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Single-walled carbon nanotubes (SWNTs) have been investigated for a variety of applications including composite materials, electronics, and drug delivery. However, these applications may be compromised depending on the negative effects of SWNTs to living systems. While reports of toxicity induced by SWNTs vary, means to alleviate or quell these effects are in small abundance. We have reported recently the degradation of carboxylated SWNTs through enzymatic catalysis with horseradish peroxidase (HRP). In this full Article, we investigated the degradation of both carboxylated and pristine SWNTs with HRP and compared these results with chemical degradation by hemin and FeCl3. The interaction between pristine and carboxylated SWNTs with HRP was further studied by computer modeling, and the products of the enzymatic degradation were identified. By examining these factors with both pristine and carboxylated SWNTs through a variety of techniques including atomic force microscopy (AFM), transmission electron microscopy (TEM), Raman spectroscopy, ultraviolet-visible-near-infrared (UV-vis-NIR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS), degradation pathways were elucidated. It was observed that pristine SWNTs demonstrate no degradation with HRP incubation but display significant degradation when incubated with either hemin or FeCl3. Such data signify a heterolytic cleavage of H2O2 with HRP as pristine nanotubes do not degrade, whereas Fenton catalysis results in the homolytic cleavage of H2O2 producing free radicals that oxidize pristine SWNTs. Product analysis shows complete degradation produces CO2 gas. Conversely, incomplete degradation results in the formation of different oxidized aromatic hydrocarbons. [ABSTRACT FROM AUTHOR]

The effect of all possible mutations at position 178 on the enantioselectivity of yeast surface- bound horseradish peroxidase (HRP) toward chiral phenols has been investigated. In contrast to their wild- type predecessor, most HAP mutants are enantioselective, with the Arg178Glu variant exhibiting the greatest, 25-fold, (S)/(R) preference. Using kinetic analysis of enzymatic oxidation of various substrate analogues and molecular modeling of enzyme-substrate complexes, this enantioselectivity enhancement is attributed to changes in the transition state energy due to electrostatic repulsion between the carboxylates of the enzyme's Glu178 and the substrate's (R)-enantiomer. [ABSTRACT FROM AUTHOR]

Femtosecond coherence spectroscopy is used to probe the low-frequency (20-200 cm-1) vibrational modes of heme proteins in solution. Horseradish peroxidase (HRP), myoglobin (Mb), and Campylobacter jejuni globin (Cgb) are compared and significant differences in the coherence spectra are revealed. It is concluded that hydrogen bonding and ligand charge do not strongly affect the low-frequency coherence spectra and that protein-specific deformations of the heme group lower its symmetry and control the relative spectral intensities. Such deformations potentially provide a means for proteins to tune heme reaction coordinates, so that they can perform a broad array of specific functions. Native HRP displays complex spectral behavior above ~50 cm-1 and very weak activity below `-~50 cm-1. Binding of the substrate analog, benzhydroxamic acid, leads to distinct changes in the coherence and Raman spectra of HRP that are consistent with the stabilization of a heme water ligand. The ON derivatives of the three proteins are studied to make comparisons under conditions of uniform heme coordination and spin-state. MbCN is dominated by a doming mode near 40 cm-1, while HRPCN displays a strong oscillation at higher frequency (96 cm') that can be correlated with the saddling distortion observed in the X-ray structure. In contrast, CgbCN displays low-frequency coherence spectra that contain strong modes near 30 and 80 cm-1, probably associated with a combination of heme doming and ruffling. HRPNO displays a strong doming mode near 40 cm-1 that is activated by photolysis. The damping of the coherent motions is significantly reduced when the heme is shielded from solvent fluctuations by the protein material and reduced still further when T ≲ 50 K, as pure dephasing processes due to the protein-solvent phonon bath are frozen out. [ABSTRACT FROM AUTHOR]

A new class of materials was prepared from aniline-containing oligomers that are covalently linked to the nucleobases of duplex DNA. Oligomers composed of repeating aniline (PANI) or 4-aminobiphenyl (PAB) units having the properties of conducting polymers conjoined to the DNA were prepared by the reaction of horseradish peroxidase and H2O2 with DNA having the appropriate monomers aligned within the major groove. These oligomers exhibit the spectral and chemical properties typical of para-linked polyanilines. This method of preparation enables utilization of the unique self-recognizing properties and sequence programmability of DNA to create tailored oligomers. This ability was demonstrated experimentally by preparation of PAB oligomers from alternating benzene and aniline monomers. Conjoined conducting polymers carrying the sequence information of DNA may have applicability as nanowires. [ABSTRACT FROM AUTHOR]

Quantum mechanics/molecular mechanics calculations were utilized to study the process of oxidation of a native substrate (ferulic acid) by the active species of horseradish peroxidase (Dunford, H. B. Heme Peroxidases; Wiley-VCH: New York, 1999), Compound I and Compound II, and the manner by which the enzyme returns to its resting state. The results match experimental findings and reveal additional novel features. The calculations demonstrate that both oxidation processes are initiated by a proton-coupled electron-transfer (PCET) step, in which the active species of the enzyme participate only as electron-transfer partners, while the entire proton-transfer event is being relayed from the substrate to and from the His42 residue by a water molecule (W402). The reason for the observed (Henriksen, A; Smith, A. T.; Gajhede, M. J. Biol. Chem. 1999, 274, 35005–35011) similar reactivities of Compound I and Compound II toward ferulic acid is that the reactive isomer of Compound II is the, hitherto unobserved, Por+FeIIIOH isomer that resembles Compound I. The PCET mechanism reveals that His42 and W402 are crucial moieties and they determine the function of the HRP enzyme and account for its ability to perform substrate oxidation (Poulos, T. L. Peroxidases and Cytochrome P450. In The Porphyrin Handboolc,, Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press: New York, 2000; Vol. 4, pp 189). In view of the results, the possibility of manipulating substrate oxidation by magnetic fields is an intriguing possibility. [ABSTRACT FROM AUTHOR]

The mammalian peroxidases eosinophil peroxidase, lactoperoxidase (LPO), and myeloperoxidase oxidize thiocyanate to the antimicrobial agents hypothiocyanous acid (HOSCN) and (SCN)2 and are part of a defense system that protects the host from infections. Horseradish peroxidase (HRP), a plant enzyme, also oxidizes thiocyanate. We report here that the prosthetic heme vinyl groups of HAP react with the catalytically generated HOSCN and (SCN)2 to form at least nine vinyl-modified heme adducts. Mass spectrometry combined with analysis of the equivalent reactions of HRP reconstituted with 2- or 4-cyclopropylheme, or mesoheme-d4, shows that all of the prosthetic heme modifications result from addition of oxidized thiocyanate to the heme vinyl groups. No δ-meso-substitution of the heme was observed, in contrast to what is observed with radical agents. Model studies show that incubation of either HRP with preformed HOSCN or a solution of heme with preformed (SCN)2 gives rise to the same products obtained in the HRP-catalyzed reaction. Model studies also demonstrate that the SCN· radical, if formed, should add to a meso-carbon. These findings implicate an electrophilic addition mechanism. In contrast, oxidation by LPO of thiocyanate, the normal substrate of this enzyme, does not result in heme modification. In view of the demonstrated intrinsic reactivity of the heme group, LPO must actively suppress heme modification. As the key difference between LPO (and other mammalian peroxidases) and HRP is the presence of two covalent ester links between the heme and the protein, we propose that these links contribute to steric protection of the adjacent heme vinyl groups. [ABSTRACT FROM AUTHOR]

In this paper an in depth study is presented of the use of the horseradish peroxidase (HRP) enzyme as a self-indicating biorecognition reagent in UV--vis molecular absorption spectrometry. The HRP/H2O2 reaction mechanism in the absence of an external substrate has been clarified, and the interaction between HRP and glucose oxidase (GOx) has been studied. It has been demonstrated that GOx can act as a substrate of HRP; in both cases the kinetic constants have been obtained and mathematical models have been developed. Second, the HRP/H2O2 reaction is used to follow a H2O2-producing enzymatic reaction, the glucose reaction with GOx being used as a model. As an application of this, two methodologies have been proposed for glucose determination: with or without previous incubation of glucose with GOx. In both cases mathematical models relating HRP absorbarice changes to glucose concentration have been developed and tested; both methods have been optimized, analytically characterized, and tested for glucose determination in samples. The methodology described could be applied to other heme-proteins and to other H2O2-producing enzymatic reactions. The models permit the reaction constants to be calculated. From the analytical chemistry point of view the models allow the prediction of the method sensitivity for other analytes involved in this type of reaction if the kinetic constants are known and can be used in the design of optical sensors. [ABSTRACT FROM AUTHOR]

Umecyanin (UMC) is a type 1 copper-containing protein which originates from horseradish roots and belongs to the stellacyanin subclass of the phytocyanins, a ubiquitous family of plant cupredoxins. The crystal. structures of Cu(II) and Cu(I) UMC have been determined at 1.9 and 1.8 Å, respectively. The protein has an overall fold similar to those of other phytocyanins. At the active site the cupric ion is coordinated by the Nδ1 atoms of His44 and His90, the Sγ of Cys85, and the Oϵ1 of Gln95 in a distorted tetrahedral geometry. Both His ligands are solvent exposed and are surrounded by nonpolar and polar side chains on the protein surface. Thus, UMC does not possess a distinct hydrophobic patch close to the active site in contrast to almost all other cupredoxins. UMC has a large surface acidic patch situated ∼ 10-30 Å from the active site. The structure of Cu(I) UMC is the first determined for a reduced phytocyanin and demonstrates that the coordination environment of the cuprous ion is more trigonal pyramidal. This subtle change in geometry is primarily due to the Cu--Nδ1(His44) and Cu--Oϵ1(Gln95) bond lengths increasing from 2.0 and 2.3 Å in Cu(II) UMC to 2.2 and 2.5 Å, respectively, in the reduced form, as a consequence of slight rotations of the His44 and Gln95 side chains. The limited structural changes upon redox interconversion at the active site of this stellacyanin are analogous to those observed in a typical type 1 copper site with an axial Met ligand and along with its surface features suggest a role for UMC in interprotein electron transfer. [ABSTRACT FROM AUTHOR]

