Activity Of Horseradish Peroxidase Research Articles

Effect of interfacial properties on the activation volume of adsorbed enzymes

We have studied the enzymatic activities of α-chymotrypsin (α-CT) and horseradish peroxidase (HRP) that are adsorbed on various chemically modified planar surfaces under aqueous solution. The enzymes were adsorbed on bare quartz, hydrophobic poly(styrene) (PS), positively charged poly(allylamine hydrochloride) (PAH), and negatively charged poly(styrene sulfonate) (PSS). Activation volumes of the enzymes at the aqueous-solid interfaces were determined by using high-pressure total internal reflection fluorescence (TIRF) spectroscopy. Apparently, the pressure response of the adsorbed enzymes strongly depends on the interfacial properties. α-CT can be activated by pressure (increasing enzymatic rate) on negatively charged surfaces like quartz and PSS, whereas HRP is activated by pressure on hydrophobic PS. Corresponding negative activation volumes of −29mLmol−1 for α-CT on quartz, −23mLmol−1 for α-CT on PSS, and −35mLmol−1 for HRP on PS are found. In addition, the absolute activities of α-CT and HRP on quartz, PS, PAH and PSS were determined by UV absorption at ambient pressure. Remarkably, large activities are found on those surfaces that are associated with negative activation volumes. However, Fourier transform infrared (FTIR) spectra collected in attenuated total reflection (ATR) mode do not indicate major adsorption induced conformational changes of the enzymes at any interface studied. Overall, the results of this study show that the activity of immobilized enzymes can largely be enhanced by the right combination of adsorbent material and applied pressure.

Catalytic activity and thermal stability of horseradish peroxidase encapsulated in self-assembled organic nanotubes.

Horseradish peroxidase (HRP) was encapsulated in self-assembled lithocholic acid (LCA) based organic nanotubes and its catalytic activity before and after thermal treatment was measured for comparison with free HRP. The apparent kcat (kcat/Km) for nanotube encapsulated HRP remained almost the same before and after thermal treatment, reporting an average value of 3.7 ± 0.4 μM(-1) s(-1). The apparent kcat value for free HRP decreased from 14.8 ± 1.3 μM(-1) s(-1) for samples stored at 4 °C to 2.4 ± 0.1 μM(-1) s(-1) after thermal treatment for 8 h at 55 °C. The Michaelis-Menten constants, Km, determined for encapsulated HRP and free HRP were relatively unperturbed by storage conditions at 4 °C or thermally treated at 55 °C for varying time periods from 2-8 h, with encapsulated HRP having a slightly higher Km than free HRP (13.4 ± 0.9 μM versus 11.7 ± 0.4 μM). The amount of HRP encapsulated in LCA nanotubes increased dramatically when the mixture of HRP and LCA nanotubes was brought to an elevated temperature. Within 4 h of thermal treatment at 55 °C, the amount of HRP encapsulated by the LCA nanotubes was more than 4 times the amount of HRP encapsulated when equilibrated at 4 °C for 7 days. Molecular dynamics (MD) simulations show that the higher degree of exposure of hydrophobic residues in HRP at elevated temperatures enhances the hydrophobic interaction between HRP and the nanotube wall, resulting in the increased amount of HRP surface adsorption and, hence, the overall amount of encapsulation inside the nanotubes.

Injectable hydrogel systems crosslinked by horseradish peroxidase

Hydrogels are widely used as reservoirs in drug delivery and scaffolds for tissue engineering. In particular, injectable hydrogel systems, which are formed by physical, chemical, or enzyme-mediated crosslinking reactions in situ, offer the advantages of minimal invasiveness, ease of application, and void-filling property. Examples of these hydrogels are provided in the first part of this paper. In the second part, hydrogels that are formed by the enzymatic activity of horseradish peroxidase (HRP) are highlighted. HRP catalyzes the crosslinking reaction of polymer-phenol conjugates in the presence of hydrogen peroxide (H2O2), resulting in hydrogels with tunable gelation rate and crosslinking density. The catalytic mechanism of the HRP-mediated crosslinking reaction is discussed in detail, and the recent biomedical applications of the HRP-crosslinked hydrogels are described. Lastly, the concerns associated with HRP-mediated crosslinking and the future outlook of HRP-crosslinked hydrogels are addressed.

