Multicommutated Flow Analysis System for Determination of Horseradish Peroxidase and Its Inhibitors.

A fully mechanized multicommutated flow analysis (MCFA) system dedicated to determining horseradish peroxidase (HRP) activity was developed. Detection was conducted using a flow-through optoelectronic detector-constructed of paired LEDs operating according to the paired emitter-detector diode (PEDD) principle. The PEDD-MCFA system is dedicated to monitoring the enzyme-catalyzed oxidation of p-phenylenediamine (pPD) by a hydrogen peroxide. Under optimized conditions, the presented bioanalytical system was characterized by a linear response range (33.47-200 U/L) with a detection limit at 10.54 U/L HRP activity and 1.66 mV·L/U sensitivity, relatively high throughput (12 signals recordings per hour), and acceptable precision (RSD below 6%). Additionally, the utility of the developed PEDD-MCFA system for the determination of HRP inhibitors allowing the detection of selected thiols at micromolar levels, is demonstrated. The practical utility of the flow system was illustrated by the analysis of some dietary supplements containing L-cysteine, N-acetylcysteine, and L-glutathione.

The inhibition mechanism of luteolin on peroxidase based on multispectroscopic techniques.

Luteolin, a plant-derived flavonoid, was found to exert effective inhibitory effect to peroxidase activity in a non-competitive manner with an IC50 of (6.62 ± 0.45) × 10-5 mol L-1. The interaction between luteolin and peroxidase induced the formation of a static complex with a binding constant (Ksv) of 7.31 × 103 L mol-1 s-1 driven by hydrogen bond and hydrophobic interaction. Further, the molecular interaction between luteolin and peroxidase resulted in intrinsic fluorescence quenching, structural and conformational alternations which were determined by multispectroscopic techniques combined with computational molecular docking. Molecular docking results revealed that luteolin bound to peroxidase and interacted with relevant amino acid residues in the hydrophobic pocket. These results will provide information for screening additional peroxidase inhibitors and provide evidence of luteolin's potential application in preservation and processing of fruit and vegetables and clinical disease remedy.

Implication and Inhibition Boolean Logic Gates Mimicked with Enzyme Reactions.

The enzyme system mimicking Implication (IMPLY) and Inhibition (INHIB) Boolean logic gates has been designed. The same enzyme system was used to operate as the IMPLY or INHIB gate simply by reformulating the input signals. The optical analysis of the logic operation confirmed the output generation as expected for the studied logic gates. The conceptual approach to the IMPLY and INHIB logic gates allows their construction with many other enzymes operating in a similar way.

Amperometric determination of heavy metal using an HRP inhibition biosensor based on ITO nanoparticles-ruthenium (III) hexamine trichloride composite: Central composite design optimization.

A novel horseradish peroxidase (HRP) enzyme inhibition biosensor based on indium tin oxide (ITO) nanoparticles, hexaammineruthenium (III) chloride (RUT), and chitosan (CH) modified glassy carbon electrode (GCE) was developed. The biosensor fabrication process was investigated using scanning electron microscopy, energy-dispersive X-ray spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The amounts of ITO nanoparticles and RUT were optimized using a 22 central composite design for the optimization of electrode composition. The detection limits were determined as 8 nM, 3 nM, and 1 nM for Pb2+, Ni2+, and Cd2+, respectively. The inhibition calibration curves of the biosensor were found to be within the range of 0.009-0.301 µM with a sensitivity of 11.97 µA µM-1 cm-2 (0.85 µA µM-1) for Pb2+, 0.011-0.368 µM with a sensitivity of 10.84 µA µM-1 cm-2 (0.77 µA µM-1) for Ni2+, and 0.008-0.372 µM with a sensitivity of 10.99 µA µM-1 cm-2 (0.78 µA µM-1) for Cd2+. The type of HRP inhibition by Pb2+, Ni2+ and Cd2+ was investigated by the Dixon and Cornish-Bowden plots. The effects of possible interfering species on the biosensor response were examined. The analysis of Pb2+, Ni2+, and Cd2+ in tap water was demonstrated using the HRP/ITO-RUT-CH/GCE with satisfactory experimental results. The proposed method agreed with the atomic absorption spectrometry results.

Inhibitors in Commercially Available 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) Affect Enzymatic Assays.

ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate), is a common chromogenic substrate for peroxidase enzymes, which are widely used in biochemical research and diagnostic tests. We discovered that impurities in the commercially available ABTS significantly affect the results of peroxidase activity assays. We show that the impurities inhibit the activity of the peroxidases and the influence varies for different batches of ABTS from the same source. The inhibition of horseradish peroxidase (HRP) is uncompetitive for the substrate H2O2 while it is competitive for the substrate ABTS. By using high-resolution mass spectrometry, potential inhibitors were identified to be precursors or analogs of ABTS. The inhibitors are also capable of inhibiting the GOx-catalyzed reduction of the ABTS radical cation by glucose in anaerobic conditions. As the inhibition is found to be pH-dependent, diagnostic applications, such as ELISA tests based on the peroxidase-H2O2-ABTS system, should be carried out at pH 4.4 to minimize the inhibitory effect of potentially present impurities.

DNA Barcoding a Complete Matrix of Stereoisomeric Small Molecules.

It is challenging to incorporate stereochemical diversity and topographic complexity into DNA-encoded libraries (DELs) because DEL syntheses cannot fully exploit the capabilities of modern synthetic organic chemistry. Here, we describe the design, construction, and validation of DOS-DEL-1, a library of 107 616 DNA-barcoded chiral 2,3-disubsituted azetidines and pyrrolidines. We used stereospecific C-H arylation chemistry to furnish complex scaffolds primed for DEL synthesis, and we developed an improved on-DNA Suzuki reaction to maximize library quality. We then studied both the structural diversity of the library and the physicochemical properties of individual compounds using Tanimoto multifusion similarity analysis, among other techniques. These analyses revealed not only that most DOS-DEL-1 members have "drug-like" properties, but also that the library more closely resembles compound collections derived from diversity synthesis than those from other sources (e.g., commercial vendors). Finally, we performed validation screens against horseradish peroxidase and carbonic anhydrase IX, and we developed a novel, Poisson-based statistical framework to analyze the results. A set of assay positives were successfully translated into potent carbonic anhydrase inhibitors (IC50 = 20.1-68.7 nM), which confirmed the success of the synthesis and screening procedures. These results establish a strategy to synthesize DELs with scaffold-based stereochemical diversity and complexity that does not require the development of novel DNA-compatible chemistry.

Alleviated Inhibition of Single Enzyme in Confined and Crowded Environment.

Most proteins perform functions in intracellular milieu. The crowding, compartmentalized cytosol environment affects the protein structure, folding, conformational stability, substrate diffusion, and substrate-enzyme binding. Moreover, enzymes are available at single or very low copy numbers in a cell, and thus the conformation fluctuations of a single enzyme in a crowding environment could also greatly influence its kinetics. However, the crowding effect is poorly understood in the kinetical aspect of enzymatic reactions. In the present study, individual horseradish peroxidase (HRP) is encapsulated in a liposome containing crowding reagents as mimics of viscous cytosol. The confined crowding environment possesses a profound influence on both the catalytic activity and the product inhibition of enzymes. By analyzing the correlation between product generation and product inhibition, we find that the allosteric noncompetitive inhibition of HRP is alleviated in the crowded and confined milieu. Small-angle X-ray scattering experiments provide straightforward proofs of structural changes of enzymes in crowding environments, which are responsible for the reduced enzyme activity and increased enzyme-substrate affinity. We expect that this work may deepen the understanding of correlations between enzymatic conformations and activity performance in real cellular environments.

Single-Molecule Studies of Allosteric Inhibition of Individual Enzyme on a DNA Origami Reactor.

