Quantum Dots Meet Enzymes: Hydrophobicity of Surface Ligands and Size Do Matter.

Colloidal quantum dots (QDs) are a class of representative fluorescent nanomaterials with tunable, bright, and sharp fluorescent emission, with promising biomedical applications. However, their effects on biological systems are not fully elucidated. In this work, we investigated the interactions between QDs with different surface ligands and different particle sizes and α-chymotrypsin (ChT) from the thermodynamic and kinetic perspectives. Enzymatic activity experiments demonstrated that the catalytic activity of ChT was strongly inhibited by QDs coated with dihydrolipoic acid (DHLA-QDs) with noncompetitive inhibitions, whereas the QDs coated with glutathione (GSH-QDs) had weak effects. Furthermore, kinetics studies showed that different particle sizes of DHLA-QDs all had high suppressive effects on the catalytic activity of ChT. It was found that DHLA-QDs with larger particle sizes had stronger inhibition effects because more ChT molecules were bound onto the surface of QDs. This work highlights the importance of hydrophobic ligands and particle sizes of QDs, which should be considered as the primary influencing factors in the assessment of biosafety. Meanwhile, the results herein can also inspire the design of nano inhibitors.

Thermodynamics, Kinetics and Mechanisms of Noncompetitive Allosteric Inhibition of Chymotrypsin by Dihydrolipoic Acid-Coated Gold Nanoclusters.

Enzymes are an important class of biomacromolecules which catalyze many metabolic processes in living systems. Nanomaterials can be synthesized with tailored sizes as well as desired surface modifications, thus acting as promising enzyme regulators. Fluorescent gold nanoclusters (AuNCs) are a representative class of ultrasmall nanoparticles (USNPs) with sizes of ∼2 nm, smaller than most of proteins including enzymes. In this work, we chose α-chymotrypsin (ChT) and AuNCs as the model system. Activity assays and inhibition kinetics studies showed that dihydrolipoic acid (DHLA)-coated AuNCs (DHLA-AuNCs) had a high inhibitory potency (IC50 = 3.4 µM) and high inhibitory efficacy (>80%) on ChT activity through noncompetitive inhibition mechanism. In distinct contrast, glutathione (GSH)-coated AuNCs (GSH-AuNCs) had no significant inhibition effects. Fluorescence spectroscopy, agarose gel electrophoresis and circular dichroism (CD) spectroscopy were conducted to explore the underlying mechanisms. A two-step interaction model was proposed. First, both DHLA-AuNCs and GSH-AuNCs might be bound to the positively charged sites of ChT through electrostatic forces. Second, further hydrophobic interactions occurred between three tyrosine residues of ChT and the hydrophobic carbon chain of DHLA, leading to a significant structural change thus to deactivate ChT on the allosteric site. On the contrary, no such interactions occurred with GSH of zwitterionic characteristic, which explained no inhibitory effect of GSH-AuNCs on ChT. To the best of our knowledge, this is the first example of the allosteric inhibition of ChT by nano regulators. These findings provide a fundamental basis for the design and development of nano regulators.