Absence Of Cyanide Research Articles

The role of active transport in potassium reabsorption in the proximal convoluted tubule of the anaesthetized rat.

1. We have previously assessed the contributions of diffusion and convection to net potassium reabsorption in the rat proximal convoluted tubule (PCT). The present study was conducted to evaluate the role of active transport in convective potassium reabsorption by measuring the transepithelial potassium reflection coefficient (sigmaK) in the presence and absence of cyanide in anaesthetized rats previously prepared for in vivo microperfusion. 2. Osmotic water permeability (Pf) was measured in double-perfused tubules (lumen and peritubular capillaries) by manipulating the applied transepithelial osmotic gradient between -30 and +40 mosmol (kg H2O)(-1) using raffinose added to or subtracted from luminal perfusates. Pf was unaffected by the presence of cyanide when the estimated dissipation of osmotic gradients along each tubule were taken into account. 3. The proportion of K+ (and Na+) convectively transported with water fluxes (i.e. sigmaK) was not affected by cyanide. In the absence of active transport and following correction for any diffusive component, sigmaK was 0.56 +/- 0.13, indicating substantial solvent drag which probably occurs via the paracellular pathway. 4. However, cyanide caused a reduction in net potassium flux over the entire range of fluid fluxes used in double-perfusion experiments. Subsequent single-perfusion experiments (tubule lumen only) using the specific K+-H+-ATPase inhibitor, SCH28080, failed to reveal any direct evidence for a primary active K+ transporting mechanism involved in K+ reabsorption in the PCT.

Formation of a porphyrin pi-cation radical in the fluoride complex of horseradish peroxidase.

Horseradish peroxidase (HRP) was oxidized by IrCl6(2-) to a mixture of compounds I and II, the rate of oxidation and the ratio of the mixture being greatly affected by pH (Hayashi & Yamazaki, 1979). Oxidation of HRP by IrCl6(2-) in the presence of fluoride was significantly accelerated. This resulted in the formation of a new compound which is a ferric fluoride complex containing a porphyrin pi-cation radical. The spectrum of the new compound showed a decreased absorption band in the Soret region and a broad band at 570 nm; which was converted to that of the original ferric fluoride complex by addition of ascorbate or hydroquinone. Addition of cyanide slowed down the oxidation of HRP by IrCl6(2-), and the oxidation product was the same as that obtained in the absence of cyanide. Compound I was formed when H2O2 was added to HRP in the presence of fluoride or cyanide. The one-electron reduction potential (Eo') of the oxidized HRP-fluoride complex was measured at several pH values, the Eo' value at pH 7 being 861 +/- 4 mV. The ratio of delta Eo' to delta pH was 49 mV/pH unit.

Causes and location of non‐specific effects of SHAM on O2 uptake by wheat roots

Uptake of O2 by whole, detached, root systems of wheat (Triticum aestivum L. cv. Alexandria) was titrated with salicylhydroxamic acid (SHAM) in the presence and absence of cyanide. The resulting Qall plot was non‐linear indicating that SHAM was acting non‐specifically. The nature of the non‐specific effects was investigated in reverse titration experiments. Uptake of O2 was titrated with KCN in the presence and absence of SHAM at 1 mM and 25 mM, which yielded Qcy1 values of < 1 and > 1, respectively. The results suggest that at 25 mM, SHAM inhibits the cytochrome pathway, but at 1 mM it stimulates an O2‐consuming process which is likely to be a peroxidase. A SHAM‐stimulated peroxidase could easily be washed from these roots. In vitro, the peroxidase was stimulated to a similar extent by low (1 mM) and high (25 mM) concentrations of SHAM. Failure to inhibit with high concentrations of SHAM distinguishes this peroxidase from those bitherto eluted from root tissue. Reverse titration experiments in the presence and absence of 1 mM SHAM indicated that there were no significant side effects of SHAM in root tips. These data are supported by the negligible peroxidase activity that was washed from this root fraction. In contrast, significant side effects occurred in vivo, and substantial peroxidase activity was measured in vitro, from sections 4–6 cm and 18–20 cm behind the seminal root apex. The greatest activity was found with the 4–6 cm section which may be associated with high rates of cell wall lignification. The implications of these results for measurements of root respiration are discussed.

A New Approach to Measurement of Redox-Cycling Activity in Escherichia coli

Redox-cycling agents catalyze the flow of reducing equivalents to oxygen; this process generates superoxide ion and other reduced oxygen species. Measurements of redox-cycling activity have been performed previously by studying cyanide-resistant oxygen consumption (respiration) of Escherichia coli cells. E. coli strain GK100, lacking both terminal oxidases, has almost no measurable respiration. We show that the use of this strain eliminates the requirement for cyanide in measurements of redoxcycling activity. The addition of either menadione sodium bisulfite or plumbagin, well-known redox-cycling agents, to GK100 cells resulted in high levels of oxygen consumption. The rate of menadione bisulfite-induced oxygen consumption in this respiration-deficient strain, in the absence of cyanide, was comparable to the cyanide-resistant respiration of isogenic respiration-proficient E. coli strains. In GK100 cells, cyanide increased menadione bisulfite-induced oxygen consumption but had no effect on plumbagin-induced oxygen consumption.

The competitive adsorption of cyanide and ethyl xanthate on pyrite and pyrrhotite surfaces

Ultraviolet-visible (UV-vis) and Fourier Transform Infrared (FTIR) Spectroscopy have been employed to determine the kinetics of ethyl xanthate adsorption onto pyrite and pyrrhotite at pH 9 in the presence and absence of cyanide. In the absence of cyanide, the rate and extent of ethyl xanthate adsorption increased with the pulp electrochemical potential ( Eh). For both pyrite and pyrrhotite, there was a dramatic increase in adsorption above an Eh of +200 mV, corresponding to the oxidation potential of ethyl xanthate to diethyl dixanthogen. In-situ and ex-situ FTIR studies using attenuated total reflectance (ATR) and single-reflection modes have confirmed the presence of dixanthogen on pyrite and pyrrhotite. The influence of cyanide on the adsorption kinetics of ethyl xanthate depends on Eh as well as the order of reagent addition. In general, cyanide reduced the rate and extent of ethyl xanthate adsorption onto the iron sulphide minerals; this was more pronounced for pyrrhotite. The preadsorption of cyanide dramatically enhanced this depressant action. Furthermore, the addition of cyanide to preadsorbed ethyl xanthate led to a desorption of ethyl xanthate from the surface, but the desorption was short-lived. FTIR and XPS studies confirmed this behaviour and showed the presence of surface cyano-species, apparently as insoluble ferrous and ferric hexacyano complexes. The competitive adsorption of cyanide in the presence of ethyl xanthate is an important mechanism in the depression of pyrite and pyrrhotite.

Effects of metabolic inhibition and changes of intracellular pH on potassium permeability and contraction of rat uterus.

1. We have investigated the role of changes of potassium efflux in the inhibition of uterine force produced by cyanide. K+ efflux (86Rb) was measured from pregnant and non-pregnant rat myometrial strips during metabolic inhibition with cyanide and following manoeuvres to displace intracellular pH (pHi). 2. Cyanide greatly reduced or abolished spontaneous contractions. If the membrane was depolarized directly at this stage (by elevating external K+) then contraction redeveloped. This suggests that the initial depression of force is due to a failure of membrane excitation. 3. Cyanide reversibly increased 86Rb efflux (30-35%) in both pregnant and nonpregnant uteri and contraction was reduced. The increase in 86Rb efflux with cyanide was not secondary to changes of membrane potential as it also occurred in both high-K+ and Ca(2+)-free solutions. 4. Glibenclamide (20 microM), an antagonist of K+ATP channels, reduced the cyanide-evoked increase of 86Rb efflux by about 50%. The glibenclamide-insensitive component of efflux persisted in a Ca(2+)-free solution. Despite its action on 86Rb efflux, glibenclamide did not restore contraction. 5. Intracellular pH falls during metabolic inhibition. We therefore investigated whether reducing pHi (in the absence of cyanide) had an effect on 86Rb efflux. Application of the weak acid butyrate (60 mM, at constant external pH, 7.4) had no significant effect on 86Rb efflux. Thus it is unlikely that the acidification in hypoxia contributes to the increased K+ efflux. 6. Intracellular alkalinization produced by the weak base trimethylamine (60 mM) increased the frequency of uterine contraction and the 86Rb efflux. However, there was no effect on the 86Rb efflux in a Ca(2+)-free solution. The increased efflux is therefore presumably a consequence of the increased frequency. 7. It is concluded that metabolic inhibition produced by cyanide, produces an increase in K+ efflux from the myometrium. Part of this efflux is glibenclamide sensitive. This increased K+ efflux will lead to hyperpolarization of the myometrial membrane and thus decrease excitation. Thus reduced surface membrane excitability will contribute to the fall of force in hypoxia; specifically it may cause the initial loss of spontaneous contractions in the uterus.

