Heterogeneous inhibition of horseradish peroxidase activity by cadmium

Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd(2+) for various periods of time. The H(2)O(2)-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd(2+) was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd(2+) concentration. Furthermore, for the same inhibition type, K(i) values dropped as incubation time increased. These results suggested that Cd(2+) would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd(2+) altered the heme Soret absorption band on binding HRP and exhibited a K(d) which decreased as the incubation time of HRP with Cd(2+) increased. Hill plots suggested a cooperative binding of up to three Cd(2+) ions per molecule of HRP. Thus, Cd(2+) binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd(2+) ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them.     

Conformational changes and activity alterations induced by nickel ion in horseradish peroxidase

Conformational changes induced by the binding of nickel to horseradish peroxidase C (HRPC) were studied by electronic absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Incubation of HRPC with various concentrations of Ni(2+) for 5 minutes resulted in changes in the enzyme absorption spectrum, including variations in the intensities of the Soret, beta and charge transfer (CT1) bands absorption, shift in the Soret, beta and CT1 bands maxima and absorption increase at 275 nm. Increases in the enzyme's intrinsic fluorescence as determined by fluorescence spectroscopy, as well as changes in the alpha-helical content, as determined by circular dichroism spectroscopy, were also found. Correlatively, alterations of the enzymatic activity by Ni(2+) were studied by following the H(2)O(2)-mediated oxidation of o-dianisidine and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) by HRPC. With both reducing substrates, it was found that in the presence of sufficient amount of enzyme, 1-10 mM nickel would enhance the enzymatic activity, while higher Ni(2+) concentrations (20-50 mM) would inhibit it. The enzyme was completely inhibited after 5 minutes incubation in 50 mM Ni(2+). Prolonged incubation would induce complete inhibition at lower Ni(2+) concentrations. Spectrophotometry investigations also showed that inhibitory concentrations of Ni(2+) altered compounds I and II formation, compound II being the first affected. Based on spectrophotometry, fluorescence and circular dichroism spectroscopy, and data on compounds I and II formation, a scheme is suggested for HRPC conformational changes in different Ni(2+) concentrations. HRPC was found to have four potential attachment sites for Ni(2+) which were sequentially occupied in a dose- and time-dependent manner by the metallic ion.     

pH dependence of carbon monoxide binding to ferrous horseradish peroxidase

The kinetic parameters of the reaction of horseradish peroxidase with CO have been determined at pH values between 10 and 3. At pH 7.0 the CO binding equilibrium constant L was measured using submicromolar concentrations of horseradish peroxidase; the value obtained corresponds to the ratio of the association and dissociation kinetic constants as expected for a simple binding mechanism to a monomeric hemeprotein. The CO association rate constant is pH-independent below pH 7, whereas in going from pH 7 to pH 11 a 2-fold increase can be detected, as previously reported (Kertesz, D., Antonini, E., Brunori, M., Wyman, J., and Zito, R. (1965) Biochemistry 4, 2672-2676). On the other hand, CO dissociation displays a peculiar pH rate profile characterized by a progressive decrease from pH 10 to pH 5 and by a very marked increase as the pH is further lowered to pH congruent to 3. Furthermore, the rate of CO dissociation is markedly enhanced in peroxidase reconstituted with protoheme dimethyl ester, suggesting a role of the propionates in the regulation of this process.     

DNA template-assisted modulation of horseradish peroxidase activity

The precise alignment of DNA molecules by Watson-Crick base-pairing combined with its polymeric characteristics have allowed DNA to be used as a template or scaffold for assembling materials. In this work, we investigate the role of calf-thymus DNA as a template for enhancing the horseradish peroxidase (HRP)-mediated oxidation of phenol and phenolic derivatives. The HRP-catalyzed oxidation of phenol into polyphenolic products and in presence of 4-aminoantipyrine into quinoneimine dye complexes is studied. Visible spectroscopy reveals an increased yield of both products of the enzymatic reaction in presence of calf-thymus DNA and is attributed to the prearrangement of the corresponding substrates on the DNA. The concentrations of calf-thymus DNA and the substrates are found to affect the nature of prearrangement and subsequent formation of polymeric or co-oxidation products. Also, phenolic derivatives with different aromatic substitutions display divergent propensities towards product formation in presence of the DNA template. Our results demonstrate the ability of calf-thymus DNA to modulate the activity of HRP and exercise control on the nature of products formed. This work highlights the potential of using DNA as a template for influencing enzymatic reactions involving aromatic substrates.     

Metal coordination influences substrate binding in horseradish peroxidase

To clarify the role of metal ion coordination in horseradish peroxidase C (HRPC), the effect of pressure and of an externally applied electric field on spectral holes was compared for both metal-free and Mg-mesoporphyrin-substituted horseradish peroxidase C (MP-HRP and MgMP-HRP), as affected by the binding of 2-naphthohydroxamic acid (NHA). The data are compared to earlier studies performed on the same derivatives. Results obtained for MP-HRP show the presence of a predominant MP tautomer, as well as that of another small population with different pocket field and isothermal compressibility (0.12 vs 0.24 GPa⁻¹). Binding NHA induces the formation of two new almost equal populations of MP-HRP tautomer complexes and the protein compressibility in both forms is increased to 0.50 and 0.36 GPa⁻¹. The protein structure becomes much softer than in the absence of NHA. Binding the same substrate to MgMP-HRP resulted in MgMP adopting a single conformation with no compressibility changes, while without NHA, two forms were possible. Stark effect results show charge rearrangement upon substrate binding in both cases. We propose that it is the presence of the metal that stabilizes the structure during the reorganization of the protein matrix induced by the substrate binding event. With the metal, only one conformation is adopted, without significant structural rearrangement but with charge redistribution. The dissociation constants determined for NHA binding to both derivatives and to native HRPC show that studies using mesoporphyrin and Mg-mesoporphyrin derivatives are relevant to investigating the specificity of the substrate-binding pocket in this enzyme. Received: 15 October 1999 / Revised version: 3 April 2000 / Accepted: 5 April 2000     

Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase.

To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by (1)H NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme, are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the (1)H NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V(max)/K(m)) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.     

Calcium binding by horseradish peroxidase C and the heme environmental structure

The hyperfine shifted proton NMR spectrum of isoenzyme c of horseradish peroxidase indicated that one calcium ion is essential to the enzyme in maintaining the protein structure in the heme vicinity.     

Taurocholate-induced inhibition of hepatic lysosomal degradation of horseradish peroxidase.

Endocytosed proteins in hepatocytes are transported to lysosomes for degradation. Metabolites accumulating in these organelles are released into bile by exocytosis, a process that seems to be regulated by the bile salt taurocholate (TC). In this study we examined if TC is also involved in the control of the lysosomal degradation of endocytosed proteins. We used [(14)C]sucrose-labeled horseradish peroxidase ([(14)C]S-HRP), a probe suitable to evaluate lysosomal proteolysis. TC-infused rats as well as isolated rat hepatocytes exposed to TC showed a significant inhibition in the lysosomal degradation of [(14)C]S-HRP (approximately 30%), with no change in either the uptake or the amount of protein reaching lysosomes. Under these conditions, the in vitro assay of lysosomal cathepsins B, L, H, and D revealed no change in their activities, suggesting that a reversible inhibition (lysosomal alkalinization?) was taking place in hepatocytes. Nevertheless, lysosomal pH measured using fluorescein isothiocyanate-dextran was shown not to be altered by TC. In addition, TC was unable to inhibit proteolysis in [(14)C]S-HRP loaded lysosomes or interfere in cathepsin assays. The results suggest that TC inhibits the lysosomal degradation of endocytosed proteins in hepatocytes and that the mechanism does not involve an effect of the bile salt per se or a rise in lysosomal pH.     

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.     

Ntlim1, a PAL-box binding factor,controls promoter activity of the horseradish wound-inducible peroxidase gene

To understand molecular mechanisms underlying wound-induced expression of plant peroxidase genes, the promoter of a horseradish C2 peroxidase (prxC2) gene was analyzed. We had previously isolated a tobacco nuclear protein, Ntlim1, as a trans factor binding to a PAL-box motif of the prxC2 promoter; however, the function of the Ntlim1 trans factor and the PAL-box motif in wound-responsive expression of the prxC2 gene remains unclear. Here, we found that the prxC2 promoter without the intact PAL-box motif failed to direct a normal level of both the basal and the wound-induced expression of -glucuronidase (GUS) reporter gene in transgenic tobacco plants, indicating that the PAL-box motif functions as an essential cis element of the prxC2 promoter. We also found that antisense expression of Ntlim1 in transgenic plants carrying the prxC2 promoter::GUS chimeric construct decreased not only the level of the basal and the wound-induced expression of the GUSreporter gene but also the extent of wound inducibility of the prxC2 promoter itself. This result indicates that Ntlim1 is required for the basal level of prxC2 promoter activity as well as its up-regulation under wound stress. Moreover, consistent with the results obtained in planta, result from super-shift assay indicates that the Ntlim1 binds to the PAL-box motif independently of wound stress.     

Indoleacetate oxidase activity of horseradish and other plant peroxidase isozymes

Zymographic analyses were carried out on a commercial peroxidasepreparation of horseradish (Cochlearia armorada) and crude extractsfrom several plant materials, such as roots of turnip, radish,spinach, sweet potato and morning glory, regarding the dualcatalytic activities of peroxidase- IA oxidase isozymes. A standardreacting mixture for the oxidase stain contained IA, TCP andFast Blue BB salt as substrate, promoting agent and dye coupler,respectively. An unidentified intermediate (or intermediates)resulting from enzymatic IA degradation was demonstrated tobe coupled with the diazonium salt and to form an insolublecomplex. At least four cathodal and four anodal peroxidase bandswere located in the horseradish preparation, the former appearingas dark IA oxidase bands and the latter as very faint bands. In the extracts from plant materials, less than half of thetotal peroxidase bands appeared as heavy to faint IA oxidasebands, whereas the remaining ones did not appear in the samecondition as that for the horseradish IA oxidase stain. Furthermore,it was noted that the intensity of each of the peroxidase bandswas not always correlated with that of the corresponding oxidasebands. ¹Contribution No. 683 from the National Institute of Genetics,Misima.     

