Significant Peroxidase Activity Research Articles

In vitro assessment of thyroid peroxidase inhibition by chemical exposure: comparison of cell models and detection methods

Disruption of the thyroid hormone (TH) system is connected with diverse adverse health outcomes in wildlife and humans. It is crucial to develop and validate suitable in vitro assays capable of measuring the disruption of the thyroid hormone (TH) system. These assays are also essential to comply with the 3R principles, aiming to replace the ex vivo tests often utilised in the chemical assessment. We compared the two commonly used assays applicable for high throughput screening [Luminol and Amplex UltraRed (AUR)] for the assessment of inhibition of thyroid peroxidase (TPO, a crucial enzyme in TH synthesis) using several cell lines and 21 compounds from different use categories. As the investigated cell lines derived from human and rat thyroid showed low or undetectable TPO expression, we developed a series of novel cell lines overexpressing human TPO protein. The HEK-TPOA7 model was prioritised for further research based on the high and stable TPO gene and protein expression. Notably, the Luminol assay detected significant peroxidase activity and signal inhibition even in Nthy-ori 3-1 and HEK293T cell lines without TPO expression, revealing its lack of specificity. Conversely, the AUR assay was specific to TPO activity. Nevertheless, despite the different specificity, both assays identified similar peroxidation inhibitors. Over half of the tested chemicals with diverse structures and from different use groups caused TPO inhibition, including some widespread environmental contaminants suggesting a potential impact of environmental chemicals on TH synthesis. Furthermore, in silico SeqAPASS analysis confirmed the high similarity of human TPO across mammals and other vertebrate classes, suggesting the applicability of HEK-TPOA7 model findings to other vertebrates.

Facile one-pot synthesis of the mesoporous chitosan-coated cobalt ferrite nanozyme as an antibacterial and MRI contrast agent.

Cobalt ferrite (CoFe) nanoparticles (NPs) with appropriate physicochemical and biological properties have attracted great attention for biomedical applications. In the present study, chitosan-coated mesoporous CoFe (CoFeCH) NPs were synthesized using a facile one-step hydrothermal method and fully characterized using FE-SEM, EDS, BET, FTIR spectroscopy, DLS, TGA, XRD, and VSM. The spherical, highly colloidal, and monodispersed CoFeCH NPs with an average hydrodynamic size of 177.9 nm, PDI of 0.238 and zeta potential value of -33 represented a high saturation magnetization value of 59.37 emu g-1. N2 adsorption-desorption analysis confirmed the mesoporous structure of CoFeCH NPs with a type IV isotherm, calculated specific surface area of 89.583 m2 g-1 and total pore volume of 0.3668 cm3 g-1. CoFeCH NPs exhibited high antibacterial effects on S. aureus and E. coli, comparable with standard antibiotics, while CH-coating led to higher biocompatibility of CoFe NPs on human cells in vitro. CoFeCH NPs also showed significant peroxidase activity with a Km value of 14.37 and specific activity of 0.632 mmol min-1. CoFeCH NPs were successfully used as a MRI contrast agent with an R2 value of 91.3 mM-1 s-1. The overall results indicated the high potential of synthesized CoFeCH NPs by the present method for biomedical applications, especially as an antibacterial and MRI contrast agent.

OSCILLATING CHARACTER OF HEAT AND COLD SHOCK PROTEIN CONTENT OF OATS PLANTS IN THE CONDITIONS OF STRESSFUL FACTORS OF THE ENVIRONMENT

The article presents the results of research on the gene expression systems of adaptation of sowing oats from the different ecological and geographical origins at low and high temperatures. Specific cold and heat shock proteins were isolated. Significant peroxidase activity was found in Ivory and Neptune varieties, which characterizes their increased resistance to cold oxidative stress. It was found that the varieties Ivory, Arkan and Neptune are resistant to high temperature and water stress, due to the higher content of heat shock proteins with a molecular weight of 80, 70 and 60 kDa. The content of high-molecular proteins (250 kDa) and proteins with a molecular weight of 110, 100, 80 and 70 kDa has decreased in the Hesser variety, which makes it possible to classify this variety as a low-resistance variety. Higher peroxidase activity was found in Ivory, Arkan and Neptune varieties, which characterizes their increased resistance to oxidative stress in winter and summer It is shown that Ivory and Neptune varieties have high protein expression and are highly frost- and drought-resistant, Arkan and Arthur – moderately frost- and drought-resistant, and Gesser variety has low resistance to stressful environmental conditions, which somewhat limits its use in Ukraine; It is found that the highest peroxidase activity in optimal growing conditions and under the action of water and high temperature stress have varieties Ivory grain purpose and Neptune – fodder purpose, which allows to recommend these varieties for growing in the Forest-Steppe and Steppe of Ukraine.

ЭФФЕКТИВНЫЕ БИОМАРКЕРЫ В ДИАГНОСТИКЕ ПИЩЕВОЙ АЛЛЕРГИИ НА ЯБЛОКО И ИХ СРАВНИТЕЛЬНАЯ ОЦЕНКА

Objectives. To evaluate the diagnostic efficacy of the prick-test and the levels of tryptase, cations K<sup>+</sup>, NH<sub>4</sub><sup>+</sup> and Ca<sup>2+</sup>, peroxidase activity in the oral fluid (OF) as biomarkers of apple allergy and to comparatively assess them. Material and methods. We examined 35 patients with a history of allergic reactions to a fresh apple (the study group) and 29 healthy persons (the control group). All participants underwent the prick-test with 3 varieties of fresh apples and oral-pharyngeal provocative test (OPPT) with freshly prepared apple juice with the assessment of tryptase, peroxidase activity, K<sup>+</sup>, NH<sub>4</sub><sup>+</sup> and Ca<sup>2+</sup> cations levels in the OF. Results. The prick-test was positive in 94% of patients. In 70% of patients after OPPT, there was a significant tryptase level increase in the OF compared with healthy persons (p=0.000003). After provocation, in the study group, in 74% of cases, a significant peroxidase activity level increase in the OF was revealed in comparison with the control group (p<0.0000001). A significant K<sup>+</sup> level increase in the oral fluid was found in 81% of patients, in contrast to healthy persons after OPPT (p=0.02). In 67% of cases in the study group, a significant NH<sub>4</sub><sup>+</sup> level decrease after OPPT was found in comparison with the initial cation level in this group (p=0.039). Patients with a history of apple allergy after provocation showed a higher Ca<sup>2+</sup> cations level in the OF than the control (p=0.02). Conclusions. Biomarkers of hypersensitivity to apples of the varieties Golden Delicious, Red Prince and Belarusian sweet were revealed: positive prick-test, an increase of tryptase, myeloperoxidase activity, K<sup>+</sup> cations and a decrease of NH<sub>4</sub><sup>+</sup> level after OPPT. Biomarkers have a high diagnostic value and can be used both in combination and separately to identify systemic and local hypersensitivity and to diagnose food allergy to apples.

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.

The inhibition of lactoperoxidase catalytic activity through mesna (2-mercaptoethane sodium sulfonate)

Here, we show that mesna (sodium-2-mercaptoethane sulfonate), primarily used to prevent nephrotoxicity and urinary tract toxicity caused by chemotherapeutic agents such as cyclophosphamide and ifosfamide, modulates the catalytic activity of lactoperoxidase (LPO) by binding tightly to the enzyme, functioning either as a one electron substrate for LPO Compounds I and II, destabilizing Compound III. Lactoperoxidase is a hemoprotein that utilizes hydrogen peroxide (H2O2) and thiocyanate (SCN−) to produce hypothiocyanous acid (HOSCN), an antimicrobial agent also thought to be associated with carcinogenesis. Our results revealed that mesna binds stably to LPO within the SCN− binding site, dependent of the heme iron moiety, and its combination with LPO-Fe(III) is associated with a disturbance in the water molecule network in the heme cavity. At low concentrations, mesna accelerated the formation and decay of LPO compound II via its ability to serve as a one electron substrate for LPO compounds I and II. At higher concentrations, mesna also accelerated the formation of Compound II but it decays to LPO-Fe(III) directly or through the formation of an intermediate, Compound I*, that displays characteristic spectrum similar to that of LPO Compound I. Mesna inhibits LPO's halogenation activity (IC50 value of 9.08 μM) by switching the reaction from a 2e− to a 1e− pathway, allowing the enzyme to function with significant peroxidase activity (conversion of H2O2 to H2O without generation of HOSCN). Collectively, mesna interaction with LPO may serve as a potential mechanism for modulating its steady-state catalysis, impacting the regulation of local inflammatory and infectious events.

The Met80Ala and Tyr67His/Met80Ala mutants of human cytochrome c shed light on the reciprocal role of Met80 and Tyr67 in regulating ligand access into the heme pocket

The spectroscopic and functional properties of the single Met80Ala and double Tyr67His/Met80Ala mutants of human cytochrome c have been investigated in their ferric and ferrous forms, and in the presence of different ligands, in order to clarify the reciprocal effect of these two residues in regulating the access of exogenous molecules into the heme pocket. In the ferric state, both mutants display an aquo high spin and a low spin species. The latter corresponds to an OH– ligand in Met80Ala but to a His in the double mutant. The existence of these two species is also reflected in the functional behavior of the mutants. The observation that (i) a significant peroxidase activity is present in the Met80Ala mutants, (ii) the substitution of the Tyr67 by His leads to only a slight increase of the peroxidase activity in the Tyr67His/Met80Ala double mutant with respect to wild type, while the Tyr67His mutant behaves as wild type, as previously reported, suggests that the peroxidase activity of cytochrome c is linked to an overall conformational change of the heme pocket and not only to the disappearance of the Fe-Met80 bond. Therefore, in human cytochrome c there is an interplay between the two residues at positions 67 and 80 that affects the conformation of the distal side of the heme pocket, and thus the sixth coordination of the heme.

Structure–function relationships in human cytochrome c: The role of tyrosine 67

Spectroscopic and functional properties of human cytochrome c and its Tyr67 residue mutants (i.e., Tyr67His and Tyr67Arg) have been investigated. In the case of the Tyr67His mutant, we have observed only a very limited structural alteration of the heme pocket and of the Ω-loop involving, among others, the residue Met80 and its bond with the heme iron. Conversely, in the Tyr67Arg mutant the Fe–Met80 bond is cleaved; consequently, a much more extensive structural alteration of the Ω-loop can be envisaged. The structural, and thus the functional modifications, of the Tyr67Arg mutant are present in both the ferric [Fe(III)] and the ferrous [Fe(II)] forms, indicating that the structural changes are independent of the heme iron oxidation state, depending instead on the type of substituting residue. Furthermore, a significant peroxidase activity is evident for the Tyr67Arg mutant, highlighting the role of Arg as a basic, positively charged residue at pH7.0, located in the heme distal pocket, which may act as an acid to cleave the O–O bond in H2O2. As a whole, our results indicate that a delicate equilibrium is associated with the spatial arrangement of the Ω-loop. Clearly, Arg, but not His, is able to stabilize and polarize the negative charge on the Fe(III)–OOH complex during the formation of Compound I, with important consequences on cytochrome peroxidation activity and its role in the apoptotic process, which is somewhat different in yeast and mammals.

Peroxidation and redox reactions catalyzed by truncated hemoglobins

Roseobacter denitrificans is one member of widespread marine Rosoebacters and performs photosynthesis and denitrification aerobically. We report a native heme‐bound R. denitrificans truncated hemoglobin (Rd. trHb) from heterologous expression in E. coli. Rd. trHb exhibits absorbance at 412, 538 and 572 nm bands with predominately alpha‐helical secondary structure. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares important heme pocket residues with both groups I and II trHbs. The resonance Raman spectra indicate that ferric Rd. trHb is mostly 6‐coordinate low‐spin and the ferrous form is a mixture of 5‐ and 6‐coordinated states. Two Fe‐His stretching modes were detected: one at 228 cm‐1, which represents an unconstrained neutral histidine as in other trHbs, and the other at 248 cm‐1, which has been reported in peroxidases and some flavohemoglobins that contain Fe‐His‐Asp (or Glu) catalytic triad, but not in trHbs. Further, Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than bovine Hb and only one order lower than horseradish peroxidase (HRP). The pH‐rate profiles yield a pKa value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme‐bound ligands in group II trHbs from M. tuberculosis and B. subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinct features and appears equipped to help Roseobacters to cope with reactive oxygen/nitrogen species and/or operate redox biochemistry.

Peroxidase activity and involvement in the oxidative stress response of roseobacter denitrificans truncated hemoglobin.

Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (k cat/K m) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pK a value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry.

A Self-Assembled Nano-Structrured Peroxidase Based on Sodium Dodecyl Sulfate Nano-Micelle and Cytochromec

A nano-structured supermolecule or artificial enzyme was built self-assembly based on sodium dodecyl sulfate nano-micelle and cytochrome c.A significant peroxidase activity was observed and reached the maximum value at pH 10.5.Its catalytic efficiency was evaluated to be 0.0219 μmol/L.s.The immobilized nano-structured peroxidase modified electrode showed quasi-reversible electrochemical redox behaviors with a formal potential of(-38±5) mV(vs.Ag/AgCl) at a scan rate of 0.05 V/s.The cathodic transfer coefficient and electron transfer rate constant were evaluated to be 0.51 and 0.59 s-1,respectively.The apparent Michaelis-Menten constant(Kmapp) was calculated to be 0.14 mmol/L.These results suggest that the nano-structured peroxidase not only perform a high activity as peroxidase and can be used in high concentration of hydrogen peroxide,but also can be immobilized on the electrode and realize direct electrochemical behavior.

Substrate binding triggers a switch in the iron coordination in dehaloperoxidase from Amphitrite Ornate

We have explored the effect of substrate binding on the heme iron conformation in the enzyme dehaloperoxidase (DHP). DHP is a dimeric hemoglobin that also has significant peroxidase activity under physiological conditions and has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a cosubstrate. Hyperfine sublevel correlation spectroscopic (HYSCORE) analysis of the ferric form of DHP was carried out to characterize effects of the substrate 2,4,6-trifluorophenol (TFP) binding on the iron coordination in order to elucidate molecular mechanisms responsible for switching the protein function from a globin to a peroxidase. The CW EPR spectrum shows that at pH 6.0 DHP heme iron exists in a highly axial high spin (HS) state that could be interpreted as arising from two different populations of the HS iron centers. Substrate binding does not change the spin state at pH 6.0, however, affects the magnetic parameters of the signal. HYSCORE spectra recorded at magnetic field corresponding to g = 2 revealed the presence of exchangeable protons with hyperfine coupling of ca. 6 MHz, consistent with a water molecule being the sixth ligand in the iron coordination. These protons' spectral features disappeared upon substrate binding. At pH 9.6 the EPR spectrum from heme iron of DHP shows the presence of both high- and low-spin states with the low spin signal characteristic of hydroxyl form. Upon TFP binding the low spin signal disappears. HYSCORE spectra at pH 9.6 also show the presence of exchangeable protons that disappear upon substrate binding. This observation highlights the proposed role of molecules in the distal pocket to control the peroxidase function of DHP.Supported by the NSF Grant MCB-0451510 to T.I.S. and ARO grant 52278-LS to S.F.

Structural and functional characterization of osmotically inducible protein C (OsmC) from Thermococcus kodakaraensis KOD1

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.

Reinvestigation of a Selenopeptide with Purportedly High Glutathione Peroxidase Activity

A 15-amino acid long selenopeptide (15SeP) was recently reported to possess nearly the same catalytic activity as glutathione peroxidase (Gpx) for the reduction of hydrogen peroxide by glutathione (Sun, Y., Li, T. Y., Chen, H., Zhang, K., Zheng, K. Y., Mu, Y., Yan, G. L., Li, W., Shen, J. C., and Luo, G. M. (2004) J. Biol. Chem. 279, 37235-37240). Such a finding is startling considering the high efficiency of the natural enzyme and the modest catalytic properties of most short peptides. As 15SeP had been subjected only to limited chemical characterization, we prepared it by a new route involving selenocysteine-mediated native chemical ligation. High resolution matrix-assisted laser desorption ionization mass spectrometry confirmed the identity of the reaction product, whereas circular dichroism spectroscopy showed that 15SeP assumes a random coil conformation in solution. Although low levels of peroxidase activity were detectable under standard assay conditions, the peptide is >5 orders of magnitude less active than native Gpx. Our observations are incompatible with claims ascribing remarkable catalytic properties to 15SeP and suggest that the efficiency of Gpx derives from its well defined three-dimensional structure.

Tyrosine–heme ligation in heme–peptide complex: design based on conserved motif of catalase

On the basis of evolutionary conservation of sequence in catalases, we have designed a heme-binding peptide (Ac-RLKSYTDTQISR12-(GGGG)-CRIVHC22-NH2) for the 'redox activity modulation' of heme. Heme-binding studies showed a blue-shifted Soret (369 nm) in the presence of TFE and a red-shifted Soret (418 nm) in the absence of TFE. These blue- and red-shifted Sorets suggest ligation through tyrosinate and histidine, respectively. This is the first designed peptide ligating to heme through tyrosine. NMR studies have confirmed that tyrosine ligation to heme in this heme-peptide complex occurs only in the presence of TFE. We suggest that TFE induces helicity in the peptide and brings the arginine and tyrosine in proximity, resulting in ionization of the phenolic side chain of tyrosine. In the absence of TFE, the unstructured peptide lacks the intra-molecular Arg(+)Tyr(-) ion pair, allowing heme binding to histidine. This peptide has significant peroxidase activity though it does not have catalase activity.

Decolorization kinetics of the azo dye drimaren blue X3LR by laccase

An extracellular Drimaren Blue X3LR decolorizing enzymatic activity was found in the crude filtrate of Funalia trogii grown by solid-state fermentation using wheat bran and soya bean waste. Decolorization of the azo dye Drimaren Blue X3LR by the crude filtrate and partially purified enzyme of Funalia trogii were investigated and compared. In the absence of additional redox mediator, maximum decolorization ratios of 81.33 % and 77.4 % were observed for Drimaren Blue X3LR using crude filtrate and partially purified enzyme respectively. Decolorization yield was found to be higher with crude enzyme preparations. Na2S2O5 inhibited laccase and dye decolorizing enzyme activities but a significant peroxidase activity inhibition was not observed. Since the reaction was catalyzed in the absence of H2O2 as co-substrate, it could be concluded that this enzyme is not a peroxidase but may be a laccase.. The kinetic parameters of decolorization were calculated according to Michaelis constant (Km of 1.700 x 10-5 mol dm-3 and Wmax = 8.02 x10-7 mol dm-3 sec-1).

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286].

Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension cells. SAP1 and SAP2 were resolved during cation-exchange FPLC of culture media proteins from 8-day-old Pi-starved cells, and purified to homogeneity and final p-nitrophenylphosphate hydrolyzing specific activities of 246 and 940 micro mol Pi produced.min-1 mg.protein-1, respectively. SDS/PAGE, periodic acid-Schiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers. SAP1 and SAP2 are purple acid phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by l-tartrate. The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs. CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct. Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable. Although they exhibited wide substrate specificities, the Vmax of SAP2 with various phosphate-esters was significantly greater than that of SAP1. SAP1 and SAP2 were activated by up to 80% by 5 mm Mg2+, and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively. Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg2+ or molybdate. This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.

