Luminol oxidation catalyzed by royal palm leaf peroxidase

We optimized the conditions for oxidation of luminol by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3–8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM Tris solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

Suicide inactivation of peroxidase from Chamaerops excelsa palm tree leaves

The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine and o-phenylenediamine). The turnover number (r) of H₂O₂ required to complete the inactivation of the enzyme varied for the different substrates, the enzyme most resistant to inactivation (r = 4844) with ABTS being the most useful substrate for biotechnological applications, opening a new avenue of enquiry with this peroxidase.     

Peroxidase from leaves of royal palm tree Roystonea regia: purification and some properties

Screening of tropical plants demonstrated high peroxidase activity in leaves of some species of palms. Using the leaves of royal palm Roystonea regia as a source, the peroxidase has been isolated to homogeneity. The enzyme purification steps included homogenization, (NH₄)₂SO₄ precipitation, extraction of palm leaf colored compounds and consecutive chromatography on Phenyl-Sepharose, Sephacryl S100 and DEAE-Toyopearl. The novel peroxidase was characterized as having a specific activity of 6170 U/mg, RZ 3.0, molecular weight of 51 kDa and isoelectric point pI 3.5. The electronic spectrum of RPP is characteristic for plant peroxidases with a Soret maximum at 403 nm and maxima in a visible region at 492 and 633 nm, respectively. The substrate specificity of royal palm tree peroxidase (RPTP) is distinct from the specificity of other plant peroxidases. The best substrates for RPTP are ferulic acid and 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid). The palm peroxidase exhibits an unusually high thermostability inactivating at 90 °C with k_inac of 1.5×10⁻² min⁻¹.     

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H₂O₂, and organic solvents. Thus, RPTP is a promising candidate for developing H₂O₂-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85 Å. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP.     

Purification and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.)

Peroxidase from date palm (Phoenix dactylifera L.) leaves was purified to homogeneity and characterized biochemically. The enzyme purification included homogenization, extraction of pigments followed by consecutive chromatographies on DEAE-Sepharose and Superdex 200. The purification factor for purified date palm peroxidase was 17 with 5.8% yield. The purity was checked by SDS and native PAGE, which showed a single prominent band. The molecular weight of the enzyme was approximately 55 kDa as estimated by SDS–PAGE. The enzyme was characterized for thermal and pH stability, and kinetic parameters were determined using guaiacol as substrate. The optimum activity was between pH 5–6. The enzyme showed maximum activity at 55 °C and was fairly stable up to 75 °C, with 42% loss of activity. Date palm leaves peroxidase showed K_m values of 0.77 and 0.045 mM for guaiacol and H₂O₂, respectively. These properties suggest that this enzyme could be a promising tool for applications in different analytical determinations as well as for treatment of industrial effluents at low cost.     

Alkaline pulping with additives of date palm rachis and leaves from Sudan

Soda-anthraquinone (soda-AQ), alkaline sulphite-anthraquinone (AS-AQ) and alkaline sulphite-anthraquinone-methanol (ASAM) pulping of date palm rachis and leaves from Sudan was carried under different conditions, and pulps with variable yields and mechanical properties were obtained. The date palm rachis gave best yields and mechanical properties with the AS-AQ or the ASAM process, while the leaves were best pulped with the soda method with low yield, but very good strength properties. Blending with 10% and 30% kenaf bark pulp was beneficial, especially for the AS-AQ pulps. Totally chlorine free (TCF) bleached rachis pulps were obtained of high brightness and strength properties suitable for use in writing and printing papers.     

Free fatty acid determination by peroxidase catalysed luminol chemiluminescence

A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H₂O₂ formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C₁₀-C₁₈) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H₂O₂ standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.     

Nonuniform variation in band pattern with luminol/horseradish peroxidase western blotting

When optimized, the peroxidase-catalyzed oxidation of luminol can provide a useful, sensitive detection system for Western blotting. However, while the luminescence from intense bands appears rapidly, faint bands require at least 30 min after removal of the membrane from reaction buffer for maximum luminescence to develop. This can result in the detection of a variant band pattern if films are exposed to the blotted membrane too soon after reaction, while exposure later after reaction can result in the preferential detection of faint bands. As a consequence, in order to detect a range of bands similar to that seen using autoradiographic or chromogenic systems, it is necessary to determine the correct time after the initiation of the luminol reaction for film exposure. These effects are due to enhancement of luminescence as a result of the peroxidase-immunoglobulin conjugate binding to a solid phase.     

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

Purification and properties of peroxidase from tea leaves]

Purification of fractions of tea leaves peroxidase is described. During ion-exchange chromatography on DEAE- and CM-cellulose peroxidase is eluted into six fractions, differing in their electrophoretic properties. The enzyme showed optimal activity at pH 4.1-5.0, when the enzyme fractions of guaiacol adsorbed on DEAE-cellulose were used as a substrate; in case of enzyme fractions adsorbed on CM-cellulose it was observed within pH range of 5.4-6.2. The dependence curves of the initial rate of the reaction on the substrate concentration were S-shaped in case of the latter fractions. Peroxidase is shown to catalyze the oxidation of tea catechins; its activity is inhibited by the products of their condensation. The catalytic effect of the enzyme on the oxidation of phenolic acids, e.g. chlorogenic, caffeic and gallic, was far stronger than on that of tea catechins, pyrogallol and pyrocatechin. It was established that two fractions of the enzyme possess predominantly the phloroglucinol oxidase activity, whereas the other fractions do not catalyze the oxidation of phloroglucin. The molecular weights of some peroxidase fractions estimated by polyacryl amide gel electrophoresis are 26.000+/-1.100, 45.00+/-1.200 and 50.000+/-1.500.     

Oxidation states of peroxidase

Summary Five oxidation states of horseradish peroxidase, ferrous, ferric, Compounds I and II, oxy-ferrous, are known. Various reactions and plausible structures of these states are reported. Mechanisms of peroxidase-oxidase reactions are discussed in terms of the five oxidation states of the enzyme.an invited article     

Partial characterization of peroxidase isoenzymes from rust-affected wheat leaves

Four anodic peroxidase isoenzymes from wheat leaves were purified by column chromatography and their kinetic behavior with common substrates were examined. One isoenzyme is more active in wheat resistant to stem rust fungi and differed from the others in carbohydrate content and also by a specific activity 2–4-fold higher with non-physiological electron donors. As a substrate, eugenol exhibited kinetic behavior different from p-phenylenediamine, guaiacol or o-dianisidine with all isoenzymes. All four isoenzymes showed similar pH and temperature optima and kinetic behavior and apparent K_m values for both H₂O₂ and non-physiological electron donors.     

Mathematical model for the luminol chemiluminescence reaction catalyzed by peroxidase

A kinetic model that accurately describes intensity vs. time reaction profiles for the chemiluminescence reaction between luminol and hydrogen peroxide, as catalyzed by horseradish perioxdase, is derived and evaluated. A set of three differential equations is derived and solved to provide intensity time information for the first 200 seconds of the reaction. The model accurately predicts intensity-time profiles when literature values are used for all but one of the reaction rate constants. Furthermore, the model predicts a nonlinear curve for plots of light intensity versus the initial hydrogen peroxide concentration. Experimental data confirm that such plots are nonlinear. Finally, a linear double-reciprocal plot is predicted by the model and the experimental data verify this relationship. (c) 1993 Wiley & Sons, Inc.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Substrate specificity of african oil palm tree peroxidase

The optimal conditions for catalysis by the peroxidase isolated from leaves of African oil palm tree (AOPTP) have been determined. The pH optimum for oxidation of the majority of substrates studied in the presence of AOPTP is in the interval of 4.5-5.5. A feature of AOPTP is low pH value (3.0) at which the peroxidase shows its maximal activity toward 2,2"-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS). Increasing the buffer concentration changes the AOPTP activity, the degree of the effect depending upon the chemical structure of the substrate. Under optimal conditions of AOPTP catalysis, the values of second order rate constant characterizing efficiency of enzymatic oxidation of substrates have been calculated. It was shown that among 12 peroxidase substrates studied, ABTS and ferulic acid are the best substrates for AOPTP. The results show that substrate specificities of AOPTP and royal palm tree peroxidase are similar, but different from substrate specificity of other plant peroxidases.     

