modeling study indicated that AML was linked differently depending on the pH; for example, at pH 7.4, the bond between AML and HSA existed in site IIB and IIIA, and Trp214 was at a distance of 1.8 and 3.05 nm relative to AML, which was in agreement with the experimental analysis.