Colorimetric determination of melamine based on the reversal of the mercury(II) induced inhibition of the light-triggered oxidase-like activity of gold nanoclusters

We describe a colorimetric assay for the quantitation of melamine (MA). It relies on the observation that MA reverses the inhibition of the light-triggered activity of horseradish peroxidase (HRP) functionalized gold nanoclusters (HRP-Au NCs) by Hg(II) ions. The HRP-Au NCs catalyze the oxidation of the substrate 3,3’,5,5’-tetramethylbenzidine (TMB) by dissolved oxygen if exposed to visible light provided by a 300 W Xe lamp (λ ≥ 400 nm). The addition of Hg(II) suppresses the enzymatic activity due to the metallophilic interaction between Hg(II) and Au(I) present on the surface of the HRP-Au NCs. MA with its triazole ring can restore the catalytic activity of the HRP-Au NCs by inhibiting the metallophilic interaction. As a result of these findings, a colorimetric “turn-on” assay for MA was worked out that can be used to determine MA in the 0.2 to 15 μmol·L−1 concentration range and with a 72 nmol·L−1 detection limit, which is much lower than the established safety limit. The method was successfully applied to the quantitation of MA in (spiked) raw milk and milk powder, with recoveries ranging from 98.5 % to 101.5 %.

Alternate hot and cold electrodes

Alternate hot and cold electrodes, actively heated and chilled, were facilely fabricated for the first time. It consists of two symmetrically placed semiconductor thermoelectric coolers and an embedded interlayer with a Pt-PtRh thermocouple. The tip of Pt-PtRh thermocouple can be used as both an electrochemical electrode and a temperature-sensing device simultaneously. Theoretical simulation shows that a local steady-state supercooled water within a radius of ca. 12.6mm around the electrode can be obtained. In this paper, the activity of horseradish peroxidase is evidenced at supercooled temperature (−9°C) in 0.1M PBS.

An enzyme-assisted electrochemiluminescent biosensor developed on order mesoporous carbons substrate for ultrasensitive glyphosate sensing

In this paper, a strategy of developing a late-model and sensitive electrochemiluminescence (ECL) biosensor for glyphosate detection based on enzyme-assisted in situ generation of ZnS quantum dots (QDs) on ordered mesoporous carbons (OMC) substrate was proposed. OMC, as a typically ordered mesoporous carbon material, not only provides a protective microenvironment for enzyme to retain its structure and activity but also has a synergistic effect with chitosan for the absorption of Zn2+ ions by virtue of its high surface area and high pore volume and thus employed as the matrix of proposed biosensor. Then horseradish peroxidase (HRP) was introduced to expedite the generation of ZnS QDs via accelerating the reduction of Na2S2O3 with H2O2 to yield H2S that reacted with Zn2+ ions. Glyphosate, as a kind of organic pesticide with amine, carboxyl and phosphonate group which would coordinate strongly to metal ions, possessed the potential to inhibited the activity of HRP, because HRP contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group which would react with the amine, carboxyl and phosphonate group of glyphosate. Accordingly, the proposed ECL QDs biosensor was employed to determine the Gly and shown a wide linear range from 0.1nM to 10mM with excellent sensitivity, reproducibility and selectivity. Further, the half maximal inhibitory concentration (IC50) and the Michaelis–Menten constant were studied, and all the results indicated that the proposed strategy is practicable and provide a new opportunity to develop novel ECL sensing platforms in various applications.

Development of a novel method for determination of mercury based on its inhibitory effect on horseradish peroxidase activity followed by monitoring the surface plasmon resonance peak of gold nanoparticles

A highly sensitive and simple indirect spectrophotometric method has been developed for the determination of trace amounts of inorganic mercury (Hg2+) in aqueous media. The method is based on the inhibitory effect of Hg2+ on the activity of horseradish peroxidase (HRP) in the oxidation of ascorbic acid by hydrogen peroxide followed by the reduction of Au3+ to Au–NPs by unreacted ascorbic acid and the measurement of the absorbance of localized surface plasmon resonance (LSPR) peak of gold nanoparticles (at 530nm) which is directly proportional to the concentration of Hg2+. Under the optimum conditions, the calibration curve was linear in the concentration range of 1–220ngmL−1. Limits of detection (LOD) and quantification (LOQ) were 0.2 and 0.7ngmL−1, respectively and the relative standard deviation at 100ngmL−1 level of Hg2+ was 2.6%. The method was successfully applied to the determination of mercury in different water samples.

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris.

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins.

Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities.

Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity.

Degradation-by-design: Surface modification with functional substrates that enhance the enzymatic degradation of carbon nanotubes

Biodegradation of carbon-based nanomaterials has been pursued intensively in the last few years, as one of the most crucial issues for the design of safe, clinically relevant conjugates for biomedical applications. In this paper it is demonstrated that specific functional molecules can enhance the catalytic activity of horseradish peroxidase (HRP) and xanthine oxidase (XO) for the degradation of carbon nanotubes. Two different azido coumarins and one cathecol derivative are linked to multi-walled carbon nanotubes (MWCNTs). These molecules are good reducing substrates and strong redox mediators to enhance the catalytic activity of HRP. XO, known to metabolize various molecules mainly in the mammalian liver, including human, was instead used to test the biodegradability of MWCNTs modified with an azido purine. The products of the biodegradation process are characterized by transmission electron microscopy and Raman spectroscopy. The results indicate that coumarin and catechol moieties have enhanced the biodegradation of MWCNTs compared to oxidized nanotubes, likely due to the capacity of these substrates to better interact with and activate HRP. Although azido purine-MWCNTs are degraded less effectively by XO than oxidized nanotubes, the data uncover the importance of XO in the biodegradation of carbon-nanomaterials leading to their better surface engineering for biomedical applications.

DNA transducer-triggered signal switch for visual colorimetric bioanalysis

A simple and versatile colorimetric biosensor has been developed for sensitive and specific detection of a wide range of biomolecules, such as oligonucleotides and aptamer-recognized targets. Combining the signal transducer and catalyzed hairpin assembly (CHA)-based signal amplification, the target DNA binds with the hairpin DNA to form a new nucleic acid sequence and creates a toehold in the transducer for initiating the recycle amplification reaction of CHA. The catalyzed assembly process produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimic horseradish peroxidase activity, which catalyzes a colorimetric reaction. Under optimal conditions, the calibration curve of synthetic target DNA has good linearity from 50 pM to 200 nM with a detection limit of 32 pM. This strategy has been successfully applied to detect S. pneumoniae as low as 156 CFU mL−1, and shows a good specificity against closely related streptococci and major pathogenic bacteria. In addition, the developed method enables successful visual analysis of S. pneumoniae in clinical samples by the naked eye. Importantly, this method demonstrates excellent assay versatility for sensitively detecting oligonucleotides or aptamer-recognized targets.

Effect of PEGylation on the activity and stability of horseradish peroxidase and l-N-carbamoylase in aqueous phases

Effect of pegylation on the activities and stabilities of horseradish peroxidase (HRP) and n-carbamoyl-l-amino acid amidohydrolase (l-N-carbamoylase) was investigated. Pegylation was favored under alkaline conditions with high PEG/enzyme molar ratios. The lysine residues in l-N-carbamoylase were found to be more readily accessible for pegylation than those in HRP, leading to the formation of multi-pegylated enzymes. Pegylation and addition of mPEG as an additive improved HRP activity by 64% and 42%, respectively, via the formation of protective layers against the free radical-induced inactivation. The activity of pegylated l-N-carbamoylase, however, declined due to the additional mass transfer resistance introduced by the PEG layers. The extent of reduction in l-N-carbamoylase activity increased with the level of pegylation. In terms of thermostability, both the addition of mPEG as an additive and pegylation with PEG5000 led to a decline in thermostability. Nevertheless, pegylation of HRP with PEG2000 resulted in a PEG-HRP conjugate exhibiting a thermostability two-fold that of the control at 70°C, probably due to the capability of the relatively hydrophilic PEG domains on the conjugate in avoiding enzyme aggregation. After a 4-day incubation at room temperature, pegylated HRP retained significantly higher activity, 89%, than the control, 12%.

Enhancement of Direct Electrocatalytic Activity of Horseradish Peroxidase on Polyaniline Nanotubes

The advantages of nanotubes over compact conducting polymer films include the large surface area available for enzyme immobilization, facile diffusion of analyte molecules and enhanced conductivity. Heat treatment of PANI induces the formation of phenazine cross-links between PANI chains, which influences the electrocatalytic response of the immobilized enzymes. In this study, both polyaniline (PANI) bulk films and nanotubes were studied as a support for horseradish peroxidase (HRP). Two types of nanotubes, with diameters of 100 and 400 nm, were investigated. HRP was immobilized by physical adsorption or covalent bonding via the carbodiimide reaction. The influence of the heat treatment of PANI prior to enzyme immobilization on the electrochemical responses of the PANI/HRP electrodes was studied. The electrocatalytic current due to hydrogen peroxide (H2O2) reduction was observed in all of the studied samples. However, the current values observed for HRP immobilized on PANI nanotubes with diameters of 100 nm (PANI/100 nm) were an order of magnitude higher than that of the bulk film. The electrocatalytic currents for the PANI/400 nm nanotubes had intermediate values between PANI/100 nm and the PANI bulk film. Linear responses to H2O2 were observed over a wide concentration range for all of the HRP/PANI systems. The formation of phenazine cross-links due to the thermal treatment of PANI was confirmed from the Raman spectra. The cross-linking contributes to the enhanced mechanical stability of the PANI nanotubes. However, this cross-linking does not enhance significantly the electrocatalytic activity of the immobilized enzyme.

Purpose-Oriented Antibody Libraries Incorporating Tailored CDR3 Sequences

The development of in vitro antibody selection technologies has allowed overcoming some limitations inherent to the hybridoma technology. In most cases, large repertoires of antibody genes have been assembled to create highly diversified libraries allowing the isolation of antibodies recognizing virtually any antigen. However, these universal libraries might not allow the isolation of antibodies with specific structural properties or particular amino acid contents that are rarely found in natural repertoires. Purpose-oriented libraries specially designed to incorporate desired characteristics have been successfully used. However, the workload required for library construction has limited the attractiveness of this approach compared to the use of large universal libraries. We have developed an approach to capture synthetic or natural diversity into the complementarity determining regions 3 (CDR3) of human antibody repertoires using Type IIS restriction enzymes. In this way, we generated several libraries either biased in amino acid content or towards long CDRH3 loops. The latter were successfully used to identify antibodies inhibiting the enzymatic activity of horseradish peroxidase, whereas libraries enriched in histidines allowed for the isolation of antibodies binding to human Fc in a pH-dependent manner. These libraries indicate that tailored diversification of CDR3 is sufficient to generate purpose-oriented libraries and isolate antibodies with uncommon properties.

Ultra-high-throughput screening of an in vitro-synthesized horseradish peroxidase displayed on microbeads using cell sorter.

The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases.

Effects of different media supplements on the production of an active recombinant plant peroxidase in a Pichia pastoris Δoch1 strain

Recombinant protein production in microorganisms is one of the most studied areas of research in biotechnology today. In this respect the yeast Pichia pastoris is an important microbial production host due to its capability of secreting the target protein and performing posttranslational modifications. In a recent study, we described the development of a robust bioprocess for a glyco-engineered recombinant P. pastoris strain where the native α-1,6-mannosyltransfrease OCH1 was knocked out (Δoch1 strain). This strain produced the glycosylated enzyme horseradish peroxidase (HRP) with more homogeneous and shorter surface glycans than the respective benchmark strain. However, the recombinant Δoch1 strain was physiologically impaired and thus hard to cultivate. We faced cell cluster formation, cell lysis and consequent intensive foam formation. Thus, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on (1) cell physiology, (2) cell morphology, (3) cell lysis, (4) productivity and (5) product purity in a multivariate manner. However, not only process parameters might influence these characteristics, but also media supplements might have an impact. Here, we describe the effects of different heme-precursors as well as of a protease-inhibitor cocktail on the production of active HRP in therecombinant P. pastoris Δoch1strain.

Activity of Horseradish Peroxidase Adsorbed onto Titanate Nanowires

Immobilization of horseradish peroxidase (HRP) onto titanate nanowires (TNWs) was investigated using different strategies. TNWs were synthesized by a hydrothermal method and characterized by scanning electron microscopy, X-ray diffraction, nitrogen physisorption (77K) and Fourier transform infrared spectroscopy. Free HRP was stable and active in a wide range of pH with optimal activity at 7.0. The Km of HRP with 4-aminoantipyrine and H2O2 as substrate was 0.77 ± 0.25 mmol l-1. Immobilization strategies studied were non-specific adsorption and covalent coupling through amine groups. Adsorption isotherms were well fitted by the Langmuir-Freundlich model. The coverage of TNWs containing HRP adsorbed by covalent coupling was 1.56 mg HRP m-2 and the residual enzymatic activity was approximately 40%. The enzymatic activity of free HRP and immobilized HRP was monitored as a function of storing time. The results confirm that the enzyme is firmly attached to the TNW surface through covalent binding, constituting a...

Enzymatically crosslinked dendritic polyglycerol nanogels for encapsulation of catalytically active proteins.

The enormous potential of nanogel scaffolds for protein encapsulation has been widely recognized. However, constructing stable polymeric nanoscale networks in a facile, mild, and controllable fashion still remains a technical challenge. Here, we present a novel nanogel formation strategy using horseradish peroxidase (HRP) catalyzed crosslinking on phenolic derivatized dendritic polyglycerol (dPG) in the presence of H2O2 in an inverse miniemulsion. This "enzymatic nanogelation" approach was efficient to produce stable 200 nm dPG nanogel particles, and was performed under physiological conditions, thus making it particularly beneficial for encapsulating biological proteins. Purification of the nanogels was easy to handle and practical because there was no need for a post-quenching step. Interestingly, the use of dPG resulted in higher HRP laden nanogels than for linear polyethylene glycol (PEG) analogs, which illustrates the benefits of dendritic backbones in nanogels for protein encapsulation. In addition, the mild immobilization contributed to the enhanced thermal stability and reusability of HRP. The nanogel preparation could be easily optimized to achieve the best HRP activity. Furthermore, a second enzyme, Candida antarctica lipase B (CalB), was successfully encapsulated and optimized for activity in dPG nanogels by the same enzymatic methodology, which shows the perspective applications of such techniques for encapsulation of diverse proteins.

Reagent pencils: a new technique for solvent-free deposition of reagents onto paper-based microfluidic devices.

Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the pencils under ambient conditions, and no significant decrease in the activity of the enzyme horseradish peroxidase stored in a pencil was observed after 63 days. Reagent pencils offer a new option for preparing and customizing diagnostic tests at the point of care without the need for specialized equipment.

Size-dependent tuning of horseradish peroxidase bioreactivity by gold nanoparticles.

Molecules with diverse biological functions, such as heme peroxidases, can be useful tools for identifying potential biological effects of gold nanoparticles (AuNPs) at the molecular level. Here, using UV-Vis, circular dichroism, dynamic light scattering, and electron spin resonance spectroscopy, we report tuning of horseradish peroxidase (HRP) bioactivity by reactant-free AuNPs with diameters of 5, 10, 15, 30 and 60 nm (Au-5 nm, Au-10 nm, Au-15 nm, Au-30 nm and Au-60 nm). HRP conjugation to AuNPs was observed with only Au-5 nm and Au-10 nm prominently increasing the α-helicity of the enzyme to extents inversely related to their size. Au-5 nm inhibited both HRP peroxidase activity toward 3,3',5,5'-tetramethylbenzidine and HRP compound I/II reactivity toward 5,5-dimethyl-1-pyrroline N-oxide. Au-5 nm enhanced the HRP peroxidase activity toward ascorbic acid and the HRP compound I/II reactivity toward redox-active residues in the HRP protein moiety. Further, Au-5 nm also decreased the catalase- and oxidase-like activities of HRP. Au-10 nm showed similar, but weaker effects, while Au-15 nm, Au-30 nm and Au-60 nm had no effect. Results suggest that AuNPs can size-dependently enhance or inhibit HRP bioreactivity toward substrates with different redox potentials via a mechanism involving extension of the HRP substrate access channel and decline in the redox potentials of HRP catalytic intermediates.