Structural and Functional Characterization of Luminescent Silver−Protein Nanobioconjugates

In this contribution, we report synthesis of luminescent Ag nanoclusters (NCs, 1 nm average diameter) directly conjugated to an enzyme, bovine pancreatic α-chymotrypsin (CHT). The structural characterization of Ag−CHT nanobioconjugates was done using steady-state UV−vis absorption/photoluminescence spectroscopy and high-resolution transmission electron microscopy. By use of the spherical jellium model, the emission energy of CHT-conjugated Ag NCs was correlated with the number of atoms in the cluster and was found to be almost consistent with the experimental result. To establish the conformational changes of the enzyme before and after the conjugation of Ag NCs, circular dichroism studies were performed. The functional integrity of the enzyme conjugated to Ag NCs was investigated by monitoring the enzymatic activity of Ag−CHT conjugates by using UV−vis absorption spectroscopy and compared with the unbound enzyme under similar experimental conditions. To confirm the conjugation of Ag NCs to CHT, we carried out the Förster resonance energy transfer studies using a fluorescent probe, 4-nitrophenyl anthranilate, known to bind at the enzymatic active site of CHT, as the donor and Ag-NC-bound CHT as the acceptor. The attachment of Ag NCs to an enzyme with its structural and functional integrity may have an impact in the future research on nanobio interfaces.