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1.
The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5-25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25-40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The alpha-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.  相似文献   

2.
The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.  相似文献   

3.
The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.  相似文献   

4.
Z Karim  R Adnan  MS Ansari 《PloS one》2012,7(7):e41422
Chemical synthesis of Ag-NPs was carried out using reduction method. The reduction mechanistic approach of silver ions was found to be a basic clue for the formation of the Ag-NPs. The nanoparticles were characterized by UV-vis, FT-IR and TEM analysis. We had designed some experiments in support of our hypothesis, "low concentrations of novel nanoparticles (silver and gold) increases the activity of plant peroxidases and alter their structure also", we had used Ag-NPs and HRP as models. The immobilization/interaction experiment had demonstrated the specific concentration range of the Ag-NPs and within this range, an increase in HRP activity was reported. At 0.08 mM concentration of Ag-NPs, 50% increase in the activity yield was found. The U.V-vis spectra had demonstrated the increase in the absorbance of HRP within the reported concentration range (0.06-0.12 mM). Above and below this concentration range there was a decrease in the activity of HRP. The results that we had found from the fluorescence spectra were also in favor of our hypothesis. There was a maximum increase in ellipticity and α-helix contents in the presence of 0.08 mM concentration of Ag-NPs, demonstrated by circular dichroism (CD) spectra. Finally, incubation of a plant peroxidase, HRP with Ag-NPs, within the reported concentration range not only enhances the activity but also alter the structure.  相似文献   

5.
Fluorescence, circular dichroism (CD), and UV-visible spectroscopic studies on horseradish peroxidase (HRP) and its calcium depleted derivative (CaD-HRP) are reported. CaD-HRP with its emission maximum at 338 nm is found to be six times as fluorescent as native HRP. The red shift relative to HRP emission observed at 328 nm indicates conformation change around trp towards more hydrophilic environment. CD spectrum of HRP in the 250–700 nm range shows that CD bands of HRP in the Soret region (403 nm) and in the aromatic region (280 nm) also undergo red shift on removal of endogenous calcium, indicating a change in conformation in the vicinity of both heme as well as aromatic residues. Comparison of CD of low spin HRP cyanide in the same region shows that CaD-HRP has some intermediate-spin character. CaD-HRP reconstituted with Tb3+ ion showed recovery of the enzyme activity by 89% of the original. Fluorescence of trp sensitized Tb3+ ion bound to apo-CaD-HRP was observed in the 450–700 nm wavelength region. trp-heme distance in CaD-HRP, calculated using the Förster theory of resonance energy transfer, was found to be 29.5 Å as opposed to 20.1 Å in HRP. The trp-Tb3+ distance was similarly estimated to be 7.1 Å.  相似文献   

6.
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/30 W 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.  相似文献   

7.
Conformation change of horseradish peroxidase in lipid membrane   总被引:1,自引:0,他引:1  
The electrochemical behavior of horseradish peroxidase (HRP) in the dimyristoyl phosphatidylcholine (DMPC) bilayer on the glassy carbon (GC) electrode was studied by cyclic voltammetry. The direct electron transfer of HRP was observed in the DMPC bilayer. Only a small cathodic peak was observed for HRP on the bare GC electrode. The electron transfer of HRP in the DMPC membrane is facilitated by DMPC membrane. UV–Vis and circular dichroism (CD) spectroscopy were used to study the interaction between HRP and DMPC membrane. On binding to the DMPC membrane the secondary structure of HRP remains unchanged while there is a substantial change in the conformation of the heme active site. Tapping mode atomic force microscopy (AFM) was first applied for the investigation on the structure of HRP adsorbed on supported phospholipid bilayer on the mica and on the bare mica. HRP molecules adsorb and aggregate on the mica without DMPC bilayer. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed in the DMPC bilayer. The adsorption of HRP in the DMPC bilayer changes drastically the domains and defects in the DMPC bilayer due to a strong interaction between HRP and DMPC films.  相似文献   

8.
Guo S  Zhou Q  Lu T  Ding X  Huang X 《化学与生物多样性》2008,5(10):2050-2059
The inhibition mechanism of Tb(III) on horseradish peroxidase (HRP) in vitro was discussed. The results from MALDI-TOF/MS and X-ray photoelectron spectroscopy (XPS) showed that Tb(III) mainly interacts with the O-containing groups of the amides in the polypeptide chains of the HRP molecules and forms the complex of Tb(III)-HRP, and, in the complex, the molar ratio Tb(III)/HRP is 2 : 1. The results from CD and atomic force microscopy (AFM) indicated that the coordination effect between Tb(III) and HRP can lead to the conformation change in the HRP molecule, in which the contents of alpha-helix and beta-sheet conformation in the peptide of the HRP molecules is decreased, and the content of the random coil conformation is increased. Meanwhile, the coordination effect also leads to the decrease in the content of inter- and intrapeptide-chain H-bonds in the HRP molecules, resulting in the HRP molecular looseness and/or aggregation. Thus, the conformation change in the HRP molecules can significantly decrease the electrochemical reaction of HRP and its electrocatalytic activity for the reduction of H2O2.  相似文献   

