Comparative functional dynamics studies on the enzyme nano-bio interface

Abstract

IntroductionBiomedical applications of nanoparticles (NPs) as enzyme inhibitors have recently come to light. Oxides of metals native to the physiological environment (eg, Fe, Zn, Mg, etc.) are of particular interest—especially the functional consequences of their enzyme interaction.Materials and methodsHere, Fe2O3, zinc oxide (ZnO), magnesium oxide (MgO) and nickel oxide (NiO) NPs are compared to copper (Cu) and boron carbide (B4C) NPs. The functional impact of NP interaction to the model enzyme luciferase is determined by 2-dimensional fluorescence difference spectroscopy (2-D FDS) and 2-dimensional photoluminescence difference spectroscopy (2-D PLDS). By 2-D FDS analysis, the change in maximal intensity and in 2-D FDS area under the curve (AUC) is in the order Cu~B4C>ZnO>NiO>>Fe2O3>MgO. The induced changes in protein conformation are confirmed by tryptic digests and gel electrophoresis.ResultsAnalysis of possible trypsin cleavage sites suggest that cleavage mostly occurs in the range of residues 112–155 and 372–439, giving a major 45 kDa band. By 2-D PLDS, it is found that B4C NPs completely ablate bioluminescence, while Cu and Fe2O3 NPs yield a unique bimodal negative decay rate, −7.67×103 and −3.50×101 relative light units respectively. Cu NPs, in particular, give a remarkable 271% change in enzyme activity. Molecular dynamics simulations in water predicted that the surfaces of metal oxide NPs become capped with metal hydroxide groups under physiological conditions, while the surface of B4C becomes populated with boronic acid or borinic acid groups. These predictions are supported by the experimentally determined zeta potential. Thin layer chromatography patterns further support this conception of the NP surfaces, where stabilizing interactions were in the order ionic>polar>non-polar for the series tested.ConclusionOverall the results suggest that B4C and Cu NP functional dynamics on enzyme biochemistry are unique and should be examined further for potential ramifications on other model, physiological or disease-relevant enzymes.

Enzyme Nanoscale Interactions with Manganese Zinc Sulfide Give Insight into Potential Antiviral Mechanisms and SARS-CoV-2 Inhibition

“… 10 Current dogma suggests that nanoscale interactions with enzymes could cause competitive, uncompetitive, or noncompetitive inhibition. Initially, this shape-dependence was investigated by testing various nanorod to nanosphere ratios 11 on the well characterized ZnO-Luc system, 5 , 6 demonstrating that nanorods were preferred ( Figure S1 ). Subsequent work showed that substitution with iron, zinc, or manganese in small amounts (≤5%, 3% or 1%) 10 could increase inhibition against Luc ( Figure S2 ) and a dose–response curve comparing 1% MnZnS against Luc and β-Gal again confirms the IC 50 is <50 μg/mL ( Figure S3 ).…”

“…UV and CD spectroscopy can be used to investigate changes in protein secondary structure as a function of binding to nanoparticles. 6 These methods were used to investigate the impact of 3% MnZnS nanoparticle interactions with hACE2 protein ( Figure 2 ).…”

“…A great deal of work by the authors’ lab and many others supports the interaction of zinc oxide nanoparticles (ZnO NPs) to proteins. , Early model biochemical studies focused on the luciferase (Luc) enzyme which combines both transferase and oxido-reductase activity; the exquisite sensitivity of its bioluminescence reaction makes it ideal for measuring the effects of nanoparticle enzyme activation or inhibition. , ZnO NP activity against drug-resistant bacteria was shown to be shape-dependent and to correlate with the inhibition of a specific class of hydrolase enzyme, β-galactosidase (β-Gal) . Another example is targeted medicines against cancer kinases that have been developed, and it has been shown that treating cancer cells or injecting melanoma tumors in mice with ZnO NPs inhibits the phosphorylation of some of these cancer-related kinases. , …”

Comparative Molecular Immunological Activity of Physiological Metal Oxide Nanoparticle and its Anticancer Peptide and RNA Complexes

“…This assay was conducted as we previously described [27]. Briefly 8000 B16F10 cells were added per well of a 96-well plate and treated with 20 mg/mL ZnO-NP loaded in the presence of 3 mM LL37 or carboplatin sedimented from those suspensions and taken up in 10% FBS/DMEM and added to the cells.…”

Interaction of Ras Binding Domain (RBD) by chemotherapeutic zinc oxide nanoparticles: Progress towards RAS pathway protein interference

“…Intraperitoneal administration of ZnO has been associated with an increase in superoxide dismutase activity and total antioxidant status in male rats [ 9 ]. Its unique surface chemistry allows formation of ZnOH+ species which likely underlies its interaction to anionic membrane of cancer cells and its drug delivery activity [ 10 , 11 ]. Recent work from our research group suggests ZnO NP inhibition of experimental melanoma [ 12 ] can be improved by its interaction to poly I:C RNA [ 13 ].…”