Hemoabzymes: towards new biocatalysts for selective oxidations

Catalytic antibodies with a metalloporphyrin cofactor or «hemoabzymes», used as models for hemoproteins like peroxidases and cytochrome P450, represent a promising route to catalysts tailored for selective oxidation reactions. A brief overview of the literature shows that until now, the first strategy for obtaining such artificial hemoproteins has been to produce antiporphyrin antibodies, raised against various free-base, N-substituted Sn-, Pd- or Fe-porphyrins. Five of them exhibited, in the presence of the corresponding Fe-porphyrin cofactor, a significant peroxidase activity, with k cat/ K m values of 3.7×10 3–2.9×10 5 M −1 min −1. This value remained, however, low when compared to that of peroxidases. This strategy has also led to a few models of cytochrome P450. The best of them, raised against a water-soluble tin(IV) porphyrin containing an axial α-naphtoxy ligand, was reported to catalyze the stereoselective oxidation of aromatic sulfides by iodosyl benzene using a Ru(II)-porphyrin cofactor. The relatively low efficiency of the porphyrin–antibody complexes is probably due, at least in part, to the fact that no proximal ligand of Fe has been induced in those antibodies. We then proposed to use, as a hapten, microperoxidase 8 (MP8), a heme octapeptide in which the imidazole side chain of histidine 18 acts as a proximal ligand of the iron atom. This led to the production of seven antibodies recognizing MP8, the best of them, 3A3, binding it with an apparent binding constant of 10 −7 M. The corresponding 3A3–MP8 complex was found to have a good peroxidase activity characterized by a k cat/ K m value of 2×10 6 M −1 min −1, which constitutes the best one ever reported for an antibody–porphyrin complex. Active site topology studies suggest that the binding of MP8 occurs through interactions of its carboxylate substituents with amino acids of the antibody and that the protein brings a partial steric hindrance of the distal face of the heme of MP8. Consequently, the use of the 3A3–MP8 complexes for the selective oxidation of substrates, such as sulfides, alkanes and alkenes will be undertaken in the future.

Physiological concentrations of homocysteine inhibit the plasma GSH peroxidase: evidence for a role of oxidant damage in the etiology of the accelerated atherosclerosis seen in hyperhomocysteinemia

The plasma GSH-selenoperoxidase is a highly conserved enzyme which reduces lipid hydroperoxides. A small clinical study found that patients with severe atherosclerosis had low peroxidase activities suggesting that the peroxidase prevents atherosclerosis. Yet others have reported that when assayed in Tris buffer, there was no activity with the reported plasma GSH concentrations (4-20 (M). Yet, we previously reported that the peroxidase was active with low GSH concentrations when the assay was run in phosphate buffered saline. Second, homocysteinemia increases the rate of atherogenesis. We have sought to determine whether homocysteine inhibits the GSH peroxidase. The homocysteine inhibition of the purified, human plasma peroxidase was determined by two assays: 1) a coupled assay in which GSH is oxidized to GSSG by tert.-butyl hydroperoxide and reduced back to GSH by NADPH and 2) a substrate disappearance chemiluminescence assay. There was significant peroxidase activity with the reported plasma GSH concentrations with both assays. Physiologically relevant free homocysteine concentrations inhibited the peroxidase but not the GSH-reductase. These data demonstrate that the peroxidase may be active in vivo and that homocysteine inhibits it. Our results imply that the peroxidase prevents atherosclerosis by protecting the endothelium from oxidant injury. They also suggest a biochemical basis for the association between hyperhomocysteinemia and cardiovascular disease. These studies suggest that low peroxidase activity together with hyperhomocysteinemia may be a combined risk factor for increased atherosclerosis.