Oxidative inactivation of rhodanese by hydrogen peroxide produces states that show differential reactivation

Controlled conditions have been found that give complete reactivation and long term stabilization of rhodanese (EC 2.8.1.1) after oxidative inactivation by hydrogen peroxide. Inactivated rhodanese was completely reactivated by reductants such as thioglycolic acid (TGA) (100 mM) and dithiothreitol (DTT) (100 mM) or the substrate thiosulfate (100 mM) if these reagents were added soon after inactivation. Reactivability fell in a biphasic first order process. At pH 7.5, in the presence of DTT inactive rhodanese lost 40% of its reactivability in less than 5 min, and the remaining 60% was lost more gradually (t 1/2 = 3.5 h). TGA reactivated better than DTT, and the rapid phase was much less prominent. If excess reagents were removed by gel filtration immediately after inactivation, there was time-independent and complete reactivability with TGA for at least 24 h, and the resulting samples were stable. Reactivable enzyme was resistant to proteolysis and had a fluorescence maximum at 335 nm, just as the native protein. Oxidized rhodanese, Partially reactivated by DTT, was unstable and lost activity upon further incubation. This inactive enzyme was fully reactivated by 200 mM TGA. Also, the enzyme could be reactivated by arsenite and high concentrations of cyanide. Addition of hydrogen peroxide (40-fold molar excess) to inactive rhodanese after column chromatography initiated a time-dependent loss of reactivability. This inactivation was a single first order process (t 1/2 = 25 min). Sulfhydryl titers showed that enzyme could be fully reactivated after the loss of either one or two sulfhydryl groups. Irreversibly inactivated enzyme showed the loss of one sulfhydryl group even after extensive reduction with TGA. The results are consistent with a two-stage oxidation of rhodanese. In the first stage there can form sulfenyl and/or disulfide derivative(s) at the active site sulfhydryl that are reducible by thioglycolate. A second stage could give alternate or additional oxidation states that are not easily reducible by reagents tried to date.     

Oxidative inactivation of the enzyme rhodanese by reduced nicotinamide adenine dinucleotide

The enzyme rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is inactivated with a half-time of approximately 3 min when incubated with 50 mM NADH. NAD+, however, has virtually no effect on the activity. Inactivation can be prevented by the inclusion of the substrate thiosulfate. The concentration of thiosulfate giving half-protection is 0.038 mM. In addition, NADH, but not NAD+, is a competitive inhibitor with respect to thiosulfate in the catalyzed reaction (Ki = 8.3 mM). Fluorescence studies are consistent with a time-dependent oxidation of NADH in the presence of rhodanese. The sulfur-free form of rhodanese is more rapidly inactivated than the sulfur-containing form. Spectrophotometric titrations show that inactivation is accompanied by the loss of two free SH groups per enzyme molecule. Inactivation is prevented by the exclusion of air and the inclusion of EDTA (1 mM), and the enzyme activity can be largely protected by incubation with superoxide dismutase or catalase. Rhodanese, inactivated with NADH, can be reactivated by incubation with the substrate thiosulfate (75 mM) for 48 h or more rapidly, but only partially, by incubating with 180 mM dithiothreitol. It is concluded that, in the presence of rhodanese, NADH can be oxidized by molecular oxygen and produce intermediates of oxygen reduction, such as superoxide and/or hydrogen peroxide, that can inactivate the enzyme with consequent formation of an intraprotein disulfide. In addition, NADH, but not NAD+, can reversibly bind to the active site region in competition with thiosulfate. These data are of interest in view of x-ray studies that show structural similarities between rhodanese and nucleotide binding proteins.     

alpha-Crystallin facilitates the reactivation of hydrogen peroxide-inactivated rhodanese

