In this work, a new protein binding agent based on multifunctional calix[4]arene CP has been synthesized by a one‐pot four‐component reaction. Protein binding affinity of designed ligand (CP) was investigated in physiological buffer (pH = 7.4) by fluorescence quenching method and revealed considerable binding affinity between CP and β‐lactoglobulin (BLG). The three‐dimensional (3D) fluorescence measurements indicated that binding of CP to BLG did not influence on BLG conformation. Furthermore, the effect of various biologically important metal ions on the binding constant of CP with BLG was investigated.
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