We have examined the electrochemical reduction of NO by myoglobin (Mb) contained within a dimethyldidodecylammonium bromide (ddab) film on pyrolytic graphite electrodes. Immersion of the (FeIII)Mb-doped electrode into aqueous solutions of NO results in a bulk chemical reductive nitrosylation forming NO−Mb−FeII, as indicated by the dissappearance of the FeIII/II−Mb couple in voltammograms. At more negative potentials, a catalytic reduction wave appears at ca. −0.7 V/SCE, which remains catalytic in solutions from pH 5.5 to pH 10 and is NO-concentration dependent. Bulk electrolysis at −0.8 V/SCE of 15NO solutions by Mb/ddab yields 15N2O as gaseous product. Cyclic voltammograms of films made of preformed nitrosyl myoglobin, MbFeII−NO, demonstrate a single, reversible reduction to the nitroxyl state, MbFeII−NO-, at E° = −0.87 V/SCE. The reversibility of the nitrosyl reduction is pH dependent; digital simulation yields a rate of 22.5 s-1 for the irreversible loss of a nitroxyl group at pH 7 and 0.7 s-1 at pH 10. The catalytic formation of N2O during reduction of MbFeII−NO in the presence of exogenous NO implies that an N−N coupling reaction occurs at the active site between the Fe-bound nitroxyl and a free NO. A mechanism is proposed for the catalysis involving decomposition of an (N2O2 -)−FeII−Mb intermediate.

Previous investigations of nitrite and nitric oxide reduction by myoglobin in surfactant film modified electrodes characterized several distinct steps in the denitrification pathway, including isolation of a nitroxyl adduct similar to that proposed in the P450nor catalytic cycle. To investigate the effect of the axial ligand on these biomimetic reductions, we report here a comparison of the electrocatalytic activity of myoglobin (Mb) with a thermophilic cytochrome P450 CYP119. Electrocatalytic nitrite reduction by CYP119 is very similar to that by Mb: two catalytic waves at analogous potentials are observed, the first corresponding to the reduction of nitric oxide, the second to the production of ammonia. CYP119 is a much more selective catalyst, giving almost exclusively ammonia during the initial half-hour of reductive electrolysis of nitrite. More careful investigations of specific steps in the catalytic cycle show comparable rates of nitrite dehydration and almost identical potentials and lifetimes for ferrous nitroxyl intermediate (Fe(II)-NO(-)) in CYP119 and Mb. The catalytic efficiency of nitric oxide reduction is reduced for CYP119 as compared to Mb, attributable to both a lower affinity of the protein for NO and a decreased rate of N-N coupling. Isotopic labeling studies show ammonia incorporation into nitrous oxide produced during nitrite reduction, as has been termed co-denitrification for certain bacterial and fungal nitrite reductases. Mb has a much higher co-denitrification activity than CYP119. Conversely, CYP119 is shown to be slightly more efficient at the two-electron reduction of N(2)O to N(2). These results suggest that thiolate ligation does not significantly alter the catalytic reactivity, but the dramatic difference in product distribution may suggest an important role for protein stability in the selectivity of biocatalysts.

Carbon nanotubes have been proposed as support materials for numerous applications, including the development of DNA sensors. One of the challenges is the immobilization of DNA or other biological molecules on the sidewall of carbon nanotubes. This paper introduces a new fabrication of DNA-carbon nanotubes particles using the layer-by-layer (LBL) technique on single-walled carbon nanotubes (SWCNTs). Poly(diallyldimethylammonium) (PDDA), a positively charged polyelectrolyte, and DNA as a negatively charged counterpart macromolecule are alternatively deposited on the water-soluble oxidized SWCNTs. Pure DNA/PDDA/SWCNTs particles can be prepared and separated by simple unltracentrifugation. The characterization of DNA/PDDA/SWCNTs particles was carried out by scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-visible spectroscopy, Raman spectroscopy, and thermogravimetric analysis (TGA). An electrode modified by the DNA/PDDA/SWCNTs particles shows a dramatic change of the electrochemical signal in solutions of tris(2,2'-bipyridyl)ruthenium(II) ((Ru(bpy)(3)2+) as a reporting redox probe. A preliminary application of the DNA-modified carbon nanotubes in the development of DNA sensors used in the investigation of DNA damage by nitric oxide is presented.

