Structure Of Horseradish Peroxidase Research Articles

The effect of radio frequency heating on the inactivation and structure of horseradish peroxidase

The effects of radio frequency (RF) heating on horseradish peroxidase (HRP) activity and its structure were investigated in this paper. The HRP was heated to 50 °C, 70 °C and 90 °C at different electrode gaps (100, 110 and 120 mm). The relative enzyme activity was 105.33 %–113.73 % at 50 °C, 91.11 %–93.05 % at 70 °C and 47.05 %–68.17 % at 90 °C. Ultraviolet–visible, circular dichroism and fluorescence spectra were used to monitor the variation in secondary and tertiary structure. The results showed that RF heating at the electrode gaps of 120 mm contributed to more severe enzyme inactivation and conformational destruction, which can be explained by the changes in Soret band, secondary structure content and tryptophan fluorescence intensity. This study revealed that enzyme inactivation by RF heating was associated with loss of helical structure, unfolding of enzyme protein and ejection of heme group.

The Structure and Function of Horseradish Peroxidase (HRP) under the Influence of Faradarmani Consciousness Field

The study of biological enzymes with their unique properties, such as substrate interactions, conformational changes, and catalytic rates, is a key to basic and applied enzymology research. Among enzymes, horseradish peroxidase (HRP) has been widely used in studying the metabolism of biological systems. In this study, we measure changes in the activity and structure of horseradish peroxidase in both laboratory grade and isoforms extracted from the plant under the influence of Faradarmani Consciousness Field (FCF). Our objectives were first to test the effect of FCF on the structure and function of HRP and, secondly, to analyze whether or not changes in the maximum velocity and Michaelis constant were reproducible under the influence of FCF. We observed that the laboratory-grade HRP had a 5% decrease in maximum velocity and an 18% increase in Michaelis constant as a result of the FCF treatment when compared to the non-treated controls. On the other hand, the HRP extracted from the plant tissue showed a 6% and 40% increase in maximum velocity and Michaelis constant, respectively, under FCF. Additionally, changes in the fluorescence emission intensity spectrum suggest that both structural and functional changes in this enzyme result from the FCF treatment.

Biomimic Nanozymes with Tunable Peroxidase-like Activity Based on the Confinement Effect of Metal-Organic Frameworks (MOFs) for Biosensing.

Biomimic nanozymes coassembled by peptides or proteins and small active molecules provide an effective strategy to design attractive nanozymes. Although some promising nanozymes have been reported, rational regulation for higher catalytic activity of biomimic nanozymes remains challenging. Hence, we proposed a novel biomimic nanozyme by encapsulating the coassembly of hemin/bovine serum albumin (BSA) in zeolite imidazolate frameworks (ZIF-8) to achieve controllable tailoring of peroxidase-like activity via the confinement effect. The assembly of Hemin@BSA was inspired by the structure of horseradish peroxidase (HRP), in which hemin served as the active cofactor surrounded by BSA as a blocking pocket to construct a favorable hydrophobic space for substrate enrichment. Benefiting from the confinement effect, ZIF-8 with a porous intracavity was identified as the ideal outer layer for Hemin@BSA to accelerate substrate transport and achieve internal circulation of peroxidase-like catalysis, significantly enhancing its peroxidase-like activity. Especially, the precise encapsulation of Hemin@BSA in ZIF-8 could also prevent it from decomposition in harsh environments by rapid crystallization around Hemin@BSA to form a protective shell. Based on the improved peroxidase-like activity of Hemin@BSA@ZIF-8, several applications were successfully performed for the sensitive detection of small molecules including H2O2, glucose, and bisphenol A (BPA). Satisfactory results highlight that using a ZIF-8 outer layer to encapsulate Hemin@BSA offers a very effective and successful strategy to improve the peroxidase-like activity and the stability of biomimic nanozymes, broadening the potential application of biocatalytic metal-organic frameworks (MOFs).

Effect of high hydrostatic pressure on activity, thermal stability and structure of horseradish peroxidase

The mechanism of the high hydrostatic pressure (HHP) effect on horseradish peroxidase (HRP) is still unclear. The activity, thermal stability and structural changes of HRP after HHP treatments were studied in this work. Compared with the untreated sample, the enzyme activity reduces by 36% after 800 MPa processing. The results indicated that the conformation of the enzyme active center changes under pressure. Furthermore, HHP also changes the conformation of disulfide bonds and some secondary structures in HRP. These structural and conformational changes induce decreased activity. In addition, differential thermal scanning (DSC) results showed that the thermal denaturation temperature decreased from 103.74 °C to 85.78 °C after pressure treatment, suggesting HRP molecules formed large aggregates after pressure treatment. In this study, the interaction mechanism between pressure and enzyme was studied as well, and the results can provide some guidance for the application of HHP technology in fruit and vegetable products processing.