Mechanism of automated alkaline methods for the determination of hemoglobin in whole blood based on the micellization of ligated heme in the presence and absence of cyanide

Conventional (cyanide-based) and cyanide-free methods for the determination of hemoglobin (Hb) in whole blood have been developed for the Miles H * 1 TM hematology instrument. Concerns with hazards associated with manufacture, transportation and disposal of cyanide-based solutions favor cyanide-free reagents. The cyanide-based method reaches completion in 3 s. The reaction mechanism of whole blood Hb or hemin with a reagent consisting of 20 mM KCN, 20 g l −1 of N,N-dimethylaurylamine N-oxide (LO), in 50 mM Na borate at pH 11.3 was investigated. The red-brown reaction product is micellized dicyanoiron(III)porphyrin based on optical spectra. Deletion of cyanide from this reagent yields a green product, micellized monoaquomonohydroxyiron(III)porphyrin. Proton magnetic resonance line broadening of LO by heme or Hb is consistent with the presence of the heme product distributed within the interior of the micelle. A scheme is presented which shows relationships between alkaline cyanide-based and cyanide-free reagents for the determination of Hb in whole blood.

The kinetics of the organic elution of gold cyanide from activated granular carbon using an aqueous caustic acetone solution

The kinetics of the elution of adsorbed gold cyanide from granular activated carbon in an aqueous caustic acetone solution in the absence of cyanide were investigated in a batch stirred reactor. The equilibrium and rate experiments were performed with a 1:1 by volume mixture of acetone and 4g/1 NaOH aqueous solution at temperatures ranging between 20 and 60°C. In the rate experiments the effect of different solution to carbon mass ratios, carbon particle size and rate of stirring were investigated. The elution equilibrium was described in terms of Freundlich isotherms. These isotherms were significantly more favourable for elution than those for inorganic solutions used in the Zadra process. The elution rate was described mathematically by a first-order desorption reaction rate equation at the outer surface of the carbon granule. The proposed model accurately desribes the batch elution process in terms of the parameters investigated. The rate of mass transfer across the hydrodynamic boundary layer does not affect the overall rate of elution under the conditions investigated and was therefore not included in the model. High rates of elution were obtained with equilibrium being approached within two hours at 50°C. The elution rate was very sensitive to variations in temperature with an apparent activation energy of 56 kJ/mol. This indicated that the rate of elution was indeed limited by the rate of the desorption reaction.

Reduction reactions of water soluble cyano-cobalt(III)-porphyrins: Metal versus ligand centered processes

Reduction reactions of dicyano-cobalt(III)-porphyrins [potential in vivo cyanide scavenger drugs] were studied by radiolytic and electrochemical methods using the water soluble tetrakis(4-sulfonatophenyl)porphyrin (TPPS) and tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP). For [(CN) 2Co IIITPPS] −, reduction occurs stepwise to the Co II, Co I, and finally to the phlorin anion. This behavior is similar to that of the cobalt porphyrins in the absence of cyanide, except that the cyanide ligand shifts the reduction potentials to much more negative values. On the other hand, under radiolytic conditions, [(CN) 2Co IIITMPyP] − is reduced on the porphyrin macrocycle by one electron to give the Co III π-radical anion, which disproportionates into the initial complex and the two-electron ring reduced Co III phlorin. The radical anion is also formed by intramolecular electron transfer subsequent to the reaction of Co IITMPyP and cyanide. The results are compared with the chemistry of Vitamin B-12.

Kinetics and mechanism of the reaction of cyanide with molybdenum nitrogenase from Azotobacter vinelandii.

The steady-state kinetic behavior of the six-electron reduction of N2 by nitrogenase is known to differ markedly from the six-electron reduction of cyanide in two ways. First, on extrapolation to infinite concentration of cyanide, the H2 evolution reaction is almost completely suppressed whereas at extrapolated infinite concentration of N2, H2 evolution continues. Second, as the ratio of the Fe protein to the MoFe protein increases, the reduction of N2 is favored over H2 evolution, whereas the reduction of cyanide becomes less favored relative to H2 evolution. We have extended these steady-state experiments with Azotobacter vinelandii nitrogenase to include a third observation, that the six-electron reduction of N2 is favored over H2 evolution at high total protein concentrations whereas cyanide reduction is less favored over H2 evolution at high total protein concentrations. All three steady-state observations can be explained by a model whereby cyanide is proposed to bind to a redox state of the MoFe protein more oxidized than that reactive toward H2 evolution and N2 reduction. To test this model, we have examined the pre-steady-state kinetic behavior of both cyanide reduction by A. vinelandii nitrogenase and cyanide inhibition of total electron flow through nitrogenase. The data show that in the presence or absence of cyanide there is a short lag of 100 ms before H2 is detected, followed by a linear phase of H2 evolution lasting for about 3 s, during which time no effects of cyanide are observable. After 3 s electron flow is finally inhibited by cyanide, and the cyanide reduction product CH4 is finally formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteins associated with somatogenic and lactogenic receptors in microsomal membranes and intact rat hepatocytes

Lactogenic and somatogenic receptors present in rat liver have been examined by cross-linking with a derivative of human somatotropin (AP-hGH1) followed by SDS-polyacrylamide gel electrophoresis. AP-hGH1, which has a content of 2.2 azidophenacyl groups per molecule, mainly linked to half Cys-182 and half Cys-189, exerted a specificity similar to that of the native hormone (hGH), with an ability of 46% with respect to hGH to compete with the radiolabelled hormone for the binding sites of microsomal preparations. Photolysis of the 125I-labelled derivative bound to the lactogenic receptors present in either microsomal membranes or Triton X-100 solubilized preparations gave rise to a 63 kDa species. In addition, 30% of the covalent complexes formed in microsomal membranes belonged to a species with a molecular mass of 70 kDa. Incubation of viable rat hepatocytes with the radiolabelled derivative at either 0°C for 3 h or 15°C for 1.5 h and subjection to irradiation, yielded covalent complexes of molecular masses estimated at 130, 73, 63, 45 and 35 kDa. Experiments performed in the presence of 1 mM NaCN, gave rise to the previous species in a similar yield as that obtained in the absence of cyanide. The 130 kDa complex is related to the somatogenic binding sites, since it was not visualized in the presence of unlabelled bovine somatotropin, while the 70–73, 63, 45 and 35 kDa bands disappeared when the incubations were performed in the presence of unlabelled ovine prolactin.

Electrical and mechanical responses to inhibition of cell respiration in vascular smooth muscle of the rat portal vein.

Metabolic regulation of contractility in vascular smooth muscle was studied in the spontaneously active rat portal vein using respiratory depression by cyanide (0.2-2.0 mM) as a model for tissue hypoxia. Intracellular recordings of electrical activity were done with concomitant registration of force development. Average membrane potential in the absence of cyanide was -61 +/- 1 mV (n = 27). Addition of cyanide to normal Krebs solution resulted in a reduction of force amplitude and the number of action potentials per burst, with a relatively more pronounced effect on the mechanical activity. At moderate levels of inhibition of force amplitude the frequency of spontaneous bursts of action potentials transiently increased concomitant with a slight depolarization, but after prolonged (15-20 min) exposure to cyanide the membrane repolarized to the level prior to cyanide addition and the burst frequency decreased to be equal to or lower than that in the absence of cyanide. Higher concentrations of cyanide totally inhibited spontaneous mechanical and electrical activity. In contrast to the results with glucose, it was found that when beta-hydroxybutyrate was used as substrate the addition of 2 mM cyanide led to a marked hyperpolarization (13 +/- 1 mV) after total inhibition of spontaneous activity. The hyperpolarization was not prevented by administration of 4-aminopyridine (2.5 mM) or tetraethylammonium (4-6 mM) prior to the addition of cyanide. To investigate the effects of increased metabolic demand on the relation between force and membrane potential in cyanide-treated muscle, high-K+ (40 mM) contractures were studied. Contractures were associated with depolarization of 34 +/- 3 mV (n = 5). 1 mM cyanide reduced the amplitude of the contractures to about 9% of control with a moderate reduction in the amount of depolarization (28 +/- 1 mV, n = 5). It is concluded that the decrease of mechanical activity during respiratory inhibition may partly reflect a reduction in the number of spikes per burst but that other mechanisms, independent of membrane activity, also contribute to the inhibition. The increase of glycolysis during respiratory inhibition seems to prevent more pronounced changes in membrane potential.

Activation by reduction of the resting form of cytochrome c oxidase: Tests of different models and evidence for the involvement of Cu B

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2 · 10 4 M −1 · s −1. In the absence of cyanide, ferrocytochrome a 3 appears at a rate ( k obs) of 0.016 s −1. Ferricytochrome a 3 maintains its 418 nm Soret maximum until reduced. The rate of a 3 reduction is independent of dithionite concentration over a range 0.9 mM–131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a 3/cyanide complex occurs at the same rate as a 3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a 3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a 3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a 3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow ( k obs ≈ 0.02 s) entry of a third electron. Above +200 mV, the number of electrons taken up in the initial fast phase drops as a redox center (presumably Cu A) titrates with an apparent mid-point potential of +240 mV. The slow phase of reduction remains at the more positive redox values. (4) The results are interpreted in terms of an initial fast reduction of cytochrome a (and Cu A at redox values more negative than +240 mV) followed by a slow reduction of Cu B. Cu B reduction is proposed to spin-uncouple cytochrome a 3 to form a cyanide sensitive center, and trigger a conformational change to an activated form of the enzyme with faster intramolecular electron transfer.

The possible role of the closed-open transition in proton pumping by cytochrome c oxidase: the pH dependence of cyanide inhibition

The rate of oxidation of reduced cytochrome c catalyzed by cytochrome oxidase in the presence and absence of cyanide has been measured spectrophotometrically at pH 5.5, 6.4, 7.4 and 8.3. At the cytochrome c concentration used (272 μM), the uninhibited rate is maximal at pH 6.4 and drops to a value about one sixth of this maximum at pH 8.3. In the presence of cyanide, the rate initially drops rapidly, but with the cyanide concentration used (5.5 μM) there is still a measurable rate of oxidation when maximal inhibition has been reached. This inhibited rate decreases as the pH increases, whereas the apparent rate constant for cyanide binding is almost independent of pH. The results have been analyzed on the basis of a model in which two-electron reduction of the oxidized enzyme triggers a transition from a closed to an open conformation. It is assumed that cyanide can only bind to the open conformation and, furthermore, that rapid internal electron transfer to the dioxygen-reducing site occurs in this state alone. The analysis shows that the true rate constant for cyanide binding decreases with decreasing pH to a constant value at low pH. It also indicates that the increase in the catalytic constant with decreasing pH is associated with an increase in the rate of the closed-open conformational transition on protonation of the enzyme, and it is proposed that this transition is operative in electron gating in the proton-pump function of the enzyme.

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity.

Cyanide-resistant respiration of [formula omitted] in course of starvation

The inhibition of respiration of Physarum polycephalum microplasmodia by KCN varies between 30% and 70% of initial rate of O 2 uptake, depending on the time of starvation. The kinetics of the development of cyanide-resistant respiration and its sensitivity toward salicylhydroxamic acid (SHAM) point out that CN-resistant respiration represents the activity of the alternative pathway of the electron transport. There is no evidence that during starvation the alternative pathway of respiration is active in the absence of cyanide.

Gold desorption from activated carbon with dilute NaOH/organic solvent mixtures

This paper presents a study of the desorption of gold adsorbed onto activated carbon using mixtures of dilute NaOH and different organic solvents. The effect of the solvent components on the desorption rate was studied and a comparative study was made of gold desorption in the presence of cyanide and thiourea. The highest gold desorption rates were obtained with water/acetone/OH − solvents in the absence of cyanide. The isopropyl alcohol/water/thiourea/OH − system also appears to be highly effective at ambient temperature. The apparent activation energy for the isopropyl alcohol/water/OH − system is 36.4 kJ/mol and for the isopropyl alcohol/water/thiourea/OH − and acetone/water/OH − systems it is 28.0 kJ/mol. In all cases, the presence of NaOH in the solvent mixture is necessary to obtain high desorption rates and good extents of desorption.

Intracellular Oxygen-metabolizing Enzymes of Phanerochaete chrysosporium

SUMMARY: The levels of catalase and superoxide dismutase present in the white-rot fungus Phanerochaete chrysosporium were investigated. The specific activities of both enzymes increased and reached a maximum after 4-6 d growth before falling to a constant low activity. Catalase was purified and found to be the typical eukaryotic enzyme. Activity staining, in the presence and absence of cyanide, revealed that the cytosol of P. chrysosporium contained only the copper/zinc superoxide dismutase commonly found in eukaryotes. These results show that P. chrysosporium meets the challenge of raised levels of activated oxygen by producing enhanced levels of the usual detoxifying enzymes rather than any novel isoenzymes.

Red blood cells generate nitric oxide from directly acting, nitrogenous vasodilators

Human red blood cells (RBC) incubated under nitrogen with methylene blue and glucose at physiological temperature and pH can be used to test for the biotransformation of nitrogenous vasodilators to nitric oxide (NO). The NO generated was trapped as nitrosylated heme by reduced subunits (hemeII) on various hemoglobin valency species and quantified by electron paramagnetic resonance spectroscopy. It was possible to separate the various valency species of hemoglobin present in the mixture as ( α 2+ β 2+) 2,( α 2+ β 3+) 2,( α 3+ β 2+) 2, or ( α 3+ β 3+) 2 by isoelectric focusing (IEF) unless cyanide (from nitroprusside) or azide was present in the mixture. These anions bind tenaciously to oxidized subunits (hemeIII) and prevent the separation of the various species by IEF. The fully oxidized tetramer, ( α 3+ β 3+) 2, does not bind NO, but the other three species have hemeII units which can be nitrosylated. In the absence of cyanide or azide the valency species could be separated by IEF, and it was possible to quantify the degree of nitrosylation on each individual species. The various agents tested (nitrite, glyceryl trinitrate, hydroxylamine, hydralazine, nitroprusside, and azide) produced different patterns of valency species and degrees of nitrosylation of hemeII. When hemeIII ligands were present or in cases of very low yields, it was still possible to quantify the total concentration of NO-hemeII in the mixture. Thus, the method could still be used to test for NO formation. All of the so-called NO vasodilators tested yielded detectable amounts of NO in the system.

Respiration of barley roots: Assessment of activity of the alternative path using SHAM

The activity of the alternative path of O2 consumption in detached and intact roots of barley [Hordeum distichum (L.) Lam. cv. Maris Mink] was determined by titration with salicylhydroxamic acid (SHAM) in the presence and absence of cyanide. In the absence of cyanide, only high concentrations were inhibititory (> 5 mM). whilst in its presence low SHAM concentrations (2.5–5.0 mM) gave maximum inhibition: the resulting ϱ Valt plots were non‐linear. A SHAM‐stimulated peroxidase could readily be washed from these roots, but non‐linearity cannot be explained in terms of SHAM‐stimulation of this peroxidase as it is not active in the absence of an exogenous supply of NADH. In detached roots the degree of inhibition of respiration with 25 mM SHAM was nearly double the capacity of the alternative path (measured as the degree of inhibition by SHAM in the presence of cyanide), suggesting non‐specific inhibition. Effects of SHAM on cytochrome path activity in intact roots were examined by reverse titration with cyanide in the presence and absence of SHAM. At 5 mM SHAM had no effect on the cytochrome path, but at 25 mM it inhibited. We conclude that the only factor causing non‐linearity of ϱValt plots in barley roots is non‐specific inhibition of the cytochrome path by high concentrations of SHAM; consequently only low concentrations of SHAM (2.5–5.0 mM) are suitable for estimating alternative path activity in barley roots.