Automated determination of asulam by enhanced chemiluminescence using luminol/peroxidase system

A flow injection system with chemiluminescence detection for the determination of asulam, enhancer of the system luminol–H₂O₂–horseradish peroxidase, is proposed. The method shows a moderate selectivity against other pesticides usually present in formulations of herbicides and in water. The procedure was applied to the determination of asulam in tap water samples and a recovery study was carried out in order to validate the method. The obtained results show acceptable recovery values (between 88.3 and 93.9%). The detection limit for asulam was 0.12 ng/mL. The precision of the method expressed as relative standard deviation was 1.55% (n = 8), at the 19 ng/mL level. Copyright © 2009 John Wiley & Sons, Ltd.     

Thermal stability of peroxidase from the african oil palm tree Elaeis guineensis.

The thermal stability of peroxidase from leaves of the African oil palm tree Elaeis guineensis (AOPTP) at pH 3.0 was studied by differential scanning calorimetry (DSC), intrinsic fluorescence, CD and enzymatic assays. The spectral parameters as monitored by ellipticity changes in the far-UV CD spectrum of the enzyme as well as the increase in tryptophan intensity emission upon heating, together with changes in enzymatic activity with temperature were seen to be good complements to the highly sensitive but integral method of DSC. The data obtained in this investigation show that thermal denaturation of palm peroxidase is an irreversible process, under kinetic control, that can be satisfactorily described by the two-state kinetic scheme, N -->(k) D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.     

Kinetic and thermodynamic behaviour of wall-bound peroxidase from wheat leaves infected with stripe rust

We have identified two types of peroxidases (POX), one ionically and one covalently bound to the particulate fraction, in stripe rust-infected and -uninfected wheat (Triticum aestivum L.) leaves. The cell walls contained a high level of POX, of which 73–76% was extractable by 1% NaCl and 24–26% by 5 mM EDTA in infected and non-infected leaves of HD 2329. The NaCl-released POX constituted the predominant fraction. Both NaCl- and EDTA-extracted POX exhibited maximum activity at pH 5.0 and had a K _m (enzyme–substrate affinity measure) value of 1.61–1.70 and 1.64–1.67 mM, respectively, with o-dianisidine as the substrate. The V _max (maximum catalytic rate) in the two extractions ranged between 7.06–7.45 and 6.65–7.82 μmol min⁻¹ g⁻¹ fresh weight. A temperature optimum of 50°C was observed for both the NaCl- and EDTA-released fractions. The two POX fractions showed a differential response to metal ions, suggesting their distinctive nature. Sodium azide inhibited POX activity markedly, which suggested the presence of heme as a prosthetic group. Inhibition of wall-bound POX by iodine and the regeneration of activity by mercaptoethanol suggested the involvement of cysteine in the active site of the enzyme. These two forms showed greater differences in terms of thermodynamic properties, such as the energy of activation (E _a) and enthalpy change (ΔH), while entropy (ΔS) and free energy changes were similar. The results further show that pathogen infection of the leaves of this susceptible wheat cultivar induces an increase in the activity and kinetics of POX, which may be critical in the response of the plant cell to infection.     

The effect of monoclonal and polyclonal antibodies to peroxidase on combined peroxidase oxidation of 4-iodophenol with luminol and 4-aminoantipyrine]

The kinetics of peroxidase-dependent cooxidation for two substrate pairs [p-iodophenol + 4-aminoantipyrine (AAP) and p-iodophenol + luminol was studied both in the absence and presence of polyclonal antibodies (polyAB), three types of peroxidase-specific monoclonal antibodies (monoAB) and their double or triple mixtures in a wide range of H2O2 concentrations (0.01-10.0 mM). MonoAB 2C, 3E and 9D at concentrations of 0.05-500 nM inhibited the cooxidation of p-iodophenol + AAP at H2O2 concentration above 1.0 mM but activated the cooxidation of p-iodophenol + luminol. The double and triple mixtures of monoAB activated the cooxidation of p-iodophenol + AAP at the same H2O2 concentrations without any effect on the p-iodophenol + luminol cooxidation. PolyAB activated the cooxidation of p-iodophenol + AAP more effectively and only slightly activated (or inhibited) that of p-iodophenol + luminol. PolyAB diminished the values of rate constants for the interaction of the peroxidase active intermediates, E1 and E2, with p-iodophenol, AAP or luminol. Possible modes of monoAB and polyAB effects on the two substrate pair cooxidation are discussed.