Absence Of Thiols Research Articles

Single phase preparation of monodispersed silver nanoclusters using a unique electron transfer and cluster stabilising agent, triethylamine.

A simple and reproducible single phase preparation of 2.5 nm silver nanoclusters is described using silver benzoate along with triethylamine (TEA) and dodecanethiol (DDT); these spontaneously self-assemble to a two-dimensional array whereas in the absence of thiol only polydispersed nanoclusters are obtained.

Thiols alter the partitioning of calicheamicin-induced deoxyribose 4'-oxidation reactions in the absence of DNA radical repair.

Cellular thiols have been proposed to play a protective role in oxidative DNA damage by quenching radical species in solution and by repairing deoxyribose and nucleobase radicals. There is also evidence for participation of thiols in reactions after formation of the DNA radical. Previous studies with neocarzinostatin, a thiol-dependent DNA-cleaving enediyne, revealed that the structure and charge of the activating thiol influence the partitioning of deoxyribose 4'-oxidation reactions between a 3'-phosphoglycolate residue and the alternative 4'-keto-1'-aldehyde abasic site [Kappen, L. S., et al. (1991) Biochemistry 30, 2034-2042; Dedon, P. C., et al. (1992) Biochemistry 31, 1917-1927]. However, interpretation of these results is confounded by the formation of a neocarzinostatin-thiol conjugate that could alter the position of the activated drug in the minor groove and quench drug radicals. Using the DNA-cleaving enediynes calicheamicin gamma(1)(I) and Ø, which are identical except for their trigger moieties, we now present a more definitive study of the role of thiol structure in the partitioning of the deoxyribose 4'-oxidation reaction. In the absence of thiols, calicheamicin Ø, which can undergo hydrolytic or reductive activation, generated 4'-oxidation products consisting of 26% 3'-phosphoglycolate residues, 33% 3'-phosphate-ended fragments, and 41% abasic sites (determined as the 3'-phosphopyridazine derivative). Using a series of thiols of varying size and charge, we found that, at concentrations that do not quench drug or DNA radicals, the negatively charged thiols glutathione and thioglycolate did not significantly affect the baseline proportions of the 4'-oxidation products. However, neutral thiols (O-ethylglutathione, methyl thioglycolate, 2-mercaptoethanol, and dithiothreitol) and, to a greater extent, the positively charged aminoethanethiol inhibited the production of 3'-phosphoglycolate residues with a proportional increase in the number of abasic sites. The effect of the thiols on the quantities of single- and double-stranded DNA lesions produced by calicheamicin gamma(1)(I) was also investigated since 3'-phosphoglycolate residues produced by calicheamicin exist only in double-stranded DNA lesions, and the thiol effects could have resulted from quenching of drug or DNA radicals. These studies revealed that, at thiol concentrations found to alter deoxyribose 4'-oxidation reactions, there was no apparent quenching of drug radicals or repair of DNA radicals. Thus, the effects of the thiols on the deoxyribose 4'-oxidation chemistry are due to reactions with a key intermediate in the phosphoglycolate- and abasic site-generating pathways. These results also suggest that cellular glutathione plays a relatively minor role in the chemistry of deoxyribose 4'-oxidation, which has implications for other oxidative reactions occurring in the minor groove of DNA (e.g., deoxyribose 5'- and 1'-oxidation).

One-pot full peptide deprotection in Fmoc-based solid-phase peptide synthesis: methionine sulfoxide reduction with Bu 4NBr

The reduction of methionine sulfoxide with Bu 4NBr in TFA is reported. The use of this reagent in conjunction with Fmoc chemistry-based acidolytic cocktails and the short reaction times that are needed for sulfoxide reduction (5 min) are the main advantages of this method, which is compatible with peptides containing aromatic amino acids. Cysteine is also stable under these conditions although, if desired, simultaneous disulfide formation can be achieved in the absence of thiols.

