Ivan Yu. Sakharov

Laccase-catalyzed synthesis of conducting polyaniline

Laccase isolated from Coriolus hirsutus was first used in the synthesis of water-soluble conducting polyaniline. The laccase-catalyzed polymerization of aniline was performed in the presence of sulfonated polystyrene (SPS) as a template. Laccase shows remarkable advantages in the synthesis of conducting polyaniline compared to the commonly used horseradish peroxidase due to its high activity and stability under acidic conditions. The characterization of the polyelectrolyte complex of polyaniline and SPS has been carried out using UV-Vis and FTIR spectroscopy. Cyclic voltammetry and dc conductivity measurements confirmed that electroactive polyaniline was synthesized by the laccase-catalyzed polymerization of aniline.

Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer

Native horseradish peroxidase (HRP) on graphite has revealed approximately 50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.

Synthesis of polyelectrolyte complexes of polyaniline and sulfonated polystyrene by palm tree peroxidase

Enzymatic synthesis of polyelectrolyte complex of polyaniline (PANI) and sulfonated polystyrene (SPS) was developed. Royal palm tree peroxidase, which is active and stable under acidic conditions, was used as a biocatalyst. The template polymerization of aniline was carried out in aqueous buffer at pH 3.5. Varying concentrations of aniline, SPS and hydrogen peroxide in feed the favorable conditions for the production of PANI were determined. Contrary to chemical polymerization of aniline, the role of enzymatic reaction is to control kinetically and proceed under environmental friendly (“green”) conditions. The production of the electroactive form of PANI–SPS complex was confirmed by Vis spectroscopy and EPR.

Biosensors based on novel plant peroxidases: a comparative study

Abstract Amperometric biosensors for hydrogen peroxide detection have been constructed using horseradish peroxidase (HRP) and two newly purified peroxidases extracted from tobacco (TOP) and sweet potato (SPP). The peroxidases were cross-linked to a redox polymer [poly(vinylimidazole) complexed with Os(4,4′dimethylbipyridine) 2 Cl] using poly(ethylene glycol) diglycidyl ether as the cross-linker. A comparative study with regard to their bioelectrochemical characteristics showed that, irrespective of peroxidase, the biosensors sensitivity was strongly influenced by hydrogel composition, curing procedure, film thickness and applied potential. The electrostatic interaction between the cationic redox polymer and the negatively charged peroxidases (TOP and SPP) enhanced the hydrogen peroxide signal. When operated in a FI system, the optimized SPP biosensor (48% redox polymer, 23% cross-linker and 29% enzyme, w/w %) displayed the highest sensitivity for H 2 O 2 (3.2 A M −1 cm −2 ), a linear range up to 220 μM, a detection limit of 25 nM (calculated as 2S/N) and a response time of about 2 min.

Extremely high stability of African oil palm tree peroxidase

A detailed kinetic study on thermal inactivation of African oil palm tree peroxidase (AOPTP) at different pHs has been carried out. The enzyme does not undergo inactivation over a broad range from pH 2 to 12 at ambient temperature. Complete inactivation of AOPTP is observed only at 70 degrees C and extremal pHs like <3.0 and >12.0, whereas under neutral conditions, its activity shows no changes. The study of AOPTP inactivation kinetics in the presence of dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) showed that calcium ions, disulfide bonds and the interaction between apo-AOPTP and heme are important structural elements responsible for the enzyme stability. The guanidium hydrochloride (GdHCl)-induced inactivation of AOPTP indicated that the hydrogen-bonding network plays also a significant role in stabilizing the active structure of the enzyme. AOPTP is stable toward hydrogen peroxide treatment, especially under neutral conditions. The comparison of AOPTP stability to that of other peroxidases shows that AOPTP is the most stable peroxidase reported so far.

Variations of peroxidase activity in cocoa (Theobroma cacao L.) beans during their ripening, fermentation and drying

Abstract An increase of peroxidase activity in the seeds of cocoa ( Theobroma cacao L.) during their ripening has been determined. An additional increase of the peroxidase activity (about 10 times) is observed during the fermentation and drying of the beans. The residual activity determined in the cocoa beans after sun-drying was higher than that in unfermented ripe seeds. The major cocoa isoperoxidase was shown to be an acidic enzyme with pI 4.7. Using isoelectrofocusing, the appearance of two basic isoenzymes of the peroxidase with pI 8.6 and 9.0 during the process of the fermentation has been detected. ©

Chemical modification and composition of tetrameric isozyme K of alkaline phosphatase from harp seal intestinal mucosa

1. The carbohydrate content of isozyme K of alkaline phosphatase (EC 3.1.3.1) from harp seal intestinal mucosa was examined. The presence of N-acetylglucosamine, N-acetylgalactosamine and considerable amounts of mannose residues was shown. 2. The amino acid content of seal alkaline phosphatase was determined. A high extent of homology (85%) between bovine and seal alkaline phosphatases was demonstrated. 3. By chemical modification lysine, dicarboxylic acids, arginine and tyrosine residues of tetrameric seal alkaline phosphatase are located near or at the active site. By contrast, the modification of either thiol or imidazole groups resulted in no alterations of the enzyme activity. 4. It has been demonstrated that inorganic phosphate is an inhibitor of alkaline phosphatase and entirely prevents the enzyme inactivation with succinic anhydride.

