Horseradish Peroxidase Preparations Research Articles

Preparation of Horseradish Peroxidase Modified Gold Nanoparticle/Coiled Carbon Nanotube Nanocomposite and its application for bromate and nitrite determination

Preparation of Horseradish Peroxidase Modified Gold Nanoparticle/Coiled Carbon Nanotube Nanocomposite and its application for bromate and nitrite determination

Co-Enzymes based nanoflowers incorporated-magnetic carbon nanotubes: A new generation nanocatalyst for superior removal of cationic and anionic dyes with great repeated use

Synthetic dyes leading to substantial discharge in various industrial areas such as textile, plastics, food, and cosmetics, have growingly threatened all livings. Although enzymes have been used for dye degradation, weak stability against changes in reaction environment, lack of reusability and high cost have strictly limited their use. Herein, we have developed co-enzymes nanoflowers incorporated-magnetic carbon nanotube nanocomposite as a novel and efficient nanocatalyst for superior degradation of malachite green (MG) and acid orange 7 (AO7) as model cationic and anionic dyes with excellent cyclic use. The HRP-Lac NF@mCNT nanocomposite (NC) containing horseradish peroxidase–laccase nanoflower (HRP-Lac NF) and iron oxide nanoparticles (Fe 3 O 4 NPs) decorated magnetic carbon nanotube (mCNT) was systematically employed in dye degradation as functions of pH, reaction time, dye type and reusability. This HRP-Lac NF@mCNT nanocomposite acted as one malfunctional nanoplatform since both HRP and Lac enzymes were used for rapid and efficient dye removal, Fe 3 O 4 NPs provided cyclic use and omitting the centrifugation step, and CNT functioned as a unique platform for NFs and Fe 3 O 4 NPs deposition. We demonstrated that HRP-Lac NF@mCNT NC induced ∼ 90 % MG and ∼ 85% AO7 decolorization in 20 min at pH 7.4 while it almost completely decolorized MG and AO7 in 60 min. In addition, ∼ 95% MG and ∼ 85% AO7 decolorization were accomplished even after 16 cycling use of HRP-Lac NF@mCNT NC. We claim that HRP-Lac NF@mCNT NC induced dye decolorization with dual mechanisms, enzymatic degradation, and physical adsorption. • Preparation of horseradish peroxidase–laccase co-enzymes nanoflower incorporated-magnetic carbon nanotubes. • Rapid and efficient dye degradation against a cationic and an anionic dye. • Magnetic property for repeated use.

Utilization of immobilized horseradish peroxidase for facilitated detoxification of a benzidine based azo dye

Horseradish peroxidase (HRP) was immobilized on the poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) [p(HEMA-GMA)] cryogel and used for the degradation of a benzidine containing azo dye (i.e., Direct Blue-6). The maximum amount of HRP loading was found to be 87.6 mg g−1 cryogel, and the retained immobilized HRP activity was 67% with respect to the same quantity of the free enzyme. Biochemical properties of the HRP preparations were also evaluated along with the stability studied under various denaturizing conditions. The toxicity of the Direct Blue-6 and its degradation products was tested using Daphnia magna. Decrease in the absorbance of the blue color of dye at 590 nm was observed during enzymatic degradation studies. Additionally, Chlorella vulgaris was used as a test organism in the algal growth inhibition studies. After 60 min reaction time, the Direct Blue-6 dye and its degradation product were enzymatically removed from the medium. Thus, after treatment with HRP approximately 60-min, the medium was non-toxic for both D. magna and C. vulgaris. The Direct Blue-6 dye and its degradation product toxicities were reduced after enzymatic treatment from 99.6 to 7.3%. It should be noted that the initial degradation products appeared to be extra toxic compared to the pristine dye.

Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris

Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible PAOX1 promoter, the strong constitutive PGAP promoter, and a novel bidirectional promoter PHTX1. Ultimately, coproduction of HRP and active Hac1 under PHTX1 control yielded a recombinant HRP titer of 132mg/L after 56h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme.

Recombinant production of a peroxidase-protein G fusion protein in Pichia pastoris

Streptococcal protein G (SpG) binds immunoglobulin G from a broad range of mammalian species with high affinity. Chemical conjugations of SpG to the reporter enzyme horseradish peroxidase (HRP) are commonly used in immunohistochemical applications. However, commercial HRP preparations are typically isolated from horseradish roots as varying mixtures of HRP isoenzymes with different biochemical properties, and chemical conjugation procedures lead to heterogeneous HRP-SpG preparations, partially including inactivated enzyme. A recombinant process allows the production of a well-defined HRP isoenzyme fused to SpG at constant 1:1 stoichiometry in a single step without the need for laborious chemical conjugation. By using state-of-the-art biotechnological tools, we produced a recombinant HRP-SpG fusion protein in Pichia pastoris in bioreactor cultivations. Purified HRP-SpG was tested successfully for functional binding of antibodies from different mammalian serums. Recombinant production of this novel well-defined fusion protein follows quality-by-design principles and facilitates the production of more reliable and cost-effective diagnostic kits.

Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen

In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP–Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP–Au NRs nanocomposites and the labeling of secondary antibody (Ab2) were performed by one-pot assembly of HRP and Ab2 on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab1) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen–antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H2O2) and 3,3′,5,5′-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1–100 ng ml−1, and the limit of detection was 30 pg ml−1 (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications.

An updated view on horseradish peroxidases: recombinant production and biotechnological applications.

Horseradish peroxidase has been the subject of scientific research for centuries. It has been used exhaustively as reporter enzyme in diagnostics and histochemistry and still plays a major role in these applications. Numerous studies have been conducted on the role of horseradish peroxidase in the plant and its catalytic mechanism. However, little progress has been made in its recombinant production. Until now, commercial preparations of horseradish peroxidase are still isolated from plant roots. These preparations are commonly mixtures of various isoenzymes of which only a small fraction has been described so far. The composition of isoenzymes in these mixed isolates is subjected to uncontrollable environmental conditions. Nowadays, horseradish peroxidase regains interest due to its broad applicability in the fields of medicine, life sciences, and biotechnology in cancer therapy, biosensor systems, bioremediation, and biocatalysis. These medically and commercially relevant applications, the recent discovery of new natural isoenzymes with different biochemical properties, as well as the challenges in recombinant production render this enzyme particularly interesting for future biotechnological solutions. Therefore, we reviewed previous studies as well as current developments with biotechnological emphasis on new applications and the major remaining biotechnological challenge—the efficient recombinant production of horseradish peroxidase enzymes.

Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris

The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.

Ultrasensitive nucleic acid biosensor based on enzyme–gold nanoparticle dual label and lateral flow strip biosensor

In this article, we describe an ultrasensitive nucleic acid biosensor (NAB) based on horseradish peroxidase (HRP)–gold nanoparticle (Au-NP) dual labels and lateral flow strip biosensor (LFSB). The results presented here expand on prior work ( Mao et al., 2009a) by optimizing the preparation of HRP–Au-NP–DNA conjugates. It was found that sodium dodecyl sulfate (SDS) and the immobilization sequence of thiolated DNA and HRP on the Au-NP surface played very important roles to improve the sensitivity of the assay. After systematic optimization, the detection limit of current approach is 1000 times lower than that in prior work. Deposition of insoluble enzymatic catalytic product (red colored chromogen) on the captured Au-NPs at the test zone of LFSB offers a dramatic visual enhancement. Combining enzyme catalytic amplification with unique optical properties of Au-NPs, the NAB was capable of detecting of 0.01-pM target DNA without instrumentation. The NAB thus provides a rapid, sensitive, low-cost tool for the detection of nucleic acid samples. It shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents.

A novel hydrogen peroxide biosensor based on horseradish peroxidase immobilized on gold nanoparticles–silk fibroin modified glassy carbon electrode and direct electrochemistry of horseradish peroxidase

The preparation of horseradish peroxidase (HRP)–gold nanoparticles (AuNPs)–silk fibroin (SF) modified glassy carbon electrode (GCE) by one step procedure was reported for the first time. The direct electrochemistry of HRP at the modified electrode was investigated. The enzyme electrode showed a quasi-reversible electrochemical redox behavior with a formal potential of −210 mV (vs. SCE) in 0.1 M phosphate buffer solution at pH 7.1. The response of the biosensor showed a surface-controlled electrochemical process with one electron transfer accompanying with one proton. The cathodic transfer coefficient was 0.42, the electron transfer rate constant was 1.84 s −1 and the surface coverage of HRP was 1.8 × 10 −9 mol cm −2. The experimental results indicated that AuNPs–SF composite matrix could not only steadily immobilize HRP, but also efficiently retain its bioactivity. The biosensor displayed an excellent and quick electrocatalytic response to the reduction of H 2O 2.

Peroxidase Oxidation of Phenols

Partially purified preparations of horseradish peroxidase were able to catalyze the effective transformation of such phenol compounds as phenol, o-chlorophenol, 2,4,6-trichlorophenol, pentachlorophenol (giving rise to the formation of polymer products insoluble in water), resorcinol, and thymol (giving rise to the formation of low-molecular-weight products). The following conditions were found to be optimal for peroxidase oxidation and provide the maximum extent of elimination of phenol compounds: temperature, 15-25 and 25-30 degrees C for phenol and chlorophenol compounds, respectively; molar ratio H2O2/phenol, 1:1; and transformation time, 1-3 h. Although effective transformation was observed within a broad range of pH, the efficiency of the process slightly increased at a pH from 6.0 to 7.5. It was suggested to carry out multiple peroxidase oxidations of phenols using partially purified peroxidase enclosed in a dialysis membrane bag placed into a solution of a phenol compound containing hydrogen peroxide.

Preparation of enzyme conjugate through adipic acid dihydrazide as linker and its use in immunoassays.

The direct coupling of the carboxylic derivative of steroids to the amino group of enzymes is a well-established method in steroid enzyme immunoassays for making enzyme conjugates (1). Horseradish peroxidase (HRP), containing six lysine residues in the sequence, is a widely used enzyme in enzyme immunoassays; in practice, however, only one or two of these are generally available for reaction (2). This variation in amino group content is caused by changes in the extraction conditions used for the isolation of HRP from the roots of the horseradish plant (2)(3). The low yield of HRP coupled to the IgG by the use of bifunctional reagents, namely glutaraldehyde, carbodiimide, cyanuric chloride, bis-diazotized O -dianisidine, and P - P ′-difluoro- m , m -dinitro-diphenyl sulfone (FNPS), and so forth, prompted Nakane and Kawaoi to investigate another method (periodate method) for the conjugation of HRP to IgG (4). Comparative coupling efficiency studies were carried out with the use of glutaraldehyde, periodate, and N -succinimidyl 3-(2-pyridyldithio) propionate (SPDP) as cross-linking reagents (5)(6) for the preparation of HRP-IgG conjugates. These studies revealed that the most efficient HRP-IgG conjugate was obtained by the periodate method. In practice, the difference in amino group availability in different batches of commercial preparations of HRP makes it difficult to establish standard reaction conditions that could be applicable for more than one batch. Therefore, adipic acid dihydrazide (ADH) has been coupled to HRP with the use of the periodate method reaction to provide the necessary amino group for the preparation of hapten enzyme conjugate with the use of the carbodiimide method. We describe for the first time the use of ADH as a linking reagent between glycoenzyme (HRP) and a steroid carboxylic derivative to prepare enzyme conjugate for ELISA. The conjugation of HRP to cortisol through ADH as …

Preparation of Horseradish Peroxidase Hydrazide and Its Use in Immunoassay

Preparation of horseradish peroxidase (HRP) hydrazide that is HRP linked to adipic acid dihydrazide (HRP-ADH) and its use in enzyme immunoassay (EIA) is described. In this new strategy, horseradish peroxidase was conjugated to adipic acid dihydrazide using a carbodiimide coupling method. The resulting HRP-ADH was then coupled to cortisol-21-hemisuccinate (Cortisol-21-HS) to prepare enzyme conjugate. The prepared cortisol-21-HS coupled ADH-HRP (Cortisol-21-HS-ADH-HRP) enzyme conjugate was used for the development of an enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol. To the cortisol antibody coated microtiter wells, standard or serum samples (50 µL), along with cortisol-21-HS-ADH-HRP enzyme conjugate (100 µL) were incubated for 1 h at 37°C. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The sensitivity of the assay was 0.05 µg/dL and the analytical recovery ranged from 92.9 to 101.7%.

A comparative study of the purity, enzyme activity, and inactivation by hydrogen peroxide of commercially available horseradish peroxidase isoenzymes A and C

Horseradish peroxidase (HRP) is a commercially important enzyme that is available from a number of supply houses in a variety of grades of purity and isoenzymic combinations. The present article describes a comparative study made on nine HRP preparations. Six of these samples were predominantly composed of basic HRP, pl 8.5, and three of acidic HRP, pl 3.5. Two of the basic preparations were of lower purity than the others. The apparent molar catalytic activity of basic HRP with 0.5 mMABTS and 0.2 mM H(2)O(2) was around 950 s(-1) (about 770 s(-1) for the less pure samples) and with a 5 mM guaiacol and 0.6 mM H(2)O(2) was about 180 s(-1) for all the samples. A similar value (approximately 1000 s(-1)) was observed for acidic HRP but only at higher concentrations of ABTS (20 mM). With 20 mM guaiacol the molar catalytic activity of the acid isoenzyme was 65 s(-1). The apparent K(M) for ABTS of the acidic isoenzyme was 4 mM whereas for the basic isoenzyme it was 0.1 mM. All the enzymes were inactivated by H(2)O(2) when it was supplied as the only substrate. Under these conditions the partition ratio (r = number of catalytic cycles given by the enzyme before its inactivation), apparent dissociation constant (K(l)), and apparent rate constant of inactivation (k(inact)) were about twice as large for the acidic samples (1350, 2.6 mM, 9 x 10(-3) s(-1)) as for the basic (650, 1.3 mM, 5 x 10(-3) s(-1)). The apparent catalytic constant (k(cat)) was 3-4 times larger, and the efficiency of catalysis (k(cat)/K(l)) was double for the acidic isoenzyme, but the efficiency of inactivation (k(inact)/K(l)) was similar. The data obtained provide useful information for those using HRP isoenzymes for biotechnological applications (e.g., biosensors, bioreactors, or assays).

Ultrastructural localization of the plasmalemmal calcium pump in cerebellar neurons.

In a previous study, fluorescence labeling of a plasmalemmal ATPase protein with the 5F10 monoclonal antibody revealed prominent antigen in the cerebellar molecular layer surrounding the somata and dendrites of Purkinje cells. In the present study, this antibody labeled with silver enhanced nano-sized gold particles on semithin plastic sections revealed a clearly demarcated plasma membrane outlining the somata and entire dendritic arbors of Purkinje cells including their spines. Ultrastructural analysis of horseradish peroxidase preparations showed reaction product along the plasmalemma and extending on to the sub-plasmalemmal endoplasmic reticulum. In the granular layer, somata of granule cells were reactive, as were their dendritic extensions into glomeruli where reactive claws surrounded voids formed by mossy fiber rosettes. Somata and dendrites of cerebellar nuclear cells also had reactive zones that were limited to the plasma membrane and a narrow zone of the sub-plasmalemmal endoplasmic reticulum. Comparative labeling of this protein and P channel protein revealed similar plasmalemmal locations. This study shows that a specific calcium ATPase pump protein is located on the plasmalemma of certain types of cerebellar neurons. The ultrastructural distribution of calcium pump and P channel antibodies occurred in punctate sites along the plasma membrane of dendrites and spines of Purkinje cells. The close association between P-type calcium channels and the plasma membrane calcium pump is consistent with rapid extrusion of intracellular calcium from neurons endowed with large numbers of voltage-gated calcium channels.

Evidence for a slow tertiary relaxation in the reaction of tert-butyl isocyanide with horseradish peroxidase.

The kinetics of tert-butyl isocyanide binding to the heme protein horseradish peroxidase (HRP) at 22 degrees C was examined on all time scales, from minutes to picoseconds, in aqueous borate buffer at pH 9.08. Unlike myoglobin (Mb) or hemoglobin, HRP shows two bimolecular ligand binding processes. For comparison, binding of the same ligand with Mb was measured under identical conditions. Ligand entry into the protein from the solvent in a mixing experiment is extremely slow in HRP: the bimolecular association constant is 0.04 M-1 s-1, while in Mb it is 4 x 10(3) M-1 s-1. Surprisingly, in view of that difference, picosecond and nanosecond photolyses reveal that once the ligand has reached the iron(II) site there is no difference in cage return or escape from the protein. The rate for the fastest cage return (from the contact pair) is close to 6 x 10(10) s-1 in both proteins. The rates of escape from the contact pair to form a secondary protein-caged pair are also similar: for Mb, 10 x 10(10) s-1, and for HRP, 8.5 x 10(10) s-1. The rate of rebinding from the protein-separated cage is near 4 x 10(6) s-1 in both proteins, and the rate of escape from protein to solvent is close to 3.7 x 10(6) s-1 in both. The difference between the two proteins lies in the low-millisecond time domain. After flash photolysis of HRP, there is a concentration-dependent recombination not seen in mixing experiments. This bimolecular rate constant varies slightly for different HRP preparations, being 2.6 x 10(4) or 4.0 x 10(4) M-1 s-1 in two cases, both of which are much faster than is observed in mixing experiments, namely, 0.04 M-1 s-1. In Mb, photolysis and mixing experiments consistently give the same combination rate, which is somewhat slower than the faster part of the HRP recombination. Similar measurements for the smaller ligand methyl isocyanide revealed no anomalous behavior. The interpretation proposed involves tertiary relaxation after ligand escape, which is significant in blocking the return of the large t-BuNC, but has no apparent effect on smaller ligands. Thus, HRP-t-BuNC reveals in dramatic fashion a phenomenon merely hinted at in earlier work involving the T-state binding kinetics of hemoglobin.

Comparison of Isoniazid Oxidation Catalyzed by Bacterial Catalase–Peroxidases and Horseradish Peroxidase

The physical properties and activities of the purified catalase–peroxidase hydroperoxidase I (HPI) ofEscherichia coli(EcHPI) and HPI with a carboxyl-terminal extension of Mycobacterium tuberculosis (MtHPI-e) are compared to those of commercial preparations of horseradish peroxidase (HRP). The catalase–peroxidase proteins had similar absorption spectra and differed primarily in that MtHPI-e has a higher peroxidatic to catalatic activity ratio than EcHPI. Trypsin cleavage of MtHPI-e resulted in the formation of an active catalase–peroxidase lacking the carboxyl-terminal extension. The three enzymes, HRP, MtHPI-e, and EcHPI, mediated the isoniazid- and H2O2-dependent production of radical species, as detected by nitroblue tetrazolium reduction. A constant flux of H2O2, generated in situ from glucose oxidase and glucose was used. MtHPI-e was more effective at isoniazid-dependent radical production than EcHPI and HRP. Similar qualitative results were obtained by staining nondenaturing polyacrylamide gels for activity with nitroblue tetrazolium in the presence of isoniazid and H2O2. The absorbance spectrum of HRP exhibited changes during incubation with isoniazid and H2O2consistent with the formation of several typical reaction intermediates, whereas the catalase–peroxidases exhibited no distinct spectral changes. The results suggest that the sensitivity of M. tuberculosis to isoniazid may be the result of isoniazid-dependent radical formation by the catalase–peroxidase in the absence of other catalase activities to remove substrate H2O2.

