Modulating effect of new potential antimelanomic agents, spin-labeled triazenes and nitrosoureas on the DOPA-oxidase activity of tyrosinase

The modulating effect of newly synthesized alkylating spin labeled triazene and spin labeled nitrosourea derivatives on the DOPA-oxidase activity of mushroom tyrosinase has been investigated by Bumett's spectrophotometric method (Burnett et al., 1967). All spin labeled triazenes have exhibited activating effect on DOPA-oxidase activity of tyrosinase, whereas clinically used triazene (DTIC), which does not contain nitroxide moiety, have showed inhibiting effect. At the same experimental conditions the spin labeled aminoacid nitrosoureas have showed dual effect - activating, in the beginning of the enzyme reaction and inhibiting later on. It is deduced that the activating effect of the spin labeled compounds is due to the nitroxide moiety and the inhibiting effect of all compounds depends on their half-life time. This study might contribute to make more clear the mechanism of action of the new compounds and on the other hand would come in quite useful as a preliminary prognosis for their antimelanomic activity.     

Progesterone-binding globulin interaction with its steroid ligands: study of the protein binding site topography using spin labeled steroids and electron spin resonance spectroscopy

The binding site topography of progesterone-binding globulin (PBG) purified from pregnant guinea pig serum was examined using synthesized spin-labeled ligands and electron spin resonance (ESR) spectroscopy. A series of deoxycorticosterone-nitroxide (DOC-NO) derivatives were prepared, bearing the free radical on the side chain at increasing distance (d) from the steroid nucleus. The ability of the spin-labeled steroids to specifically bind to PBG was assessed by measurement of their relative binding affinity as compared to progesterone. ESR spectra of the bound steroid nitroxide radical were used to calculate the rotational correlation times tau c for the nitroxides as a function of their distance d to the protein-bound steroid nucleus. The data showed that the side chain nitroxide exhibited an unrestrained rotation in a water-like environment when d reached about 18 A. This would correspond to a PBG steroid binding site depth of about 28 A and suggests that the bound steroid in the PBG site is oriented with the side chain at C-17 directed toward the outside of the protein binding crevice.     

Spatial arrangement of coenzyme and substrates bound to L-3-hydroxyacyl-CoA dehydrogenase as studied by spin-labeled analogues of NAD+ and CoA.

The synthesis of nitroxide spin-labeled derivatives of S-acetoacetyl-CoA, S-acetoacetylpantetheine, and S-acetoacetylcysteamine is described. These compounds are active substrates of L-3-hydroxyacyl-CoA dehydrogenase [(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.35] exhibiting vmax values from 20% to 70% of S-acetoacetyl-CoA itself. S-Acetoacetylpantetheine and S-acetoacetylcysteamine form binary complexes with the enzyme and exhibit ESR spectra typical for immobilized nitroxides. In the case of spin-labeled pantetheine, the radical is more mobile. When spin-labeled substrates are bound simultaneously to each active site of this dimeric enzyme, spin-spin interactions differentiate between two alternate orientations of the substrate [Birktoft, J.J., Holden, H.M., Hamlin, R., Xuong, N.H., & Banaszak, L.J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8262-8266]. The fatty acid moiety is thought to be located in a cleft between two domains whereas a large part of the CoA moiety probably extends into the solution. NAD+, spin-labeled at N6 of the adenine ring, is an active coenzyme of L-3-hydroxyacyl-CoA dehydrogenase (60% vmax). Complexes with the enzyme exhibit ESR spectra typical of highly immobilized nitroxides. Binding of coenzyme NAD+ causes conformational changes of the binary enzyme/substrate complex as revealed by changes in the ESR spectrum of spin-labeled S-acetoacetylpantetheine.     

Electron spin resonance melting of chemically spin-labeled nucleic acids.

Conformational transitions of nitroxide labeled and unlabeled nucleic acids were analyzed by esr and uv spectroscopy to evaluate potential perturbation effects caused by chemical modifications of nucleic acids with spin labels. The melting temperature (T_m) determined by uv or esr melting profiles of 2 → 1 or 3 → 1 transitions is similar for labeled and unlabeled polyadenylic acid [(A)_n] and polyuridylic acid [(U)_n] complexes provided spin-labeled (A)_n with a nitroxide to nucleotide ratio of 0.002 is used. Complexes formed with spin-labeled (A)_n of greater spin-labeling extent display a noticeable perturbation of their thermal melting profiles. The studies reconfirm the existence of a low temperature esr transition at about 20 °C with calf thymus and T4 DNA duplexes spin-labeled with a nitroxide to nucleotide ratio of about 0.006. The uv melting profiles of the spin-labeled duplexes reveal no low-temperature discontinuity, but the T_m values reflecting the 2 → 1 transitions were reduced by several degrees versus those of the unlabeled duplexes. Thus, these studies suggest that with homopolymers, chemically modified to a low extent with nitroxides, the monitoring of local conformational transitions of duplexes or triplexes reflect the overall 2 → 1 or 3 → 1 transitions. In the case of the heteropolymers the possibility that the chemical modification is responsible for the low-temperature phenomenon cannot be ruled out.     

Preparation and characterization of spin-labeled derivatives of epidermal growth factor (EGF) for investigations of the interactions of EGF with its receptor by electron paramagnetic resonance spectroscopy.

We prepared, purified, and characterized derivatives of epidermal growth factor (EGF) having a nitroxide spin-label attached covalently at the amino terminus. Characterization of these derivatives with regard to the positions of attachment of the spin-label was accomplished by a combination of peptide mapping, protein sequencing, and fast atom bombardment-mass spectrometry. One derivative was chosen for use in initial investigations by electron paramagnetic resonance (EPR) spectroscopy of receptor-bound EGF and its dissociation kinetics. This derivative was found to be equipotent with the native hormone in competitive binding assays, in activating the EGF receptor kinase, and in stimulating the formation of EGF receptor dimers in solubilized cell extracts. Upon binding to solubilized EGF receptor, the spin-labeled EGF derivative became immobilized, giving rise to a visually distinct slow-motion EPR spectrum. The resulting spectrum showed no detectable dipolar interaction between nitroxides, indicating that the nitroxide moieties of spin-labels reacted at the amino termini of receptor-bound spin-labeled EGF molecules are separated by a distance of at least 16 A. An EPR study of the kinetics of dissociation of spin-labeled EGF in the presence of excess unlabeled EGF revealed a rapid component with a k off approximately 2 x 10(-2) s-1 and a less well resolved slow component.     

Probing of sulfhydryl groups in the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier by maleimide spin-labels

Binding of spin-labeled maleimides to the mitochondrial ADP/ATP carrier was investigated both in mitochondria and in the detergent-solubilized carrier protein. In mitochondria, spin-label binding to the carrier was evaluated by preincubation with the inhibitor carboxyatractyloside. The membrane sidedness of SH groups in the carrier molecule was determined by chemical reduction of nitroxides on the cytosolic membrane surface by Fe2+ or by pretreatment of the mitochondria with impermeant SH reagents. These experiments suggest that each subunit of the dimeric carrier incorporates one spin-labeled maleimide. Roughly half of the carrier-bound spin-labels were found on either side of the mitochondrial membrane. The detergent-solubilized carrier protein was labeled with a series of maleimide derivatives containing a spacer of increasing length between the maleimide and nitroxide moieties. A total spin-label binding of 2-3 mol/mol of protein dimer, depending on the spin-label length, was found. The electron spin resonance spectra of the spin-labeled protein invariably showed strongly and weakly immobilized components. Increasing the distance of the nitroxide from the maleimide ring resulted in a strong increase of the contribution of the weakly immobilized component. These observations led to the conclusions that the geometrical constraint of spin-label mobility changes at a distance of about 10 A from the maleimide binding site.     

Synthesis and properties of novel porphyrin spin probes containing isoindoline nitroxides

We report the synthesis of novel spin-labeled porphyrins containing isoindoline nitroxides (TMIO-APTPP and TMIO-APTSPP) and their manganese complexes (Mn-TMIO-APTPP and Mn-TMIO-APTSPP). These compounds represent potential new tools for electron paramagnetic resonance (EPR) as well as novel spin probes. Both TMIO-APTPP and TMIO-APTSPP have characteristic UV absorption peaks of free base porphyrin, while the characteristic absorption peaks of their manganese complexes Mn-TMIO-APTPP and Mn-TMIO-APTSPP shifted to shorter wavelengths. Electron paramagnetic resonance (EPR) spectroscopy indicated that these compounds all exhibit hyperfine splittings characteristic of EPR spectra of tetramethylisoindoline nitroxides, the typical nitroxide g values of approximately 2.006, and nitrogen isotropic hyperfine coupling constants (a(N) values) of about 14 G (293 K). The observed linewidths (La) for TMIO-APTSPP (0.73 G) and Mn-TMIO-APTSPP (0.65 G) in distilled water are significantly narrower than for TMIO-APTPP (1.475 G) and Mn-TMIO-APTPP (1.55 G) in chloroform.     

Kinetics of superoxide-induced exchange among nitroxide antioxidants and their oxidized and reduced forms.

Nitroxide stable radicals generally serve for probing molecular motion in membranes and whole cells, transmembrane potential, intracellular oxygen and pH, and are tested as contrast agents for magnetic resonance imaging. Recently nitroxides were found to protect against oxidative stress. Unlike most low molecular weight antioxidants (LMWA) which are depleted while attenuating oxidative damage, nitroxides can be recycled. In many cases the antioxidative activity of nitroxides is associated with switching between their oxidized and reduced forms. In the present work, superoxide radicals were generated either radiolytically or enzymatically using hypoxanthine/xanthine oxidase. Electron paramagnetic resonance (EPR) spectrometry was used to follow the exchange between the nitroxide radical and its reduced form; whereas, pulse radiolysis was employed to study the kinetics of hydroxylamine oxidation. The results indicate that: a) The rate constant of superoxide reaction with cyclic hydroxylamines is pH-independent and is lower by several orders of magnitude than the rate constant of superoxide reaction with nitroxides; b) The oxidation of hydroxylamine by superoxide is primarily responsible for the non-enzymatic recycling of nitroxides; c) The rate of nitroxides restoration decreases as the pH decreases because nitroxides remove superoxide more efficiently than is hydroxylamine oxidation; d) The hydroxylamine reaction with oxidized nitroxide (comproportionation) might participate in the exchange among the three oxidation states of nitroxide. However, simulation of the time-dependence and pH-dependence of the exchange suggests that such a comproportionation is too slow to affect the rate of non-enzymatic nitroxide restoration. We conclude that the protective activity of nitroxides in vitro can be distinguished from that of common LMWA due to hydroxylamine oxidation by superoxide, which allows nitroxide recycling and enables its catalytic activity.     

Different effectiveness of piperidine nitroxides against oxidative stress induced by doxorubicin and hydrogen peroxide

The piperidine nitroxides Tempamine and Tempace have been studied for their effect on doxorubicin (DOX) and hydrogen peroxide (H₂O₂) cytotoxicity in immortalized B14 cells, a model for neoplastic phenotype. The significance for nitroxide performance of the substituent in position 4 of the piperidine ring was evaluated. The cells were exposed to DOX/H₂O₂ alone or in combination with the nitroxides Tempamine or Tempace. Two other piperidine nitroxides, Tempo and Tempol, were used for comparison. All the nitroxides except Tempamine modestly reduced DOX cytotoxicity. Tempamine evoked a biphasic response: at concentrations lower than 200 μmol/L the nitroxide decreased DOX cytotoxicity, while at concentrations higher than 200 μmol/L, it enhanced DOX cytotoxicity. In contrast to Tempo and Tempol, Tempamine and Tempace ameliorated hydrogen peroxide cytotoxicity, but none of the nitroxides influenced TBARS stimulated by hydrogen peroxide. The cytoprotective effect of Tempace, Tempo and Tempol in DOX-treated cells correlated with the inhibition of DOX-induced lipid peroxidation. The bioreduction rates of the investigated nitroxides differed significantly and were variously affected by DOX depending on the nitroxide substituent. In combination with DOX, Tempo and Tempol were reduced significantly more slowly, while no influence of DOX on Tempamine and Tempace bioreduction was observed. Our results suggest that the structure of the 4-position substituent is an important factor for biological activity of piperidine nitroxides. Among the investigated nitroxides, Tempace displayed the best protective properties in vitro but Tempamine was the only nitroxide that potentiated cytotoxicity of DOX and did not influence DOX-induced lipid peroxidation. However, this nitroxide showed different performance depending on its concentration and conditions of oxidative stress.     

Mechanisms underlying gastric antiulcerative activity of nitroxides in rats

Reactive oxygen-derived species and redox-active metals are implicated in mediation of the pathogenesis of gastric mucosal damage and ulceration. Therefore, common strategies of intervention employ metal chelators, antioxidative enzymes, and low-molecular-weight antioxidants (LMWA). The aim of the present study was to elaborate the mechanism(s) responsible for the protection provided by nitroxide radicals in the experimental model of gastric ulceration. Fasted male rats were treated ig with 1 ml 96% ethanol, with or without ig pretreatment with nitroxide or hydroxylamine. In several experiments, rats were injected ip or iv with iron(III) or iron(II) prior to ethanol administration. Rats were sacrificed 10 min after ethanol administration, the stomach was removed, washed and lesion area measured. Pretreatment with iron(III) complexed to nitrilotriacetate or citrate, aggravated the extent of the gastric injury. Conversely, iron(II) inhibited the formation of lesions. The nitroxides were rapidly reduced to their respective hydroxylamines and demonstrated antiulcerative activity for rats treated with iron. However, injecting the hydroxylamine resulted in a similar tissue distribution of nitroxide/hydroxylamnine but did not provide protection. The results show that: (a) the nitroxide radicals, rather than their respective non-radical reduced form, are the active species responsible for protection; (b) nitroxides protect by dismutating O2*- and possibly indirectly increasing the NO level; (c) unlike classical LMWA which are reducing agents, nitroxides inhibit gastric damage by acting as mild oxidants, oxidizing reduced metals and pre-empting the Fenton reaction; and (d) the nitroxides act catalytically as recycling antioxidants.     

The interaction of neurotoxin derivatives with either acetylcholine receptor or a monoclonal antibody. An electron-spin-resonance study

Five derivatives of Naja nigricollis toxin alpha, spin-labeled on a single amino group, were prepared. The toxin derivatives were purified to homogeneity by ion-exchange and high-pressure liquid chromatographies. The modified amino groups are localized at residue 1 and lysines 15, 27, 47 and 51. Competition data show that incorporation of spin label at residues 27 or 47 reduces the affinity of the toxin for the nicotinic acetylcholine receptor (AcChR), while incorporation at residues 1 or 15 diminishes toxin affinity for a monoclonal toxin-specific immunoglobulin (M alpha 1). Classical and/or saturation transfer electron spin resonance (ESR) analysis was carried out on each derivative, either in the free state or bound to AcChR or M alpha 1. The data obtained give the following indications. In the free state, the nitroxides incorporated at residues 1, 15, 47 and 51 have their own rapid motion, while that at residue 27 had no residual mobility and reflects the toxin rotation. Binding of AcChR to the toxin reduces the motion of the nitroxide bound to Lys47. Binding of M alpha 1 to the toxin immobilizes the two nitroxides fixed on residues 1 and 15. ESR spectra show that Lys27-bound nitroxide remains immobilized upon binding of either AcChR or M alpha 1. The change in nitroxide immobilization observed upon AcChR or M alpha 1 binding correlates well with the variation of nitroxide accessibility to a water-soluble paramagnetic N2+i ion. Binding of the labeled Lys47 toxin derivative to AcChR yields a complex ESR signal, disclosing the existence of a physical difference between the two toxin binding sites on AcChR. All the data indicate that AcChR and M alpha 1 bind at two topographically distinct sites on the toxin surface.     

Cellular metabolism of water-soluble nitroxides: Effect on rate of reduction of cell/nitroxide ratio,oxygen concentrations and permeability of nitroxides

In order to interpret more accurately studies that have used nitroxides and to improve the efficacy of the use of nitroxides in both basic studies of cells and as contrast agents for in vivo NMR, we have initiated a systematic study of the distribution and metabolism of nitroxides in biological systems. Overall, the results provide a reasonably coherent picture of some aspects of the interactions between nitroxides and cells. Reduction of the nitroxides appears to be an intracellular process, so that one of the principal variables that affects the rate of reduction is the ability of a nitroxide to enter cells. The entrance of nitroxides into cells shows considerable variability and ranges from essentially no penetration (e.g., 2,2,6,6-tetramethylpiperidine-N-oxyl-4-trimethylamine) through rates that are comparable to rates of reduction (e.g., 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-carboxylic acid), to rates that are so fast that there is complete equilibrium between intracellular and extracellular compartments (e.g., Tempone). The presence of a charged group on the nitroxide appears to be the important variable that affects their ability to enter cells. Once a nitroxides enters the cell, the structure of the nitroxide, e.g., piperidine vs. pyrrolidine ring, is major factor that affects the rate of reduction. The rates of reduction increase with increasing concentrations of nitroxides. This indicates that the principal mechanism(s) of reduction do not saturate in the concentration range we studied. We observed no abrupt changes in the rates of reduction over the entire concentration range of cells and nitroxides that we studied, which suggests that the mechanism(s) of nitroxide reduction did not change. The presence of oxygen decreased the observed rate of reduction of many of the nitroxides and this effect was independent of the concentration of nitroxide.     

Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.

The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.     

Mechanisms underlying gastric antiulcerative activity of nitroxides in rats

Reactive oxygen-derived species and redox-active metals are implicated in mediation of the pathogenesis of gastric mucosal damage and ulceration. Therefore, common strategies of intervention employ metal chelators, antioxidative enzymes, and low-molecular-weight antioxidants (LMWA). The aim of the present study was to elaborate the mechanism(s) responsible for the protection provided by nitroxide radicals in the experimental model of gastric ulceration.

Fasted male rats were treated ig with 1 ml 96% ethanol, with or without ig pretreatment with nitroxide or hydroxylamine. In several experiments, rats were injected ip or iv with iron(III) or iron(II) prior to ethanol administration. Rats were sacrificed 10 min after ethanol administration, the stomach was removed, washed and lesion area measured. Pretreatment with iron(III) complexed to nitrilotriacetate or citrate, aggravated the extent of the gastric injury. Conversely, iron(III) inhibited the formation of lesions. The nitroxides were rapidly reduced to their respective hydroxylamines and demonstrated antiulcerative activity for rats treated with iron. However, injecting the hydroxylamine resulted in a similar tissue distribution of nitroxide/hydroxylamine but did not provide protection.

The results show that: (a) the nitroxide radicals, rather than their respective non-radical reduced form, are the active species responsible for protection; (b) nitroxides protect by dismutating O^·-₂ and possibly indirectly increasing the NO level; (c) unlike classical LMWA which are reducing agents, nitroxides inhibit gastric damage by acting as mild oxidants, oxidizing reduced metals and pre-empting the Fenton reaction; and (d) the nitroxides act catalytically as recycling antioxidants.     

Reduction kinetics and electrochemistry of tetracarboxylate nitroxides

Abstract

Tetracarboxylate pyrroline nitroxides undergo very fast reduction with ascorbate/glutathione (GSH), with second-order rate constants that are five orders of magnitude greater than those for gem-diethyl pyrroline nitroxides. For tetracarboxylate nitroxides, the electrochemical reduction potentials, measured by square wave voltammetry, are much less negative (by about 0.8 V), compared with the corresponding gem-diethyl nitroxides, while the oxidation potentials become more positive (by about 0.7 V). Electrochemical potentials correlate well via simple regressions with field/inductive parameters such as Swain/Lupton F-parameters (and/or Charton σ_I-parameters). Rates of reduction with ascorbate/GSH similarly correlate well for four pyrroline nitroxides, except for the slowest reducing gem-diethyl nitroxide. These results suggest that the electron withdrawing groups adjacent to the nitroxide moiety have a strong accelerating impact on the reduction rates, and thus they are not suitable for the design of hydrophilic nitroxides for in vivo applications.     

Exchange and shuttling of electrons by nitroxide spin labels

The ability of nitroxide spin labels to act as oxidizers of reduced nitroxides (hydroxylamines) in biological and model systems was demonstrated. All of the nitroxides tested were able to act as oxidizing agents with respect to hydroxylamine derivatives of nitroxides. The rates of these reactions were first order with respect to nitroxide concentration and with respect to hydroxylamine concentration, making the reaction second order overall. The second-order rate constants are reported for a number of these reactions. These reactions proceeded to an equilibrium state and the equilibrium constants for several combinations of reactants are presented. Both the rate constants and the equilibrium constants were found to be dependent on the ring structure of the nitroxide and hydroxylamine, with piperidines being reduced more easily and pyrrolidines and oxazolidines being oxidized more easily. All of the hydroxylamine derivatives were oxidized by air to their respective nitroxides, with the rate of this oxidation greater for pyrrolidines than for piperidines. Furthermore, hydroxylamines that are permeable to lipid bilayers were able to act as shuttles of reducing equivalents to liposome-encapsulated nitroxides that were otherwise inaccessible to reducing agents. This mechanism of shuttling of electrons was able to explain the relatively rapid reduction by cells of a nonpermeable nitroxide in the presence of a permeable nitroxide.     