9.
Horseradish peroxidase (HRP, 10 mg/100 g body weight) was intravenously injected into rats in order to investigate the nature of the compartments involved in the transcellular transport of the protein through hepatocytes into bile. Double cytochemistry for HRP and the marker enzymes for cytoplasmic organelles was used. HRP was shown to be taken up by hepatocytes via vesicles at the sinusoidal surface, some of which were positive for 5'-nucleotidase activity. HRP was then found in the smooth-surfaced vesicles and tubules which were negative in 5'-nucleotidase, glucose 6-phosphatase, thiamine pyrophosphatase and acid phosphatase activity, suggesting that the tubular structures are neither the endoplasmic reticulum, the Golgi apparatus nor lysosomes. Biochemical studies revealed that the lead procedures used for the double cytochemistry did not inhibit the peroxidatic activity of HRP, and conversely that HRP did not interfere with the marker enzyme activity. Such cytochemical observations seemed to be supported by the observation that administration of monensin (3.5 mg/100 g) and chloroquine (5 mg/100 g), which markedly altered the structure of the Golgi apparatus and lysosomes, respectively, slightly altered the biliary excretion of HRP but not to a significant extent.  相似文献   

10.
Intercalation of horseradish peroxidase (HRP) into layered titanate by assembling it with titanate nano-sheets (TNS) was firstly used for fabrication of enzyme electrode (HRP-TNS electrode). XRD result revealed that HRP-TNS film featured layered structure with HRP monolayer intercalated between the titanate layers. UV-vis spectra result indicated the intercalated HRP in TNS film well retained its native structure. The HRP-TNS film was uniform with porous structures which were confirmed by SEM. The immobilized HRP in the TNS film exhibited fast direct electron transfer and showed a good electrocatalytic performance to H2O2 with high sensitivity, wide linear range and low detection. The excellent electrochemical performance of the HRP-TNS electrode was attributed to biocompatibility of the titanate sheets, porous architectures of the HRP-TNS film which retained activity of HRP to large extent, avoided aggregation of HRP, provided better mass transport and allowed more HRP loading per unit area. Thus, the simple method described here provides a novel and effective platform for immobilization of enzyme in realizing direct electrochemistry and has a promising application in fabrication of the third-generation electrochemical biosensors.  相似文献   

11.
The effect of cobalt ions (Co2+) on horseradish peroxidase (HRP) was studied in vitro by enzymatic activity assay, electronic absorption spectra, intrinsic fluorescence spectra and 8-anilo-1-naphthalenesulfonate(ANS)-binding fluorescence spectra. Co2+ at concentrations below 0.1 mM mildly increased the HRP activity, whereas higher concentrations of Co2+ significantly inactivated HRP in a time and concentration-dependent manner. Steady-state kinetic studies show that Co2+ was a noncompetitive inhibitor of o-dianisidine oxidation by HRP. The Ki value dropped as the incubation time increased. Furthermore, Co2+ was found to be an uncompetitive inhibitor of H2O2. These results suggested that Co2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectroscopic analysis showed that even for high Co2+ concentrations, the structure of HRP as a whole only changed slightly; however, there were significant conformational changes near or in the active site of HRP. Based on the above results, we suggest that Co2+ may bind with some amino acids near or in the active site of HRP and the conformational changes of HRP induced by such binding should be the main reason for activation and inactivation effect of Co2+. The potential binding sites of Co2+ were also proposed.  相似文献   

12.
Twenty hydroxylated and acetoxylated 3-phenylcoumarins were synthesized, and the structure-activity relationships were investigated by evaluating the ability of these compounds to modulate horseradish peroxidase (HRP) catalytic activity and comparing the results to four flavonoids (quercetin, myricetin, kaempferol and galangin), previously reported as HRP inhibitors. It was observed that 3-phenylcoumarins bearing a catechol group were as active as quercetin and myricetin, which also show this substituent in the B-ring. The presence of 6,2'-dihydroxy group or 6,7,3',4'-tetraacetoxy group in the 3-phenylcoumarin structure also contributed to a significant inhibitory effect on the HRP activity. The catechol-containing 3-phenylcoumarin derivatives also showed free radical scavenger activity. Molecular modeling studies by docking suggested that interactions between the heme group in the HRP active site and the catechol group linked to the flavonoid B-ring or to the 3-phenyl coumarin ring are important to inhibit enzyme catalytic activity.  相似文献   