Purification and characterization of an anionic peroxidase from sycamore maple (Acer pseudoplatanus) cell suspension cultures

A 42 kDa anionic peroxidase (EC 1.11.1.7) having a pl of 3.6 was purified from suspension cultures of cells of sycamore maple (Acer pseudoplatanus L.) grown in the dark by a combination of lectin‐affinity, anion‐exchange and gel permeation chromatography. The enzyme had an amino acid composition similar to that found for other anionic plant peroxidases, but the protein was blocked to amino‐terminal protein sequencing. Commercially available antibodies against horseradish peroxidase were shown to cross‐react with the sycamore maple enzyme on immunoblots. The purified peroxidase displayed differences in its affinity for each of the three monolignols, and these differences were compared to those found for a commercial preparation of horseradish peroxidase, as well as a laccase (p‐diphenol:O2 oxidoreductase: EC 1.10.3.1) purified from sycamore maple cell suspension cultures. These results are discussed with respect to the role played by peroxidases in lignin deposition and host‐pathogen response.

Peroxidase catalysed formation of prostaglandins from arachidonic acid

Horseradish peroxidase and bovine lactoperoxidase (EC 1.11.1.7), when incubated aerobically with arachidonate, gave rise to the formation of substances identified by bioassay as prostaglandin F 2α (PGF 2α)- and prostaglandin E 2 (PGE 2)-like compounds. Boiling of enzymes, which suppressed their capacity to peroxidise guaiacol, also destroyed their capacity to convert arachidonate into PG-like compounds. The rates of formation of PG-like compounds rapidly declined with time, approaching zero after 10 and 20 min for PGF 2α- and PGE 2-like compounds, respectively. Addition of more enzyme further promoted the reaction. Horseradish and lacto-peroxidases showed optimum pH values of 9.0 and 10.0, respectively. Both enzymes exhibited apparent K m values of about 5 × 10 −5 M for arachidonate. Some reducing agents such as ascorbic acid, NADH and adrenaline dose-dependently inhibited this reaction. The haem poison, phenylhydrazine, also inhibited, with an ic 50 of 1 × 10 −7 M. Indomethacin inhibited only the formation of PGE 2-like compounds with an ic 50 of about 3 × 10 −6M. As compared to a standard commercial preparation of horseradish peroxidase, the purified horseradish basic and acidic isoenzymes exhibited a higher activity, towards arachidonate whereas other haemoproteins, possessing peroxidase activity, were less active. TLC and GC-MS analyses performed on the reaction products led to the identification of PGF 2α, PGE 2 and PG6K 1α and other unidentified arachidonate derivatives. At 25°, pH 9.5, horseradish peroxidase, acting on saturating concentration of arachidonate, catalysed the formation of 60 μmol/min/mmole enzyme of PGE 2 + PGF 2α. This appears to be the first report of the synthesis of prostaglandins catalysed by peroxidases.

Porphobilinogen oxygenase. Purification and evidence of its hemoprotein structure.

Porphobilinogen oxygenase oxidizes porphobilinogen to 2-hydroxy-5-oxo-porphobilinogen. This enzyme isolated from wheat germ has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis under both nondenaturing and denaturing conditions. The molecular weight of the enzyme formed from two identical (or very similar) polypeptide chains is 70,000. It has a pI of 9.0 indicating its cationic nature. The pure enzyme contains 1 mol of high-spin heme and 2 mol of non-heme iron. It requires both of these as well as molecular O2 and a reducing agent for catalytic activity. Although the enzyme has many characteristics of a peroxidase, hydrogen peroxide cannot substitute for oxygen and dithionite for catalysis. The catalytic reaction is not affected by catalase, superoxide dismutase, or by hydroxyl radical scavengers. A comparison between porphobilinogen oxygenase and a commercial preparation of horseradish peroxidase was made. The latter also catalyzes aerobic porphobilinogen oxidation, with dithionite as electron donor. Here the oxidation of porphobilinogen is inhibited by superoxide dismutase and was not affected by catalase.