Beyond the passive interactions at the nano-bio interface: evidence of Cu metalloprotein-driven oxidative dissolution of silver nanoparticles

Abstract

Background: In a biological system, an engineered nanomaterial (ENM) surface is altered by adsorbed proteins that modify ENM fate and toxicity. Thus far, protein corona characterizations have focused on protein adsorption, interaction strength, and downstream impacts on cell interactions. Given previous reports of Ag ENM disruption of Cu trafficking, this study focuses on Ag ENM interactions with a model Cu metalloprotein, Cu(II) azurin. The study provides evidence of otherwise overlooked ENM-protein chemical reactivity within the corona: redox activity.

Results: Citrate-coated Ag ENMs of various sizes (10-40 nm) reacted with Cu(II) azurin resulted in an order of magnitude more dissolved ionic silver (Ag(I)(aq)) than samples of Ag ENMs only, ENMs mixed Cu(II) ions, or control proteins such as cytochrome c and horse radish peroxidase. This dramatic increase in ENM oxidative dissolution was observed even when Cu(II) azurin was combined with a diverse mixture of Escherchia coli proteins to mimic the complexity of the cellular conona. SDS PAGE results confirm that the multiprotein ENM corona includes azurin. A Cu(I)(aq) colorimetric indicator confirms Cu(II) azurin reduction upon interaction with Ag ENMs, but not with the addition of ionic silver, Ag(I)(aq).

Conclusions: Cu(II) azurin and 10-40 nm Ag ENMs react to catalyze Ag ENM oxidative dissolution and reduction of the model Cu metalloprotein. Results push the current evaluation of protein-ENM characterization beyond passive binding interactions and enable the proposal of a mechanism for reactivity between a model Cu metalloprotein and Ag ENMs.

Fig. 1

ICP-MS measurements of Ag(I)(aq) concentrations from Ag ENM oxidative dissolution. Oxidative dissolution of Ag ENMs was compared across four sizes: 10 (black), 20 (red), 30 (blue), and 40 nm (grey). Dissolution of Ag ENMs alone (labeled control) is compared to dissolution in the presence of 50 µM copper sulfate, cyt c, HRP, and Cu(II) azurin. In addition, both 5 and 50 µM Cu(II) azurin with 0.07 mg/ml SPE were reacted with Ag ENMs (labeled [low] SPE), as well as a higher concentration sample with 50 µM Cu(II) azurin with 0.7 mg/ml SPE (labeled [high] SPE). Data for samples with SPE present in solution are shown with the contribution of SPE subtracted from the total Ag(I)(aq) concentration. Raw data for samples with SPE are given in Additional file 1: Figure S1

Fig. 2

Colorimetric BCA assay of Cu(I)(aq) concentrations. a Spectra of BCA added to 50 µM Cu(II) azurin (black dashes) and added to a reacted mixture of 50 µM Cu(II) azurin with 10 (black), 20 (red), 30 (blue), and 40 nm Ag ENMs (grey). The BCA-Cu(I) complex (λmax = 562 nm) presents as a shoulder on the Cu(II)-thiol LMCT band from Cu(II) azurin (λmax = 630 nm). b Deconvolution of UV–Vis spectra enables comparison of Cu(I)(aq) and Cu(II) azurin concentrations after reaction with Ag ENM of various sizes. Control spectra for BCA assays and sample deconvoluted spectra are given in Additional file 1: Figure S2

Fig. 3

Schematic for proposed Cu(II) azurin–Ag ENM reaction mechanism. a Cu(II) azurin (blue protein with Cu(II) shown as blue sphere in active site) and Ag ENM (large grey sphere) bind to form a complex. b Redox reaction oxidizes Ag ENM surface to form Ag(I) (black sphere) and reduces Cu(II) azurin to Cu(I) azurin (light grey protein with Cu(I) azurin shown with a light grey sphere in active site). Weakly bound Cu(I) is displaced from the active site via one of two mechanisms, either via c direct displacement to form Ag(I) azurin and dissolved Cu(I)(aq) or via d two dissolution equilibria to form apo-azurin with dissolved Cu(I)(aq) and Ag ENMs with dissolved Ag(I)(aq)