Unraveling the conformational changes of enzymes together with inhibition kinetics during an enzymatic reaction has great potential in screening therapeutic candidates; however, it remains challenging due to the transient nature of each intermediate step. We report our study on the noncompetitive inhibition of horseradish peroxidase with single-turnover resolution using single-molecule fluorescence microscopy. By introducing DNA origami as an addressable nanoreactor, we observe the coexistence of nascent-formed fluorescent product on both catalytic and docking sites. We further propose a single-molecule kinetic model to reveal the interplay between product generation and noncompetitive inhibition and find three distinct inhibitor releasing pathways. Moreover, the kinetic isotope effect experiment indicates a strong correlation between catalytic and docking sites, suggesting an allosteric conformational change in noncompetitive inhibition. A memory effect is also observed. This work provides an in-depth understanding of the correlation between enzyme behavior and enzymatic conformational fluctuation, substrate conversion, and product releasing pathway and kinetics.

Genetically-encoded fragment-based discovery of glycopeptide ligands for DC-SIGN.

We have employed genetically-encoded fragment-based discovery to identify novel glycopeptides with affinity for the dendritic cell receptor DC-SIGN. Starting from libraries of 108 mannose-conjugated peptides, we identified glycopeptides that exhibited up to a 650-fold increase in multivalent binding affinity for DC-SIGN, which is also preserved in cells. Monovalently, our most potent glycopeptides have a similar potency to a Man3 oligosaccharide, representing a 15-fold increase in activity compared to mannose. These compounds represent the first examples of glycopeptide ligands that target the CRD of DC-SIGN. The natural framework of glycopeptide conjugates and the simplicity of orthogonal conjugation to make these glycopeptides anticipates a promising future for development of DC-SIGN-targeting moieties.

Molecular docking and two-dimensional quantitative structure-activity relationship studies of synthetic flavonoids on horseradish peroxidase compounds (I, II, and III).

For the first time, the enzymatic inhibition activity of 13 synthetic flavonoids was assessed by quantitative structure-activity relationship (QSAR) modeling and molecular docking with the three states of the enzyme horseradish peroxidase (HRP). The results show that apigenin, quercetin, kaempferol, fisetin, tricetin, and luteolin exerted a high competitive inhibition on HRP (Ki between 0.14 and 1.74 mM) compared with other flavonoids. The QSAR model of enzymatic activity (R2 = 0.95, RMSE = 5.48) showed that Ghose-Crippen octanol-water partition coefficient (Alog P) and lowest unoccupied molecular orbital's energy (εlumo ) correlated with 0.65 and 0.17, respectively, with Ki values. According to the docking results using Molegro Virtual Docker program, all the flavonoids have shown great binding affinity towards peroxidase. Apigenin has the largest MolDock score in the three states of HRP noting an increased affinity of these flavonoids between compound I and compound II by 2.26%. However, these affinities strongly decrease between compound II and compound III by 28.43% especially for luteolin whose MolDock score decreased by 74.7%. With the results of docking, the affinities of the flavonoids tested and translated by their Ki values are much more presentative of the inhibition of the first reaction states of HRP because their inhibitory effect is important.

Interference of carbidopa and other catechols with reactions catalyzed by peroxidases.

BACKGROUND: A number of compounds, including ascorbic acid, catecholamines, flavonoids, p-diphenols and hydrazine derivatives have been reported to interfere with peroxidase-based medical diagnostic tests (Trinder reaction) but the mechanisms of these effects have not been fully elucidated. METHODS: Reactions of bovine myeloperoxidase with o-dianisidine, bovine lactoperoxidase with ABTS and horseradish peroxidase with 4-aminoantipyrine/phenol in the presence of carbidopa, an anti-Parkinsonian drug, and other catechols, including l-dopa, were monitored spectrophotometrically and by measuring hydrogen peroxide consumption. RESULTS: Chromophore formation in all three enzyme/substrate systems was blocked in the presence of carbidopa and other catechols. However, the rates of hydrogen peroxide consumption were not much affected. Irreversible enzyme inhibition was also insignificant. CONCLUSIONS: Tested compounds reduced the oxidation products or intermediates of model substrates thus preventing chromophore formation. This interference may affect interpretation of results of diagnostic tests in samples from patients with Parkinson's disease treated with carbidopa and l-dopa. GENERAL SIGNIFICANCE: This mechanism allows prediction of interference in peroxidase-based diagnostic tests for other compounds, including drugs and natural products.

Structure-activity relationships and molecular docking of thirteen synthesized flavonoids as horseradish peroxidase inhibitors.