13.
Signal amplification in flow cytometry using biotin tyramine.   总被引:2,自引:0,他引:2  
BACKGROUND: Catalysed reporter deposition (CARD) has been successfully used as a means of signal amplification in solid-phase immunoassays. The procedure relies on the use of horseradish peroxidase (HRP)-conjugated reagents--normally antibodies-in conjunction with substituted phenolic compounds such as biotin tyramine. The HRP catalyses deposition of biotin tyramine around the site of enzyme activity, and streptavidin-HRP can then be added to generate an amplified HRP signal. The possibility of using this technique for solution-phase amplifications has been suggested but not yet demonstrated. METHODS: This paper describes the application of CARD to signal enhancement in flow cytometry. The specific examples described here are those of anti-human CD4 and anti-human CD36 antibodies binding to either human lymphocytes or mixed mononuclear cells. RESULTS: Optimum biotin tyramine concentrations were evaluated, and a fivefold increase in signal was observed over standard detection of the anti-human CD4 antibody with anti-mouse-fluorescein isothiocyanate (FITC). In the example using the anti-CD36 antibody, the biotin tyramine treatment was repeated, resulting in an additional 2.5-fold signal amplification. CONCLUSIONS: The technique described in this report provides a method of amplifying the signals achieved by standard flow cytometry detection reagents.  相似文献   

14.
Maeda Y  Fujihara M  Ikeda I 《Biopolymers》2002,67(2):107-112
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.  相似文献   

15.
In the present study, Peroxidase from date palm (Phoenix dactylifera) leaves was purified to homogeneity by three-step procedure including aqueous two-phase system, hydrophobic and Ion-exchange chromatography. The enzyme migrated as single band on SDS-PAGE giving molecular weight of 68?±?3?kDa. The purification factor for purified date palm peroxidase was 68 with high 41% yield. Enzymatic assays together with far-UV circular dichroism (CD), intrinsic and extrinsic fluorescence studies were carried out to monitor the structural stability of date palm and horseradish peroxidase (HRP) against various pH and temperatures. Activity measurements illustrated different pH stability for date palm and HRP. Both peroxidases are more susceptible to extreme acidic conditions as suggested by 4 & 15?nm red shift in date palm and HRP, respectively. Secondary structure analysis using far UV-CD exhibited predominance of α-helical (43.8%) structure. Also, pH induces loss in the secondary structure of date palm peroxidase. Thermal stability analysis revealed date palm peroxidase is more stable in comparison to HRP. In summary, date palm peroxidases could be promising enzymes for various applications where extreme pH and temperature is required.  相似文献   

16.
To recognize and control ligand-receptor interactions at the interface between cells and polymer materials, we investigated a model system with an enzyme and a substrate conjugated with a biocompatible phospholipid polymer in an aqueous medium. We explored the regulation of enzyme-substrate (ES) complexation using horseradish peroxidase (HRP) as the enzyme and 4-aminoantipyrine (AAP) and 3-(p-hydroxyphenyl) propionic acid (HPPA) as substrates. The phospholipid polymer (PMBN), composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl poly(oxyethylene)methacrylate, was prepared and conjugated with AAP (PMBN-AAP conjugate). The formation and dissociation of the ES complex were investigated using capillary electrophoresis and fluorescence spectroscopy. In the chart of the capillary electrophoresis, a much longer retention time of HRP was observed in the PMBN-AAP conjugate-coated capillary compared with that in a nontreated capillary. The retention time was significantly longer in comparison with the case of a mixed solution of HRP and AAP. This result clearly shows that HRP forms an ES complex with the immobilized PMBN-AAP conjugate and that the addition of AAP to the medium inhibits the interactions between HRP and the PMBN-AAP conjugate. Though HRP forms an ES complex with both AAP and the PMBN-AAP conjugate, the ES complex with the PMBN-AAP conjugate was easily dissociated by addition of HPPA as an alternative substrate because HRP started to react with the HPPA immediately. However, the HRP that formed an ES complex with AAP fell behind in reacting with the HPPA. The activity of HRP was maintained at the initial level in the presence of the PMBN-AAP conjugate at 25 degrees C for 1 week. Additionally, even under H(2)O(2) conditions, HRP stored with the PMBN-AAP conjugate maintained 40% of the initial activity whereas HRP was deactivated within 6 h. This result indicates that the PMBN-AAP conjugate could block the active sites by formation of an ES complex. This is due to the formation of the ES complex, which retained the structure of HRP by blocking the active sites. On the basis of these results, we considered that the reversible attachment and detachment by PMBN conjugated with specific ligands from cellular receptors will be realized.  相似文献   

17.
Encapsulation of horseradish peroxidase (HRP) inside a peptide nanotube (PNT) was demonstrated and its activity was measured. Enzyme assay verified that 0.16 μg of the enzymes were encapsulated in 1mg of PNTs. The encapsulation was also verified with TEM, UV-vis spectroscopy, and FTIR. The activity of the encapsulated HRP was examined for thermal stability, long-term storage stability, and resistance to a denaturant. They showed good storage stability, retaining its activity up to 90%, while the free HRP lost 50% of its activity over the course of 18 days. At 55 °C, the encapsulated HRP activity remained 20% higher than that of the free HRP. With the denaturant, guanidinium hydrochloride (GdmHCl), the encapsulated HRP activity was maintained around 10% higher than the free HRP. This result proves that the encapsulation of HRP inside the PNT may be an effective way to keep the enzyme activity stable in various environments.  相似文献   

18.
One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (Armoracia rusticana) treated with Tb(III) was investigated using some biophysical and biochemical methods. Firstly, it was found that a large amount of Tb(III) can be distributed on the cell wall, that some Tb(III) can enter into the horseradish cell, indicating that peroxidase was mainly distributed on cell wall, and thus that Tb(III) would interact with horseradish peroxidase (HRP) in the plant. In addition, peroxidase bioactivity was decreased in the presence of Tb(III). Secondly, a new peroxidase-containing Tb(III) complex (Tb–HRP) was obtained from horseradish after treatment with Tb(III); the molecular mass of Tb–HRP is near 44 kDa and the pI is about 8.80. Thirdly, the electrocatalytic activity of Tb–HRP is much lower than that of HRP obtained from horseradish without treatment with Tb(III). The decrease in the activity of Tb–HRP is due to the destruction (unfolding) of the conformation in Tb–HRP. The planarity of the heme active center in the Tb–HRP molecule was increased and the extent of exposure of Fe(III) in heme was decreased, leading to inhibition of the electron transfer. The microstructure change in Tb–HRP might be the result of the inhibition effect of Tb(III) on peroxidase activity in horseradish.  相似文献   

19.
Resonance Raman spectroscopy was used to interrogate the heme active site of horseradish peroxidase (HRP) lyophilized in the presence and absence of the lyoprotectant poly(ethylene glycol) (PEG; FW 5000; 0-80% w/w) suspended in acetone, chloroform, or acetonitrile. In aqueous solution, Fe(3+)HRP is characterized by a five-coordinate high-spin (5-c HS) heme system. The structure of the heme-active site of HRP in all solvents is perturbed by co-lyophilization of HRP with PEG. Heme active site structural changes are consistent with coordination of water in the distal axial coordination site of the ferric heme iron and disruption of the hydrogen-bond network when the protein is lyophilized in the presence of PEG (>or=60% w/w) in all of the solvent systems studied. Similar active site structural changes were previously observed for HRP in benzene and attributed to a change in the reaction mechanism for HRP in benzene. (Mabrouk, P. A.; Spiro, T. G. J. Am. Chem. Soc. 1998, 120, 10303-10309.) Thus, PEG is proposed to increase the catalytic activity of HRP in nonaqueous media by locking the heme active site into a structure that functions through an alternative catalytic pathway in nonaqueous media.  相似文献   

20.
The adsorption behavior of horseradish peroxidase (HRP) onto hybrid particles of poly(methylmethacrylate) (PMMA) and carboxymethylcellulose (CMC) was investigated by means of spectrophotometry. Dispersions of PMMA/CMC particles were characterized by light scattering, zeta potential measurements and scanning electron microscopy before and after HRP adsorption. HRP adsorbed irreversibly onto PMMA/CMC particles; the adsorption isotherm showed an initial step and an adsorption plateau. The enzymatic activity of free HRP and immobilized HRP (plateau region) was monitored by means of spectrophotometry as a function of storing time. Upon adsorbing HRP there is little (up to 20%) or no reduction of enzymatic activity in comparison to that observed for free HRP in solution. After storing free HRP and HRP-covered PMMA/CMC particles for 18 days the level of enzymatic activity is kept. HRP-covered PMMA/CMC particles dispersions, which were dried and re-dispersed, retained 50% of their catalytic properties. These interesting findings were discussed in the light of a beneficial effect of a hydrated microenvironment for maintenance of enzyme conformation and activity.  相似文献   

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