The type 1 copper sites of cupredoxins typically have a His2Cys equatorial ligand set with a weakly interacting axial Met, giving a distorted tetrahedral geometry. Natural variations to this coordination environment are known, and we have utilized paramagnetic ¹H NMR spectroscopy to study the active-site structure of umecyanin (UMC), a stellacyanin with an axial Gin ligand. The assigned spectra of the Cu(II) UMC and its Ni(II) derivative [Ni(II) UMC] demonstrate that this protein has the typical His2Cys equatorial coordination observed in other structurally characterized cupredoxins. The NMR spectrum of the Cu(II) protein does not exhibit any paramagnetically shifted resonances from the axial ligand, showing that this residue does not contribute to the singly occupied molecular orbital (SOMO) in Cu(II) UMC. The assigned paramagnetic ¹H NMR spectrum of Ni(II) UMC demonstrates that the axial Gin ligand coordinates in a monodentate fashion via its side-chain amide oxygen atom. The alkaline transition, a feature common to stellacyanins, influences all of the ligating residues but does not alter the coordination mode of the axial Gin ligand in UMC. The structural features which result in Cu(II) UMC possessing a classic type 1 site as compared to the perturbed type 1 center observed for other stellacyanins do not have a significant influence on the paramagnetic ¹H NMR spectra of the Cu(II) or Ni(II) proteins. [ABSTRACT FROM AUTHOR]

The active site of HRP Compound I(CpdI) is modeled Using hybrid density functional theory (UB3LYP). The effects of neighboring amino acids and of environmental polarity are included. The low-lying states have porphyrin radical cationic species (Por[SUP.+]). However, since the Por[SUP.+] species is a very good electron acceptor, other species, which can be either the ligand or side chain amino acid residues, may participate in electron donation to the Por[SUP.+] moiety, thereby making Cpd I behave like a chemical chameleon. Thus, this behavior that was noted before for Cpd I of P450 is apparently much more wide ranging than initially appreciated. Since chemical chameleonic behavior property was found to be expressed not only in the properties of Cpd I itself, but also in its reactivity, the roots of this phenomenon are generalized. A comparative discussion of Cpd I species follows for the enzymes HRP, CcP, APX, CAT (catalase), and P450. [ABSTRACT FROM AUTHOR]

Investigates the oxidation of N-cyclopropyl-N-methylaniline by horseradish peroxidase, a well-known single electron transfer (SET) enzyme. Formation of beta-hydroxypropionaldehyde, acrolein, nor cyclopropane hydrate as SET metabolites; Facilitation of the application of cyclopropylamine-containing probes to reactions catalyzed by cytochrome P450 enzymes.

Focuses on the role of protein environment in the formation of horseradish peroxidase compound I. Nature and stability of H bonds between peroxides and residues; Energy minimization and molecular dynamics simulations; Oxygen as the ligand for the iron.

Analyzes the photoinduced oxidation of horseradish peroxidase. Use of laser-flash transient spectroscopy; Presentation of distinct electronic spectral features; Steady-state photolysis; Description of typical transient kinetics; Comparison of the spectral changes and kinetics for oxidation.

Reports initial findings related to the optical spectroscopic study of pegylated horseradish peroxidase and its intermediates in organic media. Lyophilization; Determination of the extent of modification; Moisture determination; Determination of hydrogen peroxide concentration in neat benzene solution; Optical absorption and circular dichorism measurements; Raman spectroscopy.

Presents a Resonance Raman study of ferric and ferrous carbon monoxide forms of polyethylene glycolated horseradish peroxidase (HRPPEG) in benzene solution. Focus of other spectroscopic studies conducted; Identification of heme prosthetic group as an active site in a number of metalloproteins and enzymes; Results and discussion of the study.

The article focuses on a study that presents evidence on the result of very low magnetic fields on horseradish peroxidase (HRP). Evidence suggest that a magnetosensitive pace in the catalytic round of HRP is in fact existing. Moreover, a 30-fold increment on the HRP reaction rate was noted in the enzyme assay.

The article examines the effect of a water molecule in the formation of compound 0 in horseradish peroxide. The researchers have found that a single water molecule has a profound effect on the thermodynamic and kinetic acidities of hydrogen dioxide without hampering the follow-up formation of Cpd 1. Furthermore, the catalytic effect reflects the ease of partner switching in hydrogen bonding and highlights the importance of hydrogen bonding networks in enzymes.

The article presents information on a study which determines the magnetic field effects in the haem-dependent enzyme horseradish peroxidase (HRP). The study shows that the application of external magnetic fields can perturb reaction kinetics through the constraints of the radical pair mechanism. The study findings suggest that relatively modest fields, including those of environmentally realistic magnitude, can influence the yields and rates of radical pair. According to the researchers, the reaction catalyzed by HRP includes oxidative and reductive half reactions.