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Successful cryopreservation for human spermatozoa markedly influences the reproductive outcomes of assisted reproductive technologies. But in spite of its usefulness, cryopreservation significantly decreases sperm quality. l-carnitine has been found to improve the quality of spermatozoa in selected cases with male infertility. Here, we examined the efficacy of l-carnitine in improving sperm motility and vitality and reducing sperm DNA oxidation during cryopreservation. Semen samples from infertile patients (n = 22) were collected and analysed. Cryopreservation medium supplemented with l-carnitine was mixed with the semen at a ratio of 1 : 1 (v/v). The final l-carnitine concentration in each cryovial was 0.5 mg ml(-1) per 5 × 10(6) cell ml(-1) . Controls were cryopreserved without addition of l-carnitine. After 24 h of cryopreservation, thawed sperm samples were analysed for motility, vitality and DNA oxidation. Sperm vitality was assessed by the eosin-nigrosin test, while sperm DNA oxidation was measured by flow cytometry. Addition of l-carnitine significantly improved sperm motility and vitality (P < 0.05) compared with the control. The flow cytometry experiment showed no statistical difference (P > 0.05) in the levels of DNA oxidation between samples and controls. In conclusion, l-carnitine improves human sperm motility and vitality, but has no effect on sperm DNA oxidation after cryopreservation.

In vitro incubation and centrifugation is known to decrease human sperm quality. In the human body, besides its antioxidant effects, L-carnitine (LC) facilitates the transport of activated fatty acids from the cytosol to the mitochondrial matrix. In this study, we investigated the effect of LC on human sperm motility, viability and DNA oxidation after incubation and centrifugation, following the sperm preparation protocols of assisted reproduction. Normozoospermic semen samples (n = 55) were analysed according to the World Health Organization (WHO) guidelines. LC concentrations that are not toxic to spermatozoa as determined by sperm motility and viability were standardised after 2 and 4 h of incubation at 37 °C. Semen samples to which the optimal LC concentrations were added were also centrifuged for 20 min at 300 g and analysed for sperm motility, viability and DNA oxidation. Sperm motility was improved at 0.5 mg ml(-1) LC after incubation and centrifugation with 5 × 10(6) sperm ml(-1). Higher concentration of LC (50 mg ml(-1)) significantly decreased sperm motility and viability. LC did not alter the baseline of sperm DNA oxidation during both incubation and centrifugation. In conclusion, LC may enhance sperm motility following incubation and centrifugation, while it might not affect sperm viability and DNA oxidation.

SYNOPSIS The Nitric Oxide Synthases (NOS; EC 1.14.13.39) contain a C-terminal flavoprotein domain (NOSred) that binds FAD and FMN and an N-terminal oxygenase domain that binds heme. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain (FNR) and the oxygenase domain. previously we showed that three residues on the FMN domain (Glu762, Glu816 and Glu819) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred. In this study, we show that charge neutralization or reversal at each of these residues alters the setpoint (KeqA) of the NOSred conformational equilibrium to favor of the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have relatively larger effects on the conformational transition rates (from 1.5 to 4x faster) and the rate of interflavin electron transfer (from 1.5 to 2x faster) relative to wild type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations.

We investigated in this paper the sensing performance of inherently conductive polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), functionalized with hemin (iron protoporphyrin) as an electrocatalytic reporter. The sensing platform is prepared by electrodeposition of a composite film of hemin-PEDOT on a 30-microm diameter carbon fiber electrode (CFE). The polymerized films were characterized by field emission scanning electron microscopy (FESEM), which pointed to nanostructured films with tortuous pores. The electrocatalytic oxidation of peroxynitrite was characterized by cyclic voltammetry as well as other electrochemical methods. The catalytic current is proportional to the analyte's concentration. Optimized hemin-PEDOT modified CFEs were utilized for the first time to detect ONO2(-), with a response time down to 5 s and a limit of detection as low as 200 nM as evidenced by amperometry. Our hemin-PEDOT modified CFEs have a sensitivity of 13 nA/microM, ca.130 times higher than the bare CFE. More work is underway using other metalloporphyrins as electrocalalysts to improve the detection limit, the selectivity, and to further miniaturize these hemin-PEDOT modified electrodes.