Spectroscopic study on structure of horseradish peroxidase in water and dimethyl sulfoxide mixture

The structure of horseradish peroxidase (HRP) in phosphate buffered saline (PBS)/dimethyl sulfoxide (DMSO) mixed solvents at different compositions is investigated by IR, electronic absorption, and fluorescence spectroscopies. The fluorescence spectra and the amide I spectra of ferric HRP [HRP(Fe3+)] show that overall structural changes are relatively small up to 60% DMSO. Although the amide I band of HRP(Fe3+) shows a gradual change in the secondary structure and a decrease in the contents of a helices, its fluorescence spectra indicate that the distance between the heme and Trp173 is almost constant. In contrast, the changes in the positions of the Soret bands for resting HRP(Fe3+) and catalytic intermediates (compounds I and II) and the IR spectra at the C-O stretching vibration mode of carbonyl ferrous HRP [HRP(Fe2+)-CO] show that the microenvironment in the distal heme pocket is altered, even with low DMSO contents. The large reduction of the catalytic activity of HRP even at low DMSO contents can be attributed to the structural transition in the distal heme pocket. In PBS/DMSO mixtures containing more than 70 vol % DMSO, HRP undergoes large structural changes, including a large loss of the secondary structure and a dissociation of the heme from the apoprotein. The presence of the components of the amide I band that can be assigned to strongly hydrogen bonding amide C=O groups at 1616 and 1684 cm(-1) suggests that the denatured HRP may aggregate through strong hydrogen bonds.     

Effect of temperature on structure and functional properties of horseradish peroxidase

The dynamics of structural and functional changes proceeding in a peroxidase molecule under the effect of temperature was studied. It was shown that peroxidase thermoinactivation proceeds in two consequent stages. Based on the analysis of enzyme peroxidase and oxidase activity and peroxidase spectral and buffer characteristics, it was established that at temperatures from 20 to 55 degrees C reversible conformation there occur changes of the hemoprotein molecule related to consequent unfolding and folding of the protein globule. The influence of temperature of 60 degrees C and above induces the protein globule unfolding and loosing of peroxidase activity.     

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.     

Heme-environment of horseradish peroxidase as observed by oxygen-17 superhyperfine interaction in EPR.

The presence of a water ligand on heme-iron in ferric hemoproteins can, in suitable cases, be detected by observing ¹⁷O superhyperfine interaction in the EPR spectra of solutions in H₂¹⁷O. Although no significant superhyperfine interaction is detectable in the EPR spectra of horseradish peroxidase itself, benzo-hydroxamic acid, which forms an outersphere complex with the enzyme analogous to an enzyme-peracid transition state, stabilizes an innersphere water ligand on the heme, as indicated by a ～1.3 gauss Fe³⁺-¹⁷O superhyperfine interaction in the EPR signal at g = 2, in the presence of 34–39% H₂¹⁷O at 8°K. These results indicate that the predominantly pentacoordinate Fe³⁺ ion in horseradish peroxidase is accessible to the solvent and that it acquires a water or hydroxyl ligand in the presence of benzohydroxamic acid.     

Synthesis and properties of horseradish peroxidase copolymers

Conditions for copolymerization of native and sodium periodate-oxidized horseradish peroxidase (HTP; EC 1.11.1.7) have been optimized. Copolymerization products have been characterized electrophoretically, spectrally, and kinetically. Copolymers containing 2-3, 4, 5-7, and 9-10 molecules of the enzyme were found among the products of polymerization. The copolymers had lower values of D403/D280 than HRP. The copolymers had more ordered structures than the original HRP. Comparison of the thermal stability and kinetic characteristics of the fractions differing in the ratio of copolymers to the monomeric enzyme demonstrated that the polymeric products were more stable than HRP (in terms of resistance to high temperature or inhibitory effects of H202), but their kinetic activity was, on the whole, lower than that of the original enzyme.     

Synthesis and properties of horseradish peroxidase copolymers

Conditions for copolymerization of native and sodium periodate-oxidized horseradish peroxidase (HTP; EC 1.11.1.7) have been optimized. Copolymerization products have been characterized electrophoretically, spectrally, and kinetically. Copolymers containing 2–3, 4, 5–7, and 9–10 molecules of the enzyme were found among the products of polymerization. The copolymers had lower values of D ₄₀₃/D ₂₈₀ than HRP. The copolymers had more ordered structures than the original HRP. Comparison of the thermal stability and kinetic characteristics of the fractions differing in the ratio of copolymers to the monomeric enzyme demonstrated that the polymeric products were more stable than HRP (in terms of resistance to high temperature or inhibitory effects of H₂O₂), but their kinetic activity was, on the whole, lower than that of the original enzyme.     

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.     

Preparation and properties of matrix-supported horseradish peroxidase.

A new method of enzyme immobilization has been described using poly(4-methacryloxybenzoic acid) as the carrier. Activation of the polymer, prior to enzyme attachment, was achieved with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The enzyme coupling step proceeded through nucleophilic attack by the protein on a mixed carbonic anhydride. The degree of polymer activation was determined by analysis for quinoline, a by-product of the reaction. The polymer-enzyme complex was compared to the enzyme in solution in terms of pH optimum, substrate kinetics, and thermal denaturation. Potential uses of the polymerenzyme system in chemical synthesis of benzoquinone derivatives are discussed.     

Time-resolved and static resonance Raman spectroscopy of horseradish peroxidase intermediates

By using pulsed and continuous wave laser irradiation in the 350-450-nm region, we have characterized Raman scattering from horseradish peroxidase (HRP) compounds I and II and from iron porphyrin pi-cation radical model compounds. For compound II we support the suggestion [Terner, J., Sitter, A. J., & Reczek, C. M. (1985) Biochim. Biophys. Acta 828, 73-80; Proniewicz, L. M., Bajdor, K., & Nakamoto, K. (1986) J. Phys. Chem. 90, 1760-1766] that resonance enhancement of the FeIV = O vibration proceeds by way of a charge-transfer state. Our excitation profile data locate this state at approximately 400 nm. Compound I was prepared at neutral pH by rapid mixing of the resting enzyme with hydrogen peroxide. Each sample aliquot was excited by a single, 10-ns laser pulse to generate the Raman spectrum; optical spectroscopy following the Raman measurement confirmed that HRP-I was the principal product during the time scale of the measurement. The Raman spectrum of this species, however, is not characteristic of that which we observe from metalloporphyrin pi-cation radicals [Oertling, W. A., Salehi, A., Chung, Y., Leroi, G. E., Chang, C. K., & Babcock, G. T. (1987) J. Phys. Chem. 91, 5887-5898], including the iron porphyrin cation radicals reported here. Instead, the spectrum recorded for HRP-I at neutral pH is suggestive of an oxoferryl heme with the same geometric and electronic structure as that of HRP-II at high pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cobalt hexaamine mediated electrocatalytic voltammetry of dimethyl sulfoxide reductase: driving force effects on catalysis

The bacterial molybdoenzyme dimethyl sulfoxide (DMSO) reductase from Rhodobacter capsulatus catalyzes the reduction of DMSO to dimethyl sulfide in anaerobic respiration. In its native state, DMSO reductase is reduced to its active state by a pentaheme cytochrome (DorC). Alternatively, we show that DMSO reductase catalysis may be driven electrochemically using a series of homologous coordination compounds as mediating synthetic electron donors. All mediators are macrocyclic hexaaminecobalt(II) complexes in their active form, differing principally in their redox potentials over a range of about 250 mV. Thus, each complex presents a different reductive driving force to DMSO reductase and this leads to pronounced differences in the electrocatalytic behavior as measured by cyclic voltammetry. Digital simulation of the experimental voltammetry enables the critical features of the catalytic cycle to be extracted.     

Effect of dimethyl sulfoxide on phosphoryl transfer catalyzed by yeast hexokinase.

Hexokinase is a phosphotransferase that catalyzes phosphoryl transfer from ATP to glucose much more rapidly than the transfer from ATP to water (i.e., hydrolysis). Dimethyl sulfoxide has opposite effects on these two phosphotransferase activities: it enhances ATP hydrolysis and inhibits glucose phosphorylation. Xylose, a sugar that is non-phosphorylatable by hexokinase, enhances ATPase activity which is additive to activation by dimethyl sulfoxide, indicating that the mechanism of activation by dimethyl sulfoxide is different from that of xylose. These results suggest that it is possible to change the specificity of the enzyme in the presence of dimethyl sulfoxide.     

Effect of dimethyl sulfoxide on transformed rat Schwann cells

Cultured rat Schwann cells transformed by Simian Virus 40 (SV40) have previously been shown to retain their ability to synthesize myelin-associated galactosylceramide and sulfatide. Little is known about the mechanism regulating galactosphingolipid synthesis in Schwann cells. We have found that growing the transformed Schwann cells in the presence of dimethyl sulfoxide (DMSO) markedly inhibits the incorporation of [35S]sulfate into sulfatide, in a time- and dose-dependent manner. The concentration of DMSO which resulted in a half-maximal inhibition after 6 days of incubation was 0.5%, and the incubation time required for a half-maximal effect at 1.0% DMSO was approximately 4 days. In contrast, DMSC did not affect the incorporation of [35S]sulfate into glycosaminoglycans. In addition, DMSO treatment has little effect on the synthesis of cellular DNA, proteins and lipids. When transformed Schwann cells were treated with DMSO, a substantial decrease in the incorporation of [3H]galactose into galactosylceramide was observed. The concentration of DMSO which resulted in a half-maximal inhibition of galactosylceramide synthesis was approximately 0.5%, similar to the concentration required for a similar effect on sulfatide synthesis. However, the incubation time required for a half-maximal inhibitory effect on galactosylceramide synthesis at 1.0% DMSO was less than 1 day, which was substantially shorter than the time required for the inhibition of sulfatide synthesis at this concentration. This finding is consistent with the interpretation that treatment with DMSO inhibits the synthesis of galactosylceramide, a precursor of sulfatide, which results in a decrease in the synthesis of sulfatide during a prolonged incubation of DMSO.     

Effect of N-alkylimidazoles on oxidation of o-dianizidine catalyzed by horseradish peroxidase]

Effect of a number of N-alkylimidazoles (from N-methyl to N-octylimidazole) on peroxidase oxidation of o-dianizidine at pH 8.0 is studied. Alkylimidazoles studied are found to activate the reaction under given conditions, the activation type being non-competitive KA and (alpha) values are similar for all the activators, which suggests a similar mechanism of their action. Similar KA values suppose an insignificant role of hydrophobic interactions in the binding of N-alkylimidazoles with the enzyme.     

Comparison of photovoltaic behaviors for horseradish peroxidase and its mimicry by surface photovoltage spectroscopy

Surface photovoltage spectroscopy (SPS) was chosen to study the photovoltaic behavior of horseradish peroxidase (HRP), hemin and immobilized hemin (poly(NIPAAm/MBA/hemin)). Different photovoltaic behaviors were observed in these three systems. In air, similar SPS curves were found for HRP and poly(NIPAAm/MBA/hemin) with different response intensities. However, poly(NIPAAm/MBA/hemin) showed a wider changing range upon increasing the positive and negative bias to 1.0 V. The SPS of hemin showed a total different behavior when an external positive potential was applied. In vacuum, clearly different photovoltaic behaviors were found. Moreover, the response value decreased when HRP was exposed to O2, the SPS intensity was different from that in air, and could be altered by changing the external biases. On the other hand, the SPS could not be changed before and after poly(NIPAAm/MBA/hemin) was exposed to O2. These differences may result from different chemical microenvironments for hemin in HRP versus that in poly(NIPAAm/MBA/hemin). It could be concluded that H2O and O2 were important factors affecting the photovoltage response in HRP, but only H2O played this important role in poly(NIPAAm/MBA/hemin).