Interaction Of Horseradish Peroxidase Research Articles

Interaction of horseradish peroxidase with Langmuir monolayers of phospholipids

The method employed to incorporate guest molecules onto phospholipid Langmuir monolayers plays an important role in the interaction between the monolayer and the guest molecules. In this paper, we show that for the interaction between horseradish peroxidase (HRP) and a monolayer of dipalmitoylphosphatidylglycerol (DPPG) does depend on the method of HRP incorporation. The surface pressure isotherms of the mixed DPPG/HRP monolayers, for instance, were less expanded when the two materials were co-spread than in the case where HRP was injected into the subphase. Therefore, the method for incorporation affected not only the penetration of HRP but also the changes in molecular packing caused to the DPPG monolayer. With experiments with the monolayer on a pendant drop, we observed that the incorporation of HRP affects the dynamic elasticity of the DPPG monolayer, on a way that varies with the surface pressure. At low pressures, HRP causes the monolayer to be more rigid, while the converse is true for surface pressures above 8 mN/m. Taken all the results together, we conclude that HRP is more efficiently incorporated if injected into the subphase on which a DPPG monolayer had been spread and that the interaction between HRP and DPPG is maintained even at high surface pressures. This is promising for the possible transfer of mixed films onto solid substrates and for applications in biosensors and drug delivery systems.

Enthalpy analysis of horseradish peroxidase in the presence of Ni 2+: a stabilization study

The interaction of horseradish peroxidase (HRP) with Ni 2+ has been investigated by isothermal titration calorimetry (ITC), potentiometry, equilibrium dialysis, and spectrophotometry. Total enthalpy of cited interaction was measured by ITC at 27 °C, pH=7.0.The enthalpy of ionization (Δ H ion) is obtained by acid–base titration at 25 and 50 °C in 100 mM NaCl. Enthalpy of binding is obtained by equilibrium dialysis at 27 and 37 °C based on the Wyman binding potential to evaluate equilibrium constants at two temperatures. The van’t Hoff relation is used for calculation of enthalpy of binding (Δ H bin) and also a modified differential relation is applied for estimation of ionization enthalpy (Δ H ion). Denaturation profiles on HRP with and without the presence of Ni 2+ using n-dodecyl trimethylammonium bromide (DTAB) as a denaturant, has been studied. Using the Pace theory to evaluate free energy in the absence of denaturant (Δ G° H 2O ) is the best parameter for determination of protein stability. The results show 4.9 kJ mol −1 higher free energy for stabilization of HRP in the presence of Ni 2+. Enthalpy of conformational change of HRP by Ni 2+ (3.5 mM) is determined by resolving the contributions of calorimetric enthalpy, binding enthalpy and ionization enthalpy. The enthalpy of stabilization for HRP in the presence of Ni 2+ (3.5 mM) is equal to −118.62 kJ mol −1 which was obtained by differential relation between enthalpies of unfolding and conformation.

Oxic and anoxic enhancement of radiation-mediated toxicity by horseradish peroxidase/indole-3-acetic acid gene therapy

Purpose : To evaluate the interaction of horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) gene therapy with therapeutically relevant doses of radiation. Materials and methods : Human T24 bladder and FaDu nasopharyngeal carcinoma cells were transiently transfected with the HRP cDNA using a non-viral delivery method. HRP expression was confirmed by immunostain and enzyme activity assay. The cells were exposed to IAA or the analogue 1-Me-IAA in conjunction with X-rays in air or in anoxic conditions, and cytotoxicity was determined by clonogenic assay. Results : A significant and selective enhancement of radiation-mediated cytotoxicity was observed. Pre-incubation with the prodrugs induced sensitizer enhancement ratios (SER) ranging from 2.6 (0.1mM IAA) to 5.4 (0.5 mM IAA). Radiosensitization was also observed when prodrug exposure was performed immediately after irradiation (SER=2.1-5.6), or in anoxic conditions (SER=3.6). Conclusions : The use of gene therapy protocols to enhance the effect of ionizing radiation holds promise for anticancer therapy. The data suggest that the combination of HRP/IAA gene therapy with ionizing radiation could present therapeutic advantages in the treatment of solid malignancies, in particular to target the hypoxic population, which has been shown to correlate with poor outcome after radiotherapy.

Interaction of horseradish peroxidase with montmorillonite homoionic to Na+ and Ca2+: effects on enzymatic activity and microbial degradation

The adsorption, desorption, catalytic activity, and susceptibility to microbial degradation of the enzyme horseradish peroxidase (HRP E.C. 1.11.1.7), on Wyoming montmorillonite (M) homoionic to Na+ or Ca2+ were investigated. Adsorption at equilibrium was reached after 1h of contact between the clay and HRP. The adsorption isotherms were of the L type and fitted the Freundlich equation on M-Na and the Langmuir equation on M-Ca. Adsorption was greater on M-Na than on M-Ca and was maximal at pH 3.0, i.e., below the isoelectric point (pI=pH 9) of the protein. Only 10–25% of HRP was desorbed from the equilibrium M-Na-HRP complexes, whereas 20–30% was desorbed from the equilibrium M-Ca-HRP complexes with 4–7 washes with double distilled water. HRP partially penetrated the interlayers of M-Na and M-Ca, but complete intercalation was observed only at pH 3. The enzymatic activity of HRP measured immediately after the preparation of the complexes at all concentrations was greatly reduced when bound on M-Na (about 90%), regardless of the loading of HRP. The reduction in activity of M-Ca-HRP was related to the amount of bound protein (no reduction for the highest and 60% for the lowest amount bound). After 24h, pure HRP in dilute solution lost about 10% of its catalytic activity daily, whereas when bound on M-Ca, a greater reduction was observed (about 30% for the highest and 60% for the lowest amount bound). FT-IR analyses indicated only small changes in the secondary structure of HRP as a result of binding on the clays. Electronic absorption spectra in the UV region of bound HRP did not show the typical ‘red-shift’ of the Soret band that usually results from binding of HRP with its substrate. Consequently, the reduction in the activity of bound HRP was probably the result of the inaccessibility and/or of modifications of the active center of HRP for its substrate. The availability of HRP bound on M-Na as a source of carbon and/or nitrogen for soil microorganisms was reduced by 90% in comparison with the free enzyme.

Interaction of Peroxidases with Aromatic Peracids and Alkyl Peroxides: PRODUCT ANALYSIS

Abstract Two limiting structures may be assigned to the primary derivative of peroxidases (Compound I) demanding either (a) total or (b) partial retention of peroxide components. It is now shown that (a) interaction of horseradish peroxidase with m-nitroperbenzoic acid, in 15 mm phosphate, pH 6.2, 25°, leads to the stoichiometric formation of the enzyme-peroxide derivative, m-nitrobenzoate and 1 proton; and (b) in 5 mm phosphate, pH 6.6, 10°, the conversion of horseradish peroxidase into Compound I with ethyl hydrogen peroxide, is attended by the release of ethanol, but without concomitant prototropic changes. The results establish that horseradish peroxidase (H-peroxidase) Compound I is not an enzyme peroxide complex, but a derivative in which the active site is oxidized. The optical spectra of H-peroxidase and Compound I in 5 mm phosphate, pH 6.6, 25°, were re-examined and extended to include previously undefined features in the near infrared absorption region. The spectrum of Compound I is devoid of bands typical of isoporphyrins, suggesting that conversion of the enzyme into Compound I does not involve covalent modification of the prosthetic group.

Temporal resolution of individual steps in an enzymic reaction at low temperature.

Media are described which make it possible to study mechanisms of enzyme action at low temperature. The applicability of techniques developed are illustrated by results obtained on formation kinetics of complexes produced by the interaction of horseradish peroxidase with hydrogen peroxide. It is shown that the various steps in the time course of the reaction can be readily resolved with isolation of each intermediate in concentrations sufficient to permit rate studies between consecutive steps. The potential of the method for elaboration of enzyme mechanisms is discussed.