For the first time, the structure-activity relationships of thirteen synthesized flavonoids have been investigated by evaluating their ability to modulate horseradish peroxidase (HRP) catalytic activity. Indeed, a modified spectrophotometrically method was carried out and optimized using 4-methylcatechol (4-MC) as peroxidase co-substrate. The results show that these flavonoids exhibit a great capacity to inhibit peroxidase with Ki values ranged from 0.14±0.01 to 65±0.04mM. Molecular docking has been achieved using Auto Dock Vina program to discuss the nature of interactions and the mechanism of inhibition. According to the docking results, all the flavonoids have shown great binding affinity to peroxidase. These molecular modeling studies suggested that pyran-4-one cycle acts as an inhibition key for peroxidase. Therefore, potent peroxidase inhibitors are flavonoids with these structural requirements: the presence of the hydroxyl (OH) group in 7, 5 and 4' positions and the absence of the methoxy (O-CH3) group. Apigenin contributed better in HRP inhibitory activity. The present study has shown that the studied flavonoids could be promising HRP inhibitors, which can help in developing new molecules to control thyroid diseases.

Advantages of an Electrochemical Method Compared to the Spectrophotometric Kinetic Study of Peroxidase Inhibition by Boroxine Derivative.

In this study, boroxine derivative (K2[B3O3F4OH]) was tested as an inhibitor of horseradish peroxidase (HRP) by spectrophotometric and electrochemical methods. The activity of horseradish peroxidase was first studied under steady-state kinetic conditions by a spectrophotometric method which required the use of guaiacol as a second substrate to measure guaiacol peroxidation. The results of this method have shown that, by changing the concentration of guaiacol as the literature suggests, a different type of inhibition is observed than when changing the concentration of hydrogen peroxide as the substrate. This suggests that guaiacol interferes with the reaction in some way. The electrochemical method involves direct electron transfer of HRP immobilized in Nafion nanocomposite films on a glassy carbon (GC) electrode, creating a sensor with an electro-catalytic response to the reduction of hydrogen peroxide. The electrochemical method simplifies kinetic assays by removing the requirement of reducing substrates.

Real-time and in situ enzyme inhibition assay for the flux of hydrogen sulfide based on 3D interwoven AuPd-reduced graphene oxide network.

A highly sensitive enzyme inhibition analytical platform was established firstly based on paper-supported 3D interwoven AuPd-reduced graphene oxide (rGO) network (NW) for real-time and in situ analysis of H2S released from cancer cells. The novel paper working electrode (PWE) with large electric conductivity, effective surface area and unusual biocompatibility, was fabricated via controllably assembling rGO and AuPd alloy nanoparticles onto the surface of cellulose fibers and into the macropores of paper, which was employed as affinity matrix for horseradish peroxidase (HRP) loading and cells capture. It was the superior performances of AuPd-rGO-NW-PWE that made the loaded HRP exhibit excellent electrocatalytic behavior to H2O2, bring the rapid enhancement of current response. After releasing H2S, the current response would be obviously decreased due to the efficient inhibition effect of H2S on HRP activity. The inhibition degree of HRP was directly proportional to the amount of H2S, and so, the flux of H2S released from cells could be recorded availably. Thus, this proposed enzyme inhibition cyto-sensor could be applied for efficient recording of the release of H2S, which had potential utility to cellular biology and pathophysiology.

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 (Hg(2+)) in aqueous media. The method is based on the inhibitory effect of Hg(2+) on the activity of horseradish peroxidase (HRP) in the oxidation of ascorbic acid by hydrogen peroxide followed by the reduction of Au(3+) to Au-NPs by unreacted ascorbic acid and the measurement of the absorbance of localized surface plasmon resonance (LSPR) peak of gold nanoparticles (at 530 nm) which is directly proportional to the concentration of Hg(2+). Under the optimum conditions, the calibration curve was linear in the concentration range of 1-220 ng mL(-1). Limits of detection (LOD) and quantification (LOQ) were 0.2 and 0.7 ng mL(-1), respectively and the relative standard deviation at 100 ng mL(-1) level of Hg(2+) was 2.6%. The method was successfully applied to the determination of mercury in different water samples.

Stability and structural changes of horseradish peroxidase: microwave versus conventional heating treatment.

Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4-6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.

Amperometric inhibition biosensors based on horseradish peroxidase and gold sononanoparticles immobilized onto different electrodes for cyanide measurements.

New biosensors based on inhibition for the detection of cyanide and the comparison of the analytical performances of nine enzyme biosensor designs by using three different electrodes: Sonogel-Carbon, glassy carbon and gold electrodes were discussed. Three different horseradish peroxidase immobilization procedures with and without gold sononanoparticles were studied. The amperometric measurements were performed at an applied potential of -0.15V vs. Ag/AgCl in 50mM sodium acetate buffer solution pH=5.0. The apparent kinetic parameters (Kmapp, Vmaxapp) of immobilized HRP were calculated in the absence of inhibitor (cyanide) by using caffeic acid, hydroquinone, and catechol as substrates. The presence of gold sononanoparticles enhanced the electron transfer reaction and improved the analytical performance of the biosensors. The HRP kinetic interactions reveal non-competitive binding of cyanide with an apparent inhibition constant (Ki) of 2.7µM and I50 of 1.3µM. The determination of cyanide can be achieved in a dynamic range of 0.1-58.6µM with a detection limit of 0.03µM which is lower than those reported by previous studies. Hence this biosensing methodology can be used as a new promising approach for detecting cyanide.

Horseradish peroxidase inactivation: heme destruction and influence of polyethylene glycol.

Horseradish peroxidase (HRP) mediates efficient conversion of many phenolic contaminants and thus has potential applications for pollution control. Such potentially important applications suffer however from the fact that the enzyme becomes quickly inactivated during phenol oxidation and polymerization. The work here provides the first experimental data of heme consumption and iron releases to support the hypothesis that HRP is inactivated by heme destruction. Product of heme destruction is identified using liquid chromatography with mass spectrometry. The heme macrocycle destruction involving deprivation of the heme iron and oxidation of the 4-vinyl group in heme occurs as a result of the reaction. We also demonstrated that heme consumption and iron releases resulting from HRP destruction are largely reduced in the presence of polyethylene glycol (PEG), providing the first evidence to indicate that heme destruction is effectively suppressed by co-dissolved PEG. These findings advance a better understanding of the mechanisms of HRP inactivation.

Enzymatic assay for Cu(II) with horseradish peroxidase and its application in colorimetric logic gate.

We report an ultrasensitive and colorimetric assay for Cu(II) via enzymatic amplification strategy. The enzymatic activity of horseradish peroxidase (HRP) is strongly inhibited by Cu(I), which can be used indirectly to assay Cu(II). The limit of detection (LOD) is 0.37 nM, and the detection of 20 nM Cu(II) in solution can be achieved with naked eyes. This assay can be used to construct a colorimetric logic gate.

Immobilization of horseradish peroxidase by electrospun fibrous membranes for adsorption and degradation of pentachlorophenol in water.

Horseradish peroxidase (HRP) is successfully in situ encapsulated into the poly(D,L-lactide-co-glycolide) (PLGA)/PEO-PPO-PEO (F108) electrospun fibrous membranes (EFMs) by emulsion electrospinning. The adsorption and degradation of pentachlorophenol (PCP) by HRP-EFMs are investigated. The experimental results show that the sorption kinetic of PCP on EFMs follows the pseudo-second-order model, and the sorption capacity is as high as 44.69 mg g(-1). The sorption mechanisms of EFMs for PCP can be explained by hydrogen bonding interactions, hydrophobic interactions and π-π bonding interactions. Profiting from the strong adsorption, the removal of PCP can be dramatically enhanced by the interaction of adsorbed PCP and HRP on the surface of EFMs. For PCP degradation, the optimal pH values for free HRP and immobilized HRP are 4 and 2-4, respectively. As pH>4.7, no adsorption and degradation are observed due to the deprotonation of PCP. The removal percentages reach 83% and 47% for immobilized HRP and free HRP, respectively, at 25 ± 1°C. The presence of humic acid can inhibit the activity of HRP and decreases the adsorption capacity of PCP because of competitive adsorption. The operational and storage stability of immobilized HRP are highly improved through emulsion electrospinning.