The modulation of structural stability of horseradish peroxidase as a consequence of macromolecular crowding.

Macromolecular crowding plays an inevitable role in all biological processes influencing association, conformation, and other characteristics of proteins. Present study is based on the effect of macromolecular crowding on structure of horseradish peroxidase (HRP) enzyme. Concentration-dependent conformational changes induced by crowding agents, dextran 70 and polyethylene glycol (PEG)-4000, were monitored employing a range of biophysical techniques. The intrinsic fluorescence spectra showed transition of protein from native to unfolded state. Marked increase in 8-Anilino-1-naphthalene-sulphonoic acid and Thioflavin T fluorescence indicated presence of non-native moieties with 80 mg/mL dextran. Enhanced absorbance in turbidity, Soret, and Congo red in corroboration with scattering intensity at 350nm results revealed incidence of HRP aggregates. A new peak around 218 nm in CD spectra pointed towards change in secondary structure towards β-sheets. Significant loss of enzyme activity upon structural disruption was seen. Comet assay demonstrated DNA damage and genotoxic nature of HRP aggregates, supporting spectroscopic, and fluorescence results. The normalized results were obtained with 120 mg/mL PEG-4000 close to that of native HRP implying no disruptive effect on structure. It can be hypothesized that macromolecular crowding is a vital element, which can have diverse effects. In this study, dextran 70 was observed to have pro-aggregatory effect while enhanced stability of native enzyme was witnessed with PEG. Hence, it can be stated that PEG has potentially better crowder as it helps retain the native enzyme structure. Routine addition of crowding agents is recommended if biological molecules are to be studied under more physiologically appropriate environments.

Time-resolved fluorescence observation of di-tyrosine formation in horseradish peroxidase upon ultrasound treatment leading to enzyme inactivation

The application of ultrasound to a solution can induce cavitional phenomena and generate high localised temperatures and pressures. These are dependent of the frequency used and have enabled ultrasound application in areas such as synthetic, green and food chemistry. High frequency (100kHz to 1MHz) in particular is promising in food chemistry as a means to inactivate enzymes, replacing the need to use periods of high temperature. A plant enzyme, horseradish peroxidase, was studied using time-resolved fluorescence techniques as a means to assess the effect of high frequency (378kHz and 583kHz) ultrasound treatment at equivalent acoustic powers. This uncovered the fluorescence emission from a newly formed species, attributed to the formation of di-tyrosine within the horseradish peroxidase structure caused by auto-oxidation, and linked to enzyme inactivation.

Insights into the impact of deep eutectic solvents on horseradish peroxidase: Activity, stability and structure

Abstract As a new type of ionic fluids that are inexpensive and environmentally friendly, deep eutectic solvents (DESs) have recently attracted a broad interest in many fields including biocatalysis. In this study, 24 DESs were prepared by mixing two cholinium salts (cholinium chloride (ChCl) and cholinium acetate (ChAc)) with four H-bond donors (HBDs) (urea, glycerol, acetamide, ethylene glycol) at three molar ratios, and their effects on the activity, stability and structure of horseradish peroxidase (HRP) have been investigated. The ChCl-based DESs have been found to be superior to the ChAc-based ones in terms of promoting the HRP activity. For the DESs composed of the same salt and same HBD, an increase in the HRP activity has been observed with a higher salt/HBD molar ratio of 1:2

THE EFFECT OF PULSED MAGNETIC FIELD ON HORSERADISH PEROXIDASE

ABSTRACTThe effect of pulsed magnetic field (PMF) on the activity and structure of horseradish peroxidase (HRP) in buffer solution was investigated. HRP was inactivated when exposed to PMF at field strengths of 2.0, 3.0 and 4.0 tesla (T) for 5 to 40 pulses. The maximum inactivation of HRP was achieved at 4.0 T for 40 pulses, in which 67.67% of the enzyme activity was lost. Further experiments showed that the activity of HRP was not significantly affected by either storage time at 4C or the heat produced during lower intensity PMF (from 1.0 to 3.0 T). The inactivation data were adjusted to the Bigelow, Hülsheger and Weibull models. Results showed that the Weibull model successfully described the relationship between residual activity of HRP and pulse strength as well as pulse number. Ultraviolet and intrinsic fluorescence spectra analysis proved that the PMF‐induced inactivation correlated with alteration of the tertiary structure of HRP.PRACTICAL APPLICATIONSAs a kind of enzyme present in many foods, when peroxidase (POD) is exposed to air, it will catalyze some reactions which have a negative effect on sensorial and nutritional qualities of the food. In this article, pulsed magnetic field was proved to be a potential processing method to inactivate POD without destroying the sensorial and nutritional qualities. The kinetic models also offer some parameters for designing methods to prolong shelf life of agricultural products.