Differential protection by nitroxides and hydroxylamines to radiation-induced and metal ion-catalyzed oxidative damage

Modulation of radiation- and metal ion-catalyzed oxidative-induced damage using plasmid DNA, genomic DNA, and cell survival, by three nitroxides and their corresponding hydroxylamines, were examined. The antioxidant property of each compound was independently determined by reacting supercoiled DNA with copper II/1,10-phenanthroline complex fueled by the products of hypoxanthine/xanthine oxidase (HX/XO) and noting the protective effect as assessed by agarose gel electrophoresis. The nitroxides and their corresponding hydroxylamines protected approximately to the same degree (33-47% relaxed form) when compared to 76.7% relaxed form in the absence of protectors. Likewise, protection by both the nitroxide and corresponding hydroxylamine were observed for Chinese hamster V79 cells exposed to hydrogen peroxide. In contrast, when plasmid DNA damage was induced by ionizing radiation (100 Gy), only nitroxides (10 mM) provide protection (32.4-38.5% relaxed form) when compared to radiation alone or in the presence of hydroxylamines (10 mM) (79.8% relaxed form). Nitroxide protection was concentration dependent. Radiation cell survival studies and DNA double-strand break (DBS) assessment (pulse field electrophoresis) showed that only the nitroxide protected or prevented damage, respectively. Collectively, the results show that nitroxides and hydroxylamines protect equally against the damage mediated by oxidants generated by the metal ion-catalyzed Haber-Weiss reaction, but only nitroxides protect against radiation damage, suggesting that nitroxides may more readily react with intermediate radical species produced by radiation than hydroxylamines.     

The Effect of Indolinic and Quinolinic Nitroxide Radicals on Trout Erythrocytes Exposed to Oxidative Stress

The purpose of this study was to evaluate the ability of indolinic and quinolinic nitroxide radicals to protect trout (Salmo irideus) erythrocytes against oxidative stress. By using laurdan as a fluorescence probe, it was observed that the nitroxides inhibited the shift towards a gel phase of liposomes prepared with phospholipids extracted from trout erythrocyte membranes prior to the hemolytic event. In addition, the presence of 100 μM nitroxides in these liposomes protected the latter against lipid peroxidation determined by monitoring conjugated diene formation. However, the short chain analogue of the indolinic nitroxide and the quinolinic nitroxide had a negative effect on trout hemolysis, contrary to what has already been observed in previous studies on human RBCs (red blood cells). The half-time (t_1/2) of the hemolytic process was 174 ± 4.02 min for the former and 184 ± 4.30 min for the latter compared to the control, 283 ± 5.05 min. Furthermore, the nitroxides remarkably increased the autoxidation rate of both trout and human hemoglobin to met-Hb. Even though protection at the membrane level is conferred by the nitroxides during the early stages of lipid peroxidation, their antioxidative ability might be overwhelmed at a later stage by other mechanisms such as the increased autoxidation of hemoglobin in the presence of the nitroxides, thus giving a possible explanation for the early induction of hemolysis induced by the nitroxides. The superoxide scavenging ability of all the nitroxides used was also evaluated through chemiluminescence.     

Binding of spin-labeled galactosides to the lactose permease of Escherichia coli

A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label.     

Ascorbate- and dehydroascorbic acid-mediated reduction of free radicals in the human erythrocyte

Nitroxides were used as models of persistent free radicals to study the antioxidant function of ascorbic acid in the human erythrocyte. It was concluded that: 1) ascorbate and other reductant(s) derived from dehydroascorbic acid (DHA) in the presence of thiols are the only significant reducing agents for nitroxides, 2) glutathione and DHA reduce nitroxides by a process that cannot be inhibited by ascorbic acid oxidase, 3) erythrocytes can be depleted of ascorbic acid by exhaustive washing in the presence of membrane-permeable cationic nitroxides such as N,N-dimethylamino-Tempo, 4) ascorbate-depleted cells do not reduce nitroxides; however, nitroxide reduction is restored when the cells are incubated with DHA, 5) reduction of nitroxides in ascorbate-depleted, DHA-treated cells is significantly faster than in buffered solutions of DHA and glutathione, 6) several equivalents of nitroxide are reduced relative to the intracellular ascorbate pool, 7) sustained nitroxide reduction is observed even when most of the intracellular ascorbate is oxidized, 8) spin trapping of oxyradicals in tert-butyl hydroperoxide-treated cells is accelerated with ascorbate depletion and inhibited with ascorbate loading, 9) ascorbate can be quantified within intact cells by analyzing the initial reduction rates of membrane-permeable cationic nitroxides, and 10) DHA-stimulated reduction of cationic nitroxides is slower and less extensive in erythrocytes deficient in glucose-6-phosphate dehydrogenase than in normal erythrocytes.