Adsorption behavior and activity of horseradish peroxidase onto polysaccharide-decorated particles

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.     

Thermodynamic analysis of the binding of aromatic hydroxamic acid analogues to ferric horseradish peroxidase.

Peroxidases typically bind their reducing substrates weakly, with K(d) values in the millimolar range. The binding of benzhydroxamic acid (BHA) to ferric horseradish peroxidase isoenzyme C (HRPC) [K(d) = 2.4 microM; Schonbaum, G. R. (1973) J. Biol. Chem. 248, 502-511] is a notable exception and has provided a useful tool for probing the environment of the peroxidase aromatic-donor-binding site and the distal heme cavity. Knowledge of the underlying thermodynamic driving forces is key to understanding the roles of the various H-bonding and hydrophobic interactions in substrate binding. The isothermal titration calorimetry results of this study on the binding of aromatic hydroxamic acid analogues to ferric HRPC under nonturnover conditions (no H(2)O(2) present) confirm the significance of H-bonding interactions in the distal heme cavity in complex stabilization. For example, the binding of BHA to HRPC is enthalpically driven at pH 7.0, with the H-bond to the distal Arg38 providing the largest contribution (6.74 kcal/mol) to the binding energy. The overall relatively weak binding of the hydroxamic acid analogues to HRPC is due to large entropic barriers (-11.3 to -37.9 eu) around neutral pH, with the distal Arg38 acting as an "entropic gate keeper". Dramatic enthalpy-entropy compensation is observed for BHA and 2-naphthohydroxamic acid binding to HRPC at pH 4.0. The enthalpic loss and entropic gain are likely due to increased flexibility of Arg38 in the complexes at low pH and greater access by water to the active site. Since the Soret absorption band of HRPC is a sensitive probe of the binding of hydroxamic acids and their analogues, it was used to investigate the binding of six donor substrates over the pH range of 4-12. The negligible pH dependence of the K(d) values corrected for substrate ionization suggests that enthalpy-entropy compensation is operative over a wide pH range. Examination of the thermodynamics of binding of ring-substituted hyrazides to HRPC reveals that the binding affinities of aromatic donors are highly sensitive to the position and nature of the ring substituent.     

Partition of free and monoclonal-antibody-bound horseradish peroxidase in a two-phase aqueous polymer system--novel procedure for the determination of the apparent binding constant of monoclonal antibody to horseradish peroxidase.

The principle that the antigen and the antibody prefer different phases in an aqueous two-phase system is the analytical basis of the work presented here. The antigen horseradish peroxidase, which is bound to a monoclonal antibody (mAb), is separated from free Ag in an aqueous phase system (polyethylene glycol (PEG)/dextran) as a function of the concentration of mAb. The plot of the partition coefficient kappa of horseradish peroxidase versus the concentration of mAb yields a sigmoidal curve similar to the curve obtained by enzyme-linked immunosorbent assay (ELISA). Comparing the plots normally used for ELISA in order to determine the apparent binding constant of mAb and the number of epitopes on the Ag we derived a relationship between the difference in partitioning of the free Ag and the bound Ag (delta kappa) and the concentration of mAb. The new linear plot of reciprocal delta kappa versus reciprocal concentration of mAb gives the apparent binding constant of mAb, which is evaluated from the slope. From the intercept at the ordinate the maximum difference of the partition coefficient of the free and bound antigen is derived and the apparent partition coefficient of the free monoclonal antibody can be calculated.     

Interaction of aromatic donor molecules with horseradish peroxidase: identification of the binding site and role of heme iron in the binding and activity

The interaction of aromatic substrates with horseradish peroxidase (HRP) was studied. Chemical modification of HRP was performed using diethylpyrocarbonate (DEPC) and for the first time the amino acid involved in binding with these substrates has been identified. The kinetic parameters for this interaction have been calculated and the role of heme iron in the oxidation of aromatic substrates by HRP has been discussed.     

Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing

The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo-and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (alpha-, beta-, gamma-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0----S1 (0----0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by approximately 20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only beta-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0----0 transition energy for the tautomeric forms of MP and an increase in the 0----0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the delta v = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for beta-RE. In the same energy range, however, the transition into the delta nu = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA less than NHA less than HQ less than RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0----0 energies (from 60-70 to approximately 30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.     

Effect of cyclodextrin on the activity and secondary structure of horseradish peroxidase

The activity and secondary structure of horseradish peroxidase (HRP) was studied in aqueous solution containing alpha-, beta- and gamma-cyclodextrin (CD). The results showed that the activity of HRP was enhanced to different extents by the three kinds of CD. A Fourier Transform infrared (FTIR) spectroscopy study indicated that the amount of alpha-helical structure was important for the activity of HRP. This phenomenon is discussed.