Novel mitochondria-targeted antioxidants, “Skulachev-Ion” derivatives, accelerate dermal wound healing in animals

It is shown that the novel mitochondria-targeted antioxidant SkQ1, (10-(6′-plastoquinonyl) decyltriphenylphosphonium) stimulates healing of full-thickness dermal wounds in mice and rats. Treatment with nanomolar doses of SkQ1 in various formulations accelerated wound cleaning and suppressed neutrophil infiltration at the early (7 h) steps of inflammatory phase. SkQ1 stimulated formation of granulation tissue and increased the content of myofibroblasts in the beginning of regenerative phase of wound healing. Later this effect caused accumulation of collagen fibers. Local treatment with SkQ1 stimulated re-epithelization of the wound. Lifelong treatment of mice with SkQ1 supplemented with drinking water strongly stimulated skin wounds healing in old (28 months) animals. In an in vitro model of wound in human cell cultures, SkQ1 stimulated movement of epitheliocytes and fibroblasts into the “wound”. Myofibroblast differentiation of subcutaneous fibroblasts was stimulated by SkQ1. It is suggested that SkQ1 stimulates wound healing by suppression of the negative effects of oxidative stress in the wound and also by induction of differentiation. Restoration of regenerative processes in old animals is consistent with the “rejuvenation” effects of SkQ1, which prevents some gerontological diseases.

FeIII–TAML activator: A potent peroxidase mimic for chemiluminescent determination of hydrogen peroxide

Efforts to replace native peroxidase with its low molecular weight alternatives have stimulated a search for peroxidase mimetics. Herein we describe the oxidation of luminol with hydrogen peroxide catalyzed by commercially available Fe(III)-TAML activator 1a, which was shown to be a more active catalyst than hemin. At Fe(III)-TAML activator 1a use in chemiluminescent assay for H2O2 determination the detection limit value (3σ) of 5×10(-8)M was similar to the detection limit obtained with horseradish peroxidase (1×10(-7)M) and significantly lower than that obtained in the presence of hemin (6×10(-7)M). The linear ranges (R(2)=0.98) of the assay were 6×10(-8)-1×10(-6)M and 6×10(-7)-1×10(-6)M H2O2 for Fe(III)-TAML 1a and hemin, respectively. The CV values for Fe(III)-TAML 1a-based assay measured within the working range varied from 1.0% to 3.7% (n=4), whereas in the case of hemin -5.0% to 9.7% (n=4). Moreover, the sensitivity of Fe(III)-TAML 1a-based method was 56 and 5 times higher than that of hemin- and HRP-based methods, respectively. The obtained results open good perspectives to apply Fe(III)-TAML activator 1a in CL analytical methods instead of hemin, a traditionally used peroxidase mimetic.

Advantages of Soybean Peroxidase over Horseradish Peroxidase as the Enzyme Label in Chemiluminescent Enzyme-Linked Immunosorbent Assay of Sulfamethoxypyridazine

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) of sulfamethoxypyridazine (SMP) was developed. The conjugates of streptavidin with cationic horseradish peroxidase (HRP) and anionic soybean peroxidase (SbP) were used in CL-ELISA for the detection of biotinylated anti-SMP antibodies. For streptavidin-HRP conjugate-catalyzed chemiluminescence measured 20 s after the initiation of the enhanced chemiluminescence reaction (ECR), the limit of detection (IC(10)), the IC(50) value, and the working range in CL-ELISA of SMP are 0.3, 12.4, and 1.2-85.0 ng/mL, respectively. An increase in the time interval between the ECR initiation and the luminescence measurement results in the loss in the quality of analytical measurements because of the time-dependent quenching of chemiluminescence typical of the HRP-catalyzed ECR. In the case of SbP-based CL-ELISA of SMP, the limit of detection, the IC(50) value, and the working range (0.025, 0.17, and 0.045-0.63 ng/mL, respectively) are better than those for HRP-based CL-ELISA. Furthermore, the analytical parameters of SbP-based CL-ELISA remain unchanged during a long period of time (for at least 30 min). The recovery values from four spiked milk samples with different concentrations of SMP in SbP-based CL-ELISA vary from 70 to 130%.