Photoswitching of peroxidase activity by position-specific incorporation of a photoisomerizable non-natural amino acid into horseradish peroxidase.

Horseradish peroxidase mutants containing L-p-phenylazophenylalanine (azoAla) at various positions were synthesized by using an Escherichia coli in vitro translation system. Among the 15 mutants examined, four mutants containing a single azoAla unit at the 6th, 68th, 142nd, and 179th positions, respectively, retained the peroxidase activity. The activity of the Phe68azoAla mutant was higher when the azobenzene group was in the cis form than in the trans form. On the contrary, the activity of the Phe179azoAla mutant disappeared when the azobenzene group was photoisomerized to the cis form, but recovered in the trans form. In the latter mutant, therefore, an on/off photoswitching of the peroxidase activity was attained.     

Catalytic properties and stability of horseradish peroxidase immobilized in polyacrylamide gel]

Effect of polyacrylamide (PAA) gel on properties of horseradish peroxidase, immobilized by means of the incorporation into PAA gel is studied. Catalytic properties of immobilized enzyme are studied. Km value and pH-dependency of the enzyme activity are found to be close to those of soluble enzyme, kcat value is 3 times lower at pH 7.0. PH-stability of immobilized peroxidase at 20 degrees C and thermostability of soluble and immobilized peroxidases at pH 7.0 within the temperature range from 20 to 81 degrees C are studied. The stability of peroxidase in PAA gel is found to decrease (in 3 times at 20 degrees C, and in 17 times at 56 degrees C). A mechanism of the effect of PAA gel on catalytic properties and stability of peroxidase is discussed.     

Use of fluorescence to monitor the incorporation of horseradish peroxidase into a sol-gel derived medium

The solvatochromic dye nile red has been employed to monitor the incorporation of an enzyme (horseradish peroxidase) into a sol-gel derived medium. The fluorescence spectrum of the dye, when incorporated into the enzyme, was analysed as the sum of Gaussian component spectra and relative changes between these component spectra were monitored upon encapsulation of the dye-enzyme system within the host matrix. Activity of the confined enzyme was verified and the effect of temperature was also investigated, through the examination of nile red fluorescence in the sol-gel derived matrix, where a stabilising effect was noted.     

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected.     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.     

Catalytic and immunochemical properties of ferritin conjugates with horseradish peroxidase

The human spleen ferritin--horseradish peroxidase conjugate (HRP--Fer) was synthesized by periodate oxidation of the enzyme carbohydrate fragment. The protein fraction containing 1-2 peroxidase molecules and characterized by kinetic homogeneity was obtained in the peroxidatic ortho-dianisidine (o-DA) oxidation reaction. Gel diffusion precipitation of HRP--Fer with peroxidases and ferritin antibodies was carried out. The precipitation confirms the retention by peroxidase and ferritin of their antigenic properties. The kinetics of peroxidatic oxidation of o-DA by the HRP--Fer conjugate was studied within the temperature interval of 15-37 degrees C. The value of catalytic constant for this reaction exceeds that for native peroxidase 1.75-fold. A kinetic analysis of thermal inactivation of peroxidase and its conjugate was performed within the temperature range of 40-65 degrees C. The effective rate constants of inactivation obtained from the first order equation are higher for HRP--Fer than for the native enzyme. The effect of pH on the rates of inactivation of HRP--Fer and the non-modified enzyme was studied at 50 degrees C. The enzyme and its conjugate were shown to stabilize in acid media. The HRP--Fer conjugate can be used as an effective tool in immunoenzymatic assays of ferritin.     

Chemiluminescent multiassay of pesticides with horseradish peroxidase as a label

Competitive chemiluminescent immunoassay based on a combination of five antibodies was used in a combination with neural network to identify and estimate amounts of three cross-reacting s-triazines (atrazine, terbythylazine and ametryn). Antibodies with different cross-reactivity towards s-triazines were immobilized in separate wells an eight-well microtiter strip. Training of neural networks was carried out with four different learning procedures. The best topology for the data measured was a net with two hidden layers with ten neurons in the first and 15 in the second layer trained with the Schmidhuber method. s-Triazine classification of environmental samples containing various analyte mixtures was correct in 70-100% of all cases depending on the type of analyte. The test developed can be proposed as an alternative field test for multianalyte environmental monitoring.     

Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.     

Interaction of propylthiouracil and its precursors with horseradish peroxidase

Interactions of horseradish peroxidase with propylthiouracil, thiouracil, propyluracil and uracil lead to the formation of complexes that exhibit different absorption spectra which can be attributed to the perturbation of peroxidase as the result of the drug-binding on a polar site in the protein. In this paper, by dilatometry and viscometry structural alterations in horseradish peroxidase were detected from its interaction with propylthiouracil and thiouracil only, and the physiological inhibition of peroxidase for these antithyroid drugs seems to be through structural alterations in the protein.     

Heme and pH-dependent stability of an anionic horseradish peroxidase

Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T(m) values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. However ejection of the heme group leads to an irreversible unfolding behavior at pH 4, while at pH 7 and 10 refolding is still observed. This is putatively correlated with the titration state of the heme pocket. Thermal transitions of HRPA1 showed scan rate dependence at the three pH values, showing that the denaturation process was kinetically controlled. The denaturation process was interpreted in terms of the classic scheme, N<-->U-->D and fitted to far-UV CD ellipticity. A good agreement was obtained between the experimental and theoretical T(m) values and percentages of irreversibility. However the equilibrium between N and U is probably more complex than just a two-state process as revealed by the multiple T(m) values.     

Nano reengineering of horseradish peroxidase with dendritic macromolecules for stability enhancement

A simple bio-conjugation procedure to surround a single horseradish peroxidase (HRP) enzyme molecule with dendritic polyester macromolecules (polyester-32-hydroxyl-1-carboxyl bis-MPA dendron, generation 5) was proposed. The characterization of resultant nanoparticles entitled HRP dendrozyme, was performed by transmission electron microscopy, dynamic light scattering, gel permeation chromatography and Fourier transform infrared spectroscopy. The results showed that HRP nanoparticles were spherical in shape and have an average size of 14 ± 2 nm in diameter. Furthermore, bio-conformational characterization of HRP dendrozyme was performed by means of circular dichroism and fluorescence spectroscopy to evaluate the secondary and tertiary structure changes after enzyme modification. These investigations revealed that protein conformation had small changes (in secondary and tertiary structures) after bio-conjugation. We also reported here that dendritic modification did not significantly affect the kinetic parameters of free HRP. The stabilization of HRP with dendron macromolecules as single enzyme nanoparticles resulted in improvement of half-life over 70 days storage at 4 °C as well as its tolerance under different elevated temperatures up to 80 °C and in the presence of organic solvents for 15 min. These significant results promise extensive applications of HRP particularly in harsh environmental conditions.     

Activity, stability, and unfolding of reconstituted horseradish peroxidase with modified heme

Heme-propionates of horseradish peroxidase (HRP) were esterified by p-nitrophenol, phenol and p-methylphenol to change its electron character and to increase its hydrophobicity. These synthetic hemes were inserted apo-HRP to give a novel HRP, respectively. Of the three reconstituted HRPs, reconstituted HRP with p-nitrophenol-modified heme derivative had a larger initial rate, affinity, catalytic efficiency and substrate-binding efficiency than native HRP in aqueous buffer and some solvents. The reconstituted HRPs showed higher thermostability and tolerance of DMF because of the increase of the hydrophobicity of the active site. Changing the electron character of the aromatic moieties linked at each terminal of the two heme-propionates can control activity and stability of HRP. The initial rate, affinity, catalytic efficiency and substrate-binding efficiency increased with the increases of electron-withdrawing efficiency of substituents at 4-position of the phenolic used to synthesize the heme derivatives, contrariwise, the stability decreased. The modifications resulted in the increase in the temperature (T_m) at the midpoint of thermal denaturation and the decreases in both enthalpy and entropy change at T_m. The changes of catalytic properties and stabilities are related to the changes of the conformation of HRP. The modification changed the environment of heme and tryptophan, increased α-helix content of HRP. The present work demonstrates that enhancement of the hydrophobicity and the electron-withdrawing efficiency of heme improves the activity and stability of HRP.     

Stimulation of the activity of horseradish peroxidase by nitrogenous compounds

A variety of nitrogenous compounds broaden the activity versus pH profile for the peroxidation of dianisidine catalyzed by horseradish peroxidase (HRP), but not by myeloperoxidase, chloroperoxidase, Escherichia coli hydroperoxidase I, methemoglobin, or microperoxidases. The peroxidation of dianisidine catalyzed by cytochrome c peroxidase was affected by the nitrogenous compounds, but to a lesser extent than was the action of HRP. The peroxidations of a variety of phenols by HRP exhibited broad activity versus pH profiles and were unaffected by the nitrogenous compounds. The energy of activation for the peroxidation of dianisidine by HRP was unaffected by changes of pH in the range 6.5-8.5 and was unchanged by the presence of the nitrogenous compounds. The nitrogenous compounds markedly increased Vm for the peroxidation of dianisidine by HRP, but did not change the slope of Lineweaver-Burk plots of kinetic data. These results are accommodated by a mechanism in which nitrogenous compounds hydrogen-bond to the distal histidine of HRP and in so doing raise its pK alpha. Since the acid form of the distal histidine is thought to facilitate peroxidations catalyzed by HRP by hydrogen bonding to the ferryl oxygen of compound II, raising its pK alpha broadens the activity versus pH profile for the peroxidation of anilino substrates, such as dianisidine. We propose that phenolic substrates hydrogen-bond directly to the ferryl oxygen, thus displacing the distal histidine and eliminating the possibility of being influenced by nitrogenous compounds.     

Cytochemical detection of mannose-specific receptors for glycoproteins with horseradish peroxidase as a ligand

Summary Horseradish peroxidase (HRP), a glycoprotein rich in mannose groups, was used as a ligand to detect receptors for glycoproteins in formalinfixed, frozen sections of rat liver. Specific binding of HRP occurred to surface membranes of sinusoidal cells but not to those of parenchymal cells. The binding sites were visualized after the peroxidatic reaction in erythrocytes had been suppressed by methanol-H₂O₂ and phenylhydrazine, the latter reagent also decreasing the nonspecific background adsorption of HRP. Several factors influencing the reaction were studied systematically. The specific binding of HRP to sinusoidal cells was greatly decreased or abolished when tissue blocks were fixed for longer than 1–2 h in a cold 4% formaldehyde solution and the frozen sections subsequently treated for 30 min in cold methanol. The specific binding of HRP increased when the concentration of HRP in the medium was increased from 10 g/ml to 40 g/ml, when the time of incubation with HRP was increased from 1 h to 4 h, or when the temperature of incubation with HRP was increased from 4°C to 22°C, or from 22°C to 37°C. The specific binding of HRP also increased when the pH of the incubation medium was increased from 7.0 to 10.0. Little or no specific binding of HRP was observed in the absence of added Ca⁺⁺. The binding of HRP was suppressed by 10 mM mannose or 0.004% mannan whereas the suppression of the binding reaction by galactose or galactan required 30–40 times higher concentrations.This work was supported by the Morris A. Kaplan Fund     

Catalytic activity of poly(acryloyl morpholine)-immobilized horseradish peroxidase in organic/aqueous solvent mixtures

The immobilization of horseradish peroxidase by covalent coupling within an expanded poly(acryloyl morpholine) gel network is described. The activity of the immobilized horseradish peroxidase was compared with that of the native enzyme in aqueous buffer and in buffered mixtures of dimethyl-formamide/water, ethanediol/water, methanol/water and tetrahydrofuran/water of varying solvent ratios at pH 6.1. On increasing the organic solvent concentration in the substrate solution, active immobilized enzyme retained its activity much better than an equivalent amount of the native enzyme. The oxidation of ferrocene (water-insoluble) and ferrocene derivatives to the corresponding ferricinium ions, was accomplished efficiently by the immobilized enzyme in buffered 50% methanol/water solution. The immobilized enzyme exhibited superior resistance to thermal denaturation.     

Serine incorporation into the selenocysteine moiety of glutathione peroxidase

The selenium in mammalian glutathione peroxidase is present as a selenocysteine ([Se]Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the [Se]Cys moiety, we perfused isolated rat liver with 14C- or 3H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the [Se]Cys in GSH peroxidase to carboxymethylselenocysteine ([Se]Cys(Cm)), and determined the amino acid specific activity. Perfusion with [14C]cystine resulted in [14C]cystine incorporation into GSH peroxidase without labeling [Se]Cys(Cm), indicating that cysteine is not a direct precursor for [Se]Cys. [14C]Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and [Se]Cys(Cm) in purified GSH peroxidase, whereas [3-3H]serine perfusion only labeled serine and [Se]Cys(Cm), thus demonstrating that the [Se]Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and [Se]Cys(Cm) strongly suggest that the precursor pool of serine used for [Se] Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.     

The interaction of benzhydroxamic acid with horseradish peroxidase and its fluorescent analogs

Horseradish peroxidase (HRP) reconstituted with protoporphyrin IX or zinc protoporphyrin IX binds benzhydroxamic acid with affinities of 1.54 × 10⁴ and 8 × 10²m^?1, respectively. This interaction is competitive with respect to hydrogen donor substrates of peroxidase. The steady-state oxidation of benzhydroxamic acid by HRP was studied by monitoring the disappearance of the hydroxamate function and the formation of nitrite. The inhibition by benzhydroxamic acid of oxidation of HRP substrates may be classified as an inhibition by a competing substrate. In the case of HRP-catalyzed oxidation of ferrocyanide a marked activating effect of benzhydroxamic acid was observed. Mechanisms responsible for this effect are discussed.