Radical-chain reductive alkylation of electron-rich alkenes mediated by silanes in the presence of thiols as polarity-reversal catalysts

In the presence of a thiol catalyst, triphenylsilane mediates the reductive alkylation of electron-rich terminal alkenes R1R2CCH2 by organic halides R3Hal via electrophilic carbon-centred radicals R3˙. Reactions were carried out in benzene or dioxane solvent using di-tert-butyl hyponitrite (at 60 °C) or dilauroyl peroxide (at 80 °C) as initiators and good yields of the adducts R1R2CHCH2R3 were obtained with either methyl thioglycolate or triphenylsilanethiol as catalysts (5–10 mol% based on alkene). In the presence of the thiol, the slow direct abstraction of hydrogen from the silane by the nucleophilic adduct radical R1R2ĊCH2R3 is replaced by a cycle of more rapid polarity-matched reactions in which hydrogen-atom transfer to the adduct radical from the thiol is followed by abstraction of hydrogen from the silane by the derived thiyl radical, to regenerate the catalyst. In the absence of thiol, negligible yields of reductive alkylation products were obtained. The homochiral thiols, 1-thio-β-D-mannopyranose tetraacetate and 1-thio-β-D-glucopyranose tetraacetate, and the tetrapivalate and tetrabenzoate analogues of the latter were effective catalysts and reductive carboxyalkylation products with enantiomeric excesses up to 72% were obtained from prochiral alkenes. Homochiral samples of two of these adducts were obtained by recrystallisation and their absolute configurations were determined by X-ray diffraction.

Extracellular β-amylase from Syncephalastrum racemosum

Syncephalastrum racemosum RR 96, MTCC 2774, synthesized extra-cellular β-amylase (EC 3.2.1.2) when cultivated with starch as sole carbon source. The strain showed maximum β-amylolytic activity on the fourth day of cultivation, and the partly purified enzyme showed highest activity at 60° and pH 5. About 60% of the activity was found to be retained within the range pH 4-7. The enzyme activity was not increased in presence of exogenous thiols and remained unaffected in presence of heavy metals and the thiol inhibitor p-chloromercuribenzoate, indicating the absence of thiols at the active site of the enzyme. Absence of substrate cross specificity and ability to hydrolyse waste starches made the enzyme competent for industrial application.

Kinetics and Mechanism of the Reactions of Superoxochromium(III) Ion with Biological Thiols

The kinetics of the oxidation of three biological thiols (l-cysteine, glutathione, and dl-penicillamine) to their sulfinic and sulfonic acid derivatives by CrOO2+ in aqueous perchloric acid and in the presence of 2-propanol have been studied spectrophotometrically with the aid of the initial-rates method. The kinetic order of the oxidant is 2, whereas that of the reductant is not defined. The acidity of the medium has a slight effect on the initial rates (acid catalysis for both l-cysteine and dl-penicillamine and base catalysis for glutathione). An increase of the ionic strength leads to a rise of the initial rate for both l-cysteine and dl-penicillamine, whereas the initial rate for glutathione is insensitive to the ionic strength. The reactions are inhibited by both 2-propanol and dissolved O2 and catalyzed by Mn2+, whereas Ce3+ has almost no effect on them. At low 2-propanol concentration and in the absence of Mn2+ the initial rate vs temperature plots have a minimum at around 20 °C, whereas in the presence of either concentrated 2-propanol or Mn2+ the Arrhenius law is fulfilled. A single mechanism is proposed for the three reactions involving a CrOO2+/thiol complex, CrOOH2+, and CrO2+ as intermediates. The bimolecular rate constants for the reactions of the intermediate CrO2+ with l-cysteine and dl-penicillamine at 25.0 °C have been obtained (around 103 M-1 s-1 in both cases). Some kinetic data for the decomposition of CrOO2+ in the absence of thiol are also given.

Intramolecular radical-chain hydrosilylation catalysed by thiols: cyclisation of alkenyloxysilanes

Alkenyloxy(diphenyl)silanes that contain a terminal double bond undergo radical-chain cyclisation at 60–65 °C, in the presence of di-tert-butyl hyponitrite as initiator and a thiol as a catalyst. The thiol acts as a polarity-reversal catalyst and promotes the overall abstraction of hydrogen from the Si–H group in the alkenyloxysilane by the cyclic carbon-centred radical, formed by intramolecular addition of the corresponding silyl radical to the CCH2 group. Allyloxysilanes give five-membered-ring products via 5-endo-trig cyclisation of the intermediate allyloxysilyl radical. Homoallyloxysilanes give mixtures of five- and six-membered heterocycles, but the intermediate silyl radicals undergo predominantly 6-endo cyclisation, in contrast to the corresponding carbon-centred radicals which cyclise preferentially in the 5-exo mode. An analogous pentenyloxysilane gives only the seven-membered-ring product via a 7-endo radical cyclisation. Steric effects play an important part in influencing the final-product stereochemistry when this is determined in the hydrogen-atom transfer reaction between the cyclic adduct radical and the thiol catalyst. Complementary EPR spectroscopic studies of the short-lived intermediate cyclic adduct radicals have been carried out in the absence of thiol and the structures and conformations of these species have been determined. It is emphasised that, for thiol catalysis of the overall cyclisation of alkenyloxysilanes to be successful, it is necessary for the addition of the chain-carrying thiyl radical to the CCH2 group to be reversible under the reaction conditions.

Thiol-independent DNA cleavage by a leinamycin degradation product

To understand the mechanism of action of a novel antitumor antibiotic leinamycin ( 1) which induces single-strand scission of DNA in the presence of thiol, the reaction of 1 with thiol in aqueous conditions was investigated. Two major degradation products were obtained from 1 in the presence of thiol. 2 was an inactive product, while 3 caused DNA cleavage in the absence of thiol. The DNA-cleaving activity of their synthetic derivatives indicates that the DNA alkylation and subsequent strand scission by leinamycin require the conversion of leinamycin to an electrophilic episulfonium species.

Measurement of Nitrite and Nitrate in Biological Fluids by Gas Chromatography–Mass Spectrometry and by the Griess Assay: Problems with the Griess Assay—Solutions by Gas Chromatography–Mass Spectrometry

Assay methods based on the Griess reaction are frequently used to measure nitrite and nitrate in urine, plasma, and other biological fluids. With minor exceptions, careful attention has not been paid in extending the Griess assay from aqueous solutions to biological fluids. In the present study, parallel measurements of nitrite and nitrate were performed in urine, plasma, and aqueous solutions with a published batch assay based on the Griess reaction and with gas chromatography–mass spectrometry (GC–MS). We report here further interferences by free reduced thiols, proteins, and other plasma constituents in the Griess assay but not in GC–MS. The best correlation (r2= 0.985) between the Griess assay and GC–MS was observed for aqueous solutions in the absence of thiols. Unlike GC–MS, the Griess assay was not applicable to whole human plasma and urine samples. For the measurement of nitrate in diluted human urine samples, reduction by cadmium was performed both under acidic (pH 2 or 5) and alkaline (pH 8.8) conditions. The mean recovery rate of nitrate from urine samples was quantitative in the GC–MS but amounted to only 30–80% in the Griess assay. Measurement of nitrate in human urine samples (n= 33) resulted in an excellent correlation between two GC–MS techniques (r2= 0.979) but only in a poor correlation (r2< 0.64) between the Griess assay and GC–MS. Unlike GC–MS, the batch Griess assay is associated with many problems in measuring nitrate in biological fluids.

Tyrosinase‐Mediated Cytotoxicity of 4‐Substituted Phenols: Use of QSAR to Forecast Reactivities of Thiols towards the Derivedortho‐Quinones

AbstractCertain 4‐substituted phenols can behave as tyrosine analogues and undergo tyrosinase‐catalysed oxidation to cytotoxico‐quinones which react with crucial cellular thiols. Such phenols are potential melanogenesis‐specific pro‐drugs for the chemotherapy of malignant melanoma. Previously (Cookseyet al.(1995)Anti‐Cancer Drug Design,10, 119), by pulse radiolysis under oxidative conditions of the corresponding stable catechols in the presence and absence of thiols, we showed that the glutathione and cysteine reactivities of ten 4‐substitutedo‐quinones correlated well with the Hammett σpconstants of the corresponding substituents. Starting from the corresponding catechols, one of which has not previously been described, we have now investigated six further 4‐substitutedo‐quinones including two with substituent σpvalues higher than those previously studied. Extrapolation of the earlier quantitative structure ‐ activity relationships (QSAR) correctly predicted the high reactivities of these twoo‐quinones, with respective substituents OCF3and CH2NH3+, towards thiols. The data for all sixteen 4‐substitutedo‐quinones, and foro‐benzoquinone itself, have been combined to provide updated, statistically significant Hammett and Swain‐Lupton correlations, including multivariate regressions using the data for both thiols. These relationships show that in reacting the substitutedo‐quinones with thiols, the rate constants increase with the electron withdrawing capacities of the substituent groups, this being principally due to the resonance effect, with a smaller but significant contribution attributable to the field effect. Such QSAR may facilitate the design of improved melanogenesis ‐ targeted anti‐melanoma prodrugs.

Large scale isolation and characterization of the molybdenum-iron cluster from nitrogenase.

Here we report the large scale isolation and characterization of a species, designated MoFe cluster, that exhibits an S = 3/2 EPR signal, and the comparison of this entity to isolated FeMo cofactor in N-methylformamide and to the active site of the enzyme nitrogenase. MoFe cluster is isolated from purified nitrogenase by extraction into acidic methyl ethyl ketone and it is stable in that solvent in the absence of thiols. As initially isolated, MoFe cluster solutions exhibit an S = 1/2 EPR signal that arises from an oxidized species that can be reduced by dithionite or thiols to an EPR silent state and then to a state that exhibits an S = 3/2 EPR signal. The S = 3/2 signal is as sharp as the signal exhibited by the protein and much sharper than the signal exhibited by isolated FeMo cofactor. Circular dichroism experiments indicate that unlike the last two species, MoFe cluster does not contain the endogenous ligand R-homocitrate and thus, the sharpness of the S = 3/2 signal is an intrinsic property of the metal center and does not depend upon specific interactions with this organic ligand or with the protein. Metal analyses indicate that the metal core responsible for the S = 3/2 signal contains 6 Fe atoms per molybdenum. X-ray absorption spectroscopy experiments show that although the molybdenum atom in MoFe cluster retains its pseudo-octahedral geometry, its first coordination shell has one less iron atom than that of FeMo cofactor and there has been a significant change in the long range order of the cluster.

C1027 chromophore, a potent new enediyne antitumor antibiotic, induces sequence-specific double-strand DNA cleavage.

C1027, a new macromolecular antitumor antibiotic produced by a Streptomyces strain, shows highly potent cytotoxicity to cultured cancer cells and marked DNA cleaving ability. The structure of its chromophore, responsible for most of the biological activities of the antibiotic, was recently determined and found to contain a nine-membered enediyne. In contrast to other enediyne antibiotics, such as neocarzinostatin, calicheamicin, esperamicin, and recently found kedarcidin, C1027 damages duplex DNA even in the absence of thiols. The DNA damage caused by C1027 includes double-strand breaks, single-strand breaks, and abasic sites. Experiments with plasmid DNA and 32P-end-labeled restriction fragments demonstrated that the chromophore, extracted from the protein-containing holoantibiotic, interacts in the DNA minor groove and cleaves double-helical DNA with a remarkable sequence-selectivity causing direct double-strand breaks. The double-strand cleavage sites, occurring predominantly at CTTTT/AAAAG, ATAAT/ATTAT, CTTTA/TAAAG, CTCTT/AAGAG, and especially GTTAT/ATAAC, consist of five nucleotide sequences with a two-nucleotide 3'-stagger of the cleaved residues (cutting sites are underlined). The chemical structures of the damaged residues at the GTTAT/ATAAC cleavage site suggest a model in which a C1027-induced double-strand break results from abstraction, by a single molecule of the diradical form of the chromophore, of a C4' hydrogen atom from the A residue of GTTAT and a C5' hydrogen atom from the A of ATAAC on the opposite strand. Single-strand breaks, which are mainly produced at adenylate and thymidylate residues, appear to be separate events presumably resulting from different binding modes of the drug to DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of ebselen with glutathione S-transferase and papain in vitro

The interaction of ebselen(2-phenyl-1,2-benzisoselenazol-3(2 H)-one) with rat liver cytosolic glutathione S-transferases (GSTs) and the plant cysteine protease, papain, was studied as cysteine residues are important for the activity of these enzymes. The capacity of GST 1–2 and 3–4 for ebselen binding is similar (1.5 mol ebeselen/mol GST isozyme), while GST 2-2 and GST 7-7 bind 0.3 and more than 2.0 mol ebselen/mol GST isozyme, respectively. Ebselen does not bind to N-ethylmaleimide-treated GST, and its binding to GST is prevented by 5mM thiol. Ebselen irreversibly inactivates the different GST isozymes with a second order rate constant ranging from 20 to 2250 M −1 sec −1 for the different subunits. GST inhibition by ebselen is partially restored by 5 mM thiols. Ebselen binds to untreated papain and to cysteine-treated papain at a ratio of about 0.1 and 0.75 mol ebselen/mol papain, respectively. Ebselen does not bind to N-ethymaleimide-treated papain, and its binding to papain is interfered with by added thiols. Papain is inactivated by ebselen with a second order rate constant of 1800 M −1 sec −1 in the absence of thiols. However, in the presence of GSH, 2-mercaptoethanol orsodium borohydrine, ebselen exerts an activating effect on papain. The binding of ebselen by a seleno-sulfide bond to cysteine residues of GSTs and papain leads to their inactivation.

An examination of the copper requirement of phenylalanine hydroxylase from Chromobacterium violaceum.

Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) was classified as a copper metalloenzyme by virtue of a 1/1 Cu/enzyme stoichiometry and its inhibition with various chelators [Pember, S. O., Villafranca, J. J., & Benkovic, S. J. (1986) Biochemistry 25, 6611]. We have prepared "copper-free" CVPAH by extraction with DTT. These preparations retained full activity though the Cu/enzyme ratio averaged 0.015. Reconstitution by extraneous copper was disproved by measuring a Cu/enzyme ratio of 0.09 in assay mixtures after the specific activity was determined to be within 85% of a fully copper-complexed control. Several copper chelators were examined and were not inhibitory. The "copper-free" enzyme had significant activity without a thiol or other reducing agent capable of reducing the copper center whereas copper-complexed CVPAH had minimal activity under these conditions. Copper-complexed CVPAH can be activated, however, by nonreducing copper ligands such as imidazole. From these results, we conclude that copper is not a requirement of activity. Iron, cobalt, nickel, manganese, molybdenum, and chromium were not found in the "copper-free" preparation, indicating that the active hydroxylating species may not require a redox-active metal. The KdS for binding Cu2+ and Zn2+ were measured to be 0.48 and 0.85 microM, respectively. Both copper and zinc were found to be potent inhibitors of "copper-free" CVPAH in the absence of thiols. DTT reverses inhibition due to Cu2+ but not inhibition caused by Zn2+. The product stoichiometry indicates the same tightly coupled turnover found with all other pterin-dependent hydroxylases when using natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA conformation-induced activation of an enediyne for site-specific cleavage.

Neocarzinostatin chromophore (NCS chrom) was found to induce site-specific cleavage at the 3' side of a bulge in single-stranded DNA in the absence of thiol. This reaction involved the oxidative formation of a DNA fragment with a nucleoside 5'-aldehyde at its 5' terminus and generated an ultraviolet light-absorbing and fluorescent species of post-activated drug containing tritium abstracted from the carbon at the 5' position of the target nucleotide. The DNAs containing point mutations that disrupt the bulge were not cleavage substrates and did not generate this drug product. Thus, DNA is an active participant in its own destruction, and NCS chrom may be useful as a probe for bulged structures in nucleic acids.

Some characteristics of DNA strand scission by macromolecular antitumor antibiotic C-1027 containing a novel enediyne chromophore

A new macromolecular antitumor antibiotic, C-1027, shows potent cytotoxic effects and DNA cutting activity. The DNA cleaving properties of C-1027 are compared with those of other enediyne compounds such as neocarzinostatin, esperamicin A1, and calicheamicin gamma 1. Even in the absence of thiols or reductants, the antibiotic C-1027 has high DNA breakage ability. Of special interest is the fact that C-1027 causes strand breaks two base pairs apart at specific sites such as 5'-TAT/3'-ATA and 5'-AGA/3'-TCT (cleavage sites in italics) in the two strands. This novel double-stranded cleavage fashion is different from that of calicheamicin gamma 1, which is found to have a 3-bp separation between cleavage sites on the two strands. The asymmetric cleavage pattern to the 3'-side and a competitive experiment with distamycin A reveal minor-groove interaction of double-helical DNA with C-1027. This antibiotic appears to oxidize DNA through hydrogen abstraction predominantly at the C-4' carbon of deoxyribose. The activation mechanism of C-1027, which contains an enediyne chromophore of the esperamicin/calicheamicin type, has been proposed.

Purification and properties of adenosine 5′-phosphosulphate sulphotransferase from Euglena

Adenosine 5'-phosphosulphate sulphotransferase (APSST) was extracted from Euglena gracilis Klebs var. bacillaris mutant W10BSmL by freezing and thawing and was purified about 10,000-fold (to homogeneity) with 10.5% recovery by (NH4)2SO4 precipitation, Sephadex G-100 chromatography, Reactive Blue-agarose, Reactive Dye-agarose, DEAE-cellulose, preparative isoelectric focusing and non-inactivating SDS/PAGE. The active APSST, with a molecular mass of 102 kDa and multiple forms from pI 5.0 to 5.5, is a tetramer held together by covalent (probably disulphide) bonds. An apparent Km of the purified enzyme for adenosine 5'-phosphosulphate (APS) of 0.1 microM is obtained when dithiothreitol is used as the thiol. The enzyme is stimulated by Mg2+, Ca2+ or Ba2+, and uses almost any thiol; dithiothreitol and dithioerythritol give the highest activity. In the absence of APS, the enzyme is inactivated (and is rendered monomeric) by thiols but is protected from thiol inactivation by AMP, adenosine 5'-phosphoramidate (APA) or adenosine 5'-monosulphate (AMS), which also inhibit APSST activity somewhat. The enzyme resists inactivation by SDS in the absence of thiols; SDS stimulates APSST activity at low concentration, but high concentrations are inhibitory.