Effect of noble metal coated on magnetic nanoparticles on horseradish peroxidase structure

Effect of noble metal coated on magnetic nanoparticles on horseradish peroxidase structure

Effects of compressed fluids on the activity and structure of horseradish peroxidase

This work investigated the stability of commercial horseradish peroxidase in compressed carbon dioxide and propane. Effects of temperature, exposure time and pressure on the enzyme activity were evaluated. The results obtained indicated that the HRP treatment in CO 2 promotes a significant decrease in the enzyme activity, whereas HRP showed good stability in propane, indicating that this enzyme is more stable in propane than in CO 2. Far UV-circular dichroism (CD) analyses of HRP in solution indicated that treatment of the enzyme with propane did not induce changes in the secondary structure content of HRP. On the other hand, incubation with CO 2 led to significant loss of HRP secondary structure. The solid HRP did not present decrease of its activity after treatment with pressurized CO 2 or propane. The CD analysis of solid HRP treated with propane and CO 2 revealed that the enzyme once more present higher stability in propane, but the treatment in both solvents provoked loss in secondary structure.

The structure of horseradish peroxidase C characterized as a molten globule state after Ca 2+ depletion

The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca 2+ ions. Earlier studies suggested a critical role of one of the bound Ca 2+ ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca 2+-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca 2+-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca 2+, perturbs a H-bonding network connecting this Ca 2+ to the heme crevice that involves the distal water. The measured effects of Ca 2 + depletion can be interpreted as supporting a structural role for the distal Ca 2+ and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.

Structure of Poly(Ethylene Glycol)-Modified Horseradish Peroxidase in Organic Solvents: Infrared Amide I Spectral Changes upon Protein Dehydration Are Largely Caused by Protein Structural Changes and Not by Water Removal Per Se

Fourier transform infrared (FTIR) spectroscopy has emerged as a powerful tool to guide the development of stable lyophilized protein formulations by providing information on the structure of proteins in amorphous solids. The underlying assumption is that IR spectral changes in the amide I and III region upon protein dehydration are caused by protein structural changes. However, it has been claimed that amide I IR spectral changes could be the result of water removal per se. Here, we investigated whether such claims hold true. The structure of horseradish peroxidase (HRP) and poly(ethylene glycol)-modified HRP (HRP-PEG) has been investigated under various conditions (in aqueous solution, the amorphous dehydrated state, and dissolved/suspended in toluene and benzene) by UV-visible (UV-Vis), FTIR, and resonance Raman spectroscopy. The resonance Raman and UV-Vis spectra of dehydrated HRP-PEG dissolved in neat toluene or benzene were very similar to that of HRP in aqueous buffer, and thus the heme environment (heme iron spin, coordination, and redox state) was essentially the same under both conditions. Therefore, the three-dimensional structure of HRP-PEG dissolved in benzene and toluene was similar to that in aqueous solution. The amide I IR spectra of HRP-PEG in aqueous buffer and of dehydrated HRP-PEG dissolved in neat benzene and toluene were also very similar, and the secondary structure compositions (percentages of α-helices and β-sheets) were within the standard error the same. These results are irreconcilable with recent claims that water removal per se could cause substantial amide I IR spectral changes (M. van de Weert, P.I. Haris, W.E. Hennink, and D.J. Crommelin. 2001. Anal. Biochem. 297:160–169). On the contrary, amide I IR spectral changes upon protein dehydration are caused by perturbations in the secondary structure.

Spectroscopic study on structure of horseradish peroxidase in water and dimethyl sulfoxide mixture.

The structure of horseradish peroxidase (HRP) in phosphate buffered saline (PBS)/dimethyl sulfoxide (DMSO) mixed solvents at different compositions is investigated by IR, electronic absorption, and fluorescence spectroscopies. The fluorescence spectra and the amide I spectra of ferric HRP [HRP(Fe3+)] show that overall structural changes are relatively small up to 60% DMSO. Although the amide I band of HRP(Fe3+) shows a gradual change in the secondary structure and a decrease in the contents of a helices, its fluorescence spectra indicate that the distance between the heme and Trp173 is almost constant. In contrast, the changes in the positions of the Soret bands for resting HRP(Fe3+) and catalytic intermediates (compounds I and II) and the IR spectra at the C-O stretching vibration mode of carbonyl ferrous HRP [HRP(Fe2+)-CO] show that the microenvironment in the distal heme pocket is altered, even with low DMSO contents. The large reduction of the catalytic activity of HRP even at low DMSO contents can be attributed to the structural transition in the distal heme pocket. In PBS/DMSO mixtures containing more than 70 vol % DMSO, HRP undergoes large structural changes, including a large loss of the secondary structure and a dissociation of the heme from the apoprotein. The presence of the components of the amide I band that can be assigned to strongly hydrogen bonding amide C=O groups at 1616 and 1684 cm(-1) suggests that the denatured HRP may aggregate through strong hydrogen bonds.

Spectroscopic study on structure of horseradish peroxidase in water and dimethyl sulfoxide mixture

Spectroscopic study on structure of horseradish peroxidase in water and dimethyl sulfoxide mixture

Stabilization of diluted aqueous solutions of horseradish peroxidase

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55 degrees C and infinite dilution, HRP was inactivated with a rate constant of 2.86 x 10(-3) s-1. CaCl2, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the "glycerol-based" one containing 10% ethanol and 20% glycerol, or the "protein-based" one containing 0.1% Kathon CG and 0.2 g/l of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl2 were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.

How do organic solvents affect peroxidase structure and function?

The effect of organic solvents on horseradish peroxidase structure and function has been studied. Some, but not complete, enzyme denaturation occurs even in low volumes of water-miscible organic solvents (e.g., greater than 30% v/v dioxane, greater than 50% v/v methanol, and greater than 20% v/v acetonitrile) as determined by the decreased difference between the fluorescence of peroxidase's sole tryptophan residue and free L-tryptophan in solution. Absorbance and electron paramagnetic resonance spectroscopies indicate exposure of peroxidase's active site to the organic solvent. This reduces the local polarity in the enzyme's active site and results in stronger hydrogen bonding of phenolic substrates to the enzyme. In extreme cases (e.g., 95% v/v dioxane, 90% v/v acetonitrile, and ethyl and butyl acetate containing 2 and 1% v/v aqueous buffer, respectively), the transition state of the enzymic reaction is sufficiently perturbed so as to alter the magnitude of the Hammett rho value. This is most likely the result of the increased strength of hydrogen bonding between electron-donating alkoxyphenols (negative sigma values) and an electrophilic group in the enzyme's active site, thereby reducing catalytic efficiencies for such substrates relative to alkyl- and chlorophenols. Perhaps the most important effect of the organic solvent, however, is the significant ground-state stabilization of phenolic substrates in organic media as opposed to aqueous buffer. This stabilization can account for nearly 4 orders of magnitude in reduction of catalytic efficiency and is manifested in increased Km's. This study indicates that enzymes can maintain much of their native active-site structure in organic media and that the effect of solvent on substrate thermodynamics must be considered.

Investigations on the multiple components of commercial horseradish peroxidase

Investigations on the structure of horseradish peroxidase (donor: H 2O 2 oxidoreductase, EC 1.11.1.7), including its amino acid and carbohydrate composition, are described. Commercial horseradish peroxidase preparations contain multiple components which can be separated by electrophoresis on starch. The major components have been purified and their properties compared; they do not differ significantly in size, absorption spectrum, enzymatic activity, or amino acid composition. Horseradish peroxidase contains the following sugars: glucose, galactose, mannose, arabinose, xylose, fucose and hexosamine.

Investigations on the multiple components of commercial horseradish peroxidase

Investigations on the structure of horseradish peroxidase (donor: H 2 O 2 oxidoreductase, EC 1.11.1.7), including its amino acid and carbohydrate composition, are described. Commercial horseradish peroxidase preparations contain multiple components which can be separated by electrophoresis on starch. The major components have been purified and their properties compared; they do not differ significantly in size, absorption spectrum, enzymatic activity, or amino acid composition. Horseradish peroxidase contains the following sugars: glucose, galactose, mannose, arabinose, xylose, fucose and hexosamine.