It was previously shown that rhodanese, inactivated with hydrogen peroxide, could only be reactivated in the presence of a reductant or the substrate thiosulfate if these reagents were added soon after inactivation and if the oxidant was removed. Here, we report on the facilitated reactivation (75%) of hydrogen peroxide-inactivated rhodanese by the chaperone alpha-crystallin. Reactivation by the chaperone still required a reductant and thiosulfate. Without alpha-crystallin, but in the presence of the reductant and thiosulfate, the inactivated enzyme regained about 39% of its original activity. The alpha-crystallin-assisted reactivation of hydrogen peroxide-inactivated rhodanese was independent of ATP. Further, we found, that alpha-crystallin interacted transiently, but could not form a stable complex with hydrogen peroxide-inactivated rhodanese. Unlike in prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that alpha-crystallin can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein.     

Interaction of rhodanese with intermediates of oxygen reduction

Cyanide-promoted inactivation of the enzyme rhodanese [thiosulfate sulfurtransferase (EC 2.8.1.1)] in the presence of ketoaldehydes is caused by reduced forms of molecular oxygen generated during autoxidation of the reaction products. The requirement of both catalase and superoxide dismutase to prevent rhodanese inactivation indicates that hydroxyl radical could be the most efficient inactivating agent. Rhodanese, also in the less stable sulfur-free form, shows a different sensitivity towards oxygen activated species. While the enzyme is unaffected by superoxide radical, it is rapidly inactivated by hydrogen peroxide. The extent of inactivation depends on the molar ratio between sulfur-free enzyme and oxidizing agent. Fully inactive enzyme is reactivated by reduction with its substrate thiosulfate.     

A study of dithiothreitol inactivation of the enzyme rhodanese.

When air oxidized, partially inactivated rhodanese (EC 2.8.1.1) is treated with dithiothreitol (DTT) to regenerate the reduced essential sulfhydryl group there is an initial reactivation followed by an anomalous slower inactivation. Fully active enzyme shows only inactivation. The inactivated enzyme may be completely reactivated on long incubation with the substrate thiosulfate ion. None of the normal potentialities of DTT appear to be responsible for the inactivation. The results are interpreted in terms of disulfide formation between DTT and an essential enzymic sulfhydryl group with the resulting complex being stabilized by secondary interactions which are particularly favorable due to similarities between DTT and lipoic acid--a normal sulfur acceptor substrate.     

Acceptor substrate-potentiated inactivation of bovine liver rhodanese

The interaction of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) with the acceptor substrates, dithiothreitol or cyanide, was studied. When incubated in the presence of cyanide or dithiothreitol, rhodanese was inactivated in a time-dependent process. This inactivation was detectable only at low enzyme concentrations; the rate and degree of inactivation could be modulated by varying the substrate concentration or the system pH. Activity measurements and fluorescence spectroscopy techniques were used in examining the inactivation phenomenon. Sulfur transfer to dithiothreitol was measured by direct assay and was shown to involve the dequenching of enzymic intrinsic fluorescence that had been previously observed only with cyanide as the acceptor substrate. Substrate-potentiated inactivation of rhodanese (with cyanide) has been reported before, but the cause and nature of this interaction were unexplained. The results presented here are consistent with an explanation invoking oxidation of rhodanese in the course of inactivation.     

Chaperonin cpn60 from Escherichia coli protects the mitochondrial enzyme rhodanese against heat inactivation and supports folding at elevated temperatures.