The Dependence of Thiol-Inducible Radiation Resistance in Escherichia coli K12 on the Medium and Catalytic Metal

Radiation protection by thiols in procaryotes and lower eucaryotes has been demonstrated repeatedly to require a competent DNA repair phenotype, suggesting that simple chemical radical scavenging and hydrogen donation are only a portion of the mechanism of radiation protection by thiols. In the present report, thiol-induced radiation resistance--a model in which cells are pretreated with dithiothreitol and then irradiated in the absence of thiol--is shown to be a medium-dependent process. Wild-type log-phase cells treated with dithiothreitol in minimal-glucose medium are induced to radioresistance that persists after the thiol has been removed. Although the thiol pretreatment affected the antioxidants (catalase, superoxide dismutase, and glutathione) in cells at the time of irradiation, various antioxidant levels did not predict radiation resistance. Thiol-induced radioresistance is not expressed in rich medium-treated cells or in DNA repair (recA)-deficient cells. Addition of the efficient chelator, DETAPAC, to the thiol treatment medium leads to additional radioresistance in the case of minimal medium and a moderate expression of resistance in rich medium. Experiments using the intracellular chelator, 1,10-phenanthroline, in the presence of thiol led to inhibition of thiol-induced resistance in minimal medium and radiosensitization in rich medium. These results can be explained by a "site-specific" mechanism of thiol oxidation in which the chelators control the site(s) and rate of thiol oxidation, subsequently determining the type of cellular response.

Aldose reductase from human skeletal and heart muscle. Interconvertible forms related by thiol-disulfide exchange.

Aldose reductase was purified from human skeletal and heart muscle by a rapid and efficient scheme involving Red Sepharose chromatography, chromatofocusing on Pharmacia PBE 94, and hydroxylapatite high pressure liquid chromatography. The scheme afforded homogeneous enzyme, 65% recovery, in 2 days. All muscle samples express aldose reductase but not the closely related aldehyde reductase. Aldose reductase is isolated in one of two forms that are distinguishable by their kinetic patterns with glyceraldehyde as substrate and which are interconvertible by treatment with dithiothreitol. Both forms are capable of catalyzing the reduction of glucose (Km = 68 mM), and both are highly sensitive to inhibition by aldose reductase inhibitors. The reduction of glucose was shown to be nearly stoichiometric with production of sorbitol (92 +/- 2%). Dialysis of aldose reductase in the absence of thiols or NADP converts it into a form that shows markedly different kinetic properties, including very weak catalytic activity toward glucose and insensitivity to aldose reductase inhibitors. This modified form can be converted back into the native form by dithiothreitol. Thiol titration of the two forms of aldose reductase with Ellman's reagent indicated that two thiol groups were lost when the enzyme was dialyzed in the absence of dithiothreitol or NADP.

Application of Spin Traps to Biological Systems

Since 1971, when nitroxides were first reported to be bioreduced, several cellular enzymes, in addition to ascorbic acid, have been found to catalyze the reduction of nitroxides to their corresponding hydroxylamines. Numerous studies have demonstrated that cellular bioreduction of nitroxides are both dependent upon the structure of the nitroxide and cell type. For example, pyrrolidinyloxyls are considerably more resistant to bioreduction than their corresponding piperidinyloxyls. In addition, cellular levels of reductases present in freshly isolated rat hepatocytes are considerably greater than concentrations found in freshly isolated rat enterocytes. Thus, through the proper selection of a cell type and an appropriate nitroxide, one can study cellular-mediated free radical processes. With the discovery that alpha-hydrogen-containing nitroxides, including 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH) decompose rapidly in the presence of superoxide and thiols, the ability to determine if hydroxyl radical is generated during stimulation of human neutrophils, is in doubt. To explore the limits of spin trapping in this context, we have studied the effect of varying the rates of superoxide production, in the presence and absence of thiols, on the decomposition of DMPO-OH. In parallel studies, we have found that t-butyl alpha-methyl-4-pyridinyl-N-oxide nitroxide (4-POBN-CH3) will not degrade in the presence of superoxide and a thiol. From these studies, we have determined that if hydroxyl radicals were generated as an isolated event in the presence of a continual flow of superoxide, spin trapping might not be able to detect its formation. Otherwise, spin trapping should be able to measure hydroxyl radicals, if continually generated, during activation of human neutrophils.