The chaperonin protein cpn60 from Escherichia coli protects the monomeric, mitochondrial enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) against heat inactivation. The thermal inactivation of rhodanese was studied for four different states of the enzyme: native, refolded, bound to cpn60 in the form of a binary complex formed from unfolded rhodanese, and a thermally perturbed state. Thermal stabilization is observed in a range of temperatures from 25 to 48 degrees C. Rhodanese that had been inactivated by incubation at 48 degrees C, in the presence of cpn60 can be reactivated at 25 degrees C, upon addition of cpn10, K+, and MgATP. A recovery of about 80% was achieved after 1 h of the addition of those components. Thus, the enzyme is protected against heat inactivation and kept in a reactivable form if inactivation is attempted using the binary complex formed between rhodanese folding intermediate(s) and cpn60. The chaperonin-assisted refolding of urea-denatured rhodanese is dependent on the temperature of the refolding reaction. However, optimal chaperonin assisted refolding of rhodanese observed at 25 degrees C, which is achieved upon addition of cpn10 and ATP to the cpn60-rhodanese complex, is independent of the temperature of preincubation of the complex, that was formed previously at low temperature. The results are in agreement with a model in which the chaperonin cpn60 interacts with partly folded intermediates by forming a binary complex which is stable to elevated temperatures. In addition, it appears that native rhodanese can be thermally perturbed to produce a state different from that achieved by denaturation that can interact with cpn60.     

Chemical modification of bovine liver rhodanese with tetrathionate: differential effects on the sulfur-free and sulfur-containing catalytic intermediates

Sulfhydryl groups of bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) were modified by treatment with tetrathionate. There was a linear relationship between loss of enzyme activity and the amount of tetrathionate used. At a ratio of one tetrathionate per mole of rhodanese, 100% of enzyme activity was lost in the sulfur-free E-form as compared with a 70% loss for the sulfur-containing ES-form of the enzyme. Addition of up to a 100-fold molar excess of tetrathionate to ES gave no further inactivation. Addition of cyanide to the maximally inactivated ES-tetrathionate complex gave complete loss of activity. Kinetic studies of maximally inactivated ES and partially inactivated E gave Km (Ks) values that were essentially the same as native enzyme, indicating that the active enzyme, in all cases, bound thiosulfate similarly. Reactivation was faster with the ES-form than with the E-form. The substrate, thiosulfate, could reactivate the enzyme up to 70% in 1 h with ES as compared to 24 h with E. Tetrathionate modification of rhodanese could be correlated with the changes in intrinsic fluorescence and with the binding of the active site reporter 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS). Circular dichroism spectra of the protein suggested increased ordered secondary structure in the protein after reaction with tetrathionate. Cadmium chloride and phenylarsine oxide totally inactivated the enzyme at levels usually associated with their effect on enzymes containing vicinal sulfhydryl groups. Further, cadmium inhibition could be reversed by EDTA. Tetrathionate modification of rhodanese may proceed through the formation of sulfenylthiosulfate intermediates at sulfhydryl groups, close to but not identical with the active-site sulfhydryl group, which then can react further with the active-site sulfhydryl group to form disulfide bridges.     

Properties of an Escherichia coli rhodanese

A rhodanese enzyme of less than 20,000 molecular weight has been purified from Escherichia coli. The enzyme is accessible to substrates upon addition of whole cells to standard assay mixtures. This rhodanese has a Stokes radius of 17 A which for a globular protein corresponds to a molecular weight close to 14,000. It undergoes autoxidation to a polymeric form which is probably an inert dimer. Enzyme inactivated by oxidation can be reactivated by millimolar concentrations of cysteine. Steady-state initial velocity measurements indicate that the enzyme catalyzes the transfer of sulfane sulfur by way of a double displacement mechanism with formation of a covalent enzyme-sulfur intermediate. The turnover number for the enzyme-catalyzed reaction, with thiosulfate as donor substrate and cyanide ion as the sulfur acceptor, is 260 s-1. This value corresponds to a catalytic efficiency 60% of that measured for a previously characterized bovine liver enzyme of more than twice the molecular weight. Furthermore, KmCN is 24 mM which is 2 orders of magnitude higher than the value observed previously for the bovine enzyme. Evidence from chemical inactivation studies implicates an essential sulfhydryl group in the enzyme activity. It is proposed that this group is the site of substrate-sulfur binding in the obligatory enzyme-sulfur intermediate. Furthermore, a cationic site important for binding of the donor thiosulfate is tentatively identified from anion inhibition studies. Tests of alternate acceptor substrates indicate that the physiological dithiol, dihydrolipoate, is a more efficient acceptor than cyanide ion for the enzyme-bound sulfur. Of possibly greater physiological significance, it has been found that the enzyme catalyzes the formation of iron-sulfur centers. Other work indicates the E. coli rhodanese is subject to catabolite repression and suggests a physiological role for the enzyme in aerobic energy metabolism.     

Hydrogen peroxide involvement in the rhodanese inactivation by dithiothreitol.

The conditions required to obtain rhodanese inactivation in the presence of dithiothreitol indicate the involvement of hydrogen peroxide produced by metal-ion catalyzed oxidation of dithiothreitol. Inhibition of dithiothreitol oxidation by a chelating agent, or by removal of hydrogen peroxide by catalase prevents the enzyme inactivation. The inactivated enzyme contains a disulfide bond resulting from the oxidation of the catalytic sulfhydryl group and another sulfhydryl group close to it. This disulfide might be formed via a sulfenic intermediate.     

The specificity of active-site alkylation by iodoacetic acid in the enzyme thiosulfate sulfurtransferase

The active-site sulfhydryl group in the enzyme thiosulfate sulfurtransferase (rhodanese; thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) is alkylated rapidly by iodoacetic acid in the free enzyme form, E, with complete loss of sulfurtransferase activity. Iodoacetic acid is completely ineffective with the sulfur-substituted form of the enzyme, ES. Iodoacetamide, on the other hand, has no effect on either enzyme form. The competitive enzyme inhibitor, toluenesulfonic acid, protects against inactivation in a strictly competitive way and analysis gives an apparent binding constant for toluenesulfonic acid of 12.5 mM, which is in agreement with studies of its effect on the catalyzed reaction. These results are taken to indicate that iodoacetic acid is an affinity analog for the substrate, thiosulfate, and inactivates because it can use the specific thiosulfate binding interactions, correctly orient its reactive center and displace intraprotein interactions which appear to protect the active-site sulfhydryl group in the E form.     

The differential functional stability of various forms of bovine liver rhodanese

Bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was prepared in dilute solutions and subjected to conditions that led to a time-dependent loss of enzyme activity. The rate of this activity loss was found to be dependent upon the sulfur substitution state of the enzyme, and the presence or absence of the substrates, thiosulfate and cyanide. In the absence of excess substrates, free enzyme (E), and the covalent intermediate form of the enzyme bearing a divalent sulfur atom in the active site (ES), are of approximately equal functional stability. In comparison, E, in the presence of excess cyanide, was markedly more labile, while ES, supported by 10-50 mM thiosulfate, showed no significant loss of activity under any of the conditions tested. All the enzyme solutions were shown to be losing assayable protein from solution. However, it was demonstrated that, for rhodanese in the E form, the amount of protein lost was insufficient to account for the activity lost, and a marked decline in specific activity was observed. Enzyme in the ES form, whether supported by additional thiosulfate or not, did not decline in the specific activity, though comparable protein loss did occur from these solutions. Intrinsic fluorescence measurements of rhodanese in the ES form, before and after removal of the persulfide sulfur through the addition of cyanide, indicated that loss of enzymic activity was not accompanied by loss of the bound sulfur atom. Therefore, the stabilizing effect observed with thiosulfate could not be explained simply by its ability to maintain enzyme in the sulfur-substituted state. Since the concentration of thiosulfate employed in these experiments was insufficient to maintain all the enzyme in ES.S2O3 form, thiosulfate was acting as a chemical reagent rather than a substrate in stabilizing enzyme activity.     

Thermally perturbed rhodanese can be protected from inactivation by self-association

A fluorescence-detected structural transition occurs in the enzyme rhodanese between 30–40°C that leads to inactivation and aggregation, which anomalously decrease with increasing protein concentration. Rhodanese at 8 µg/ml is inactivated at 40°C after 50 min of incubation, but it is protected as its concentration is raised, such that above 200 µg/ml, there is only slight inactivation for at least 70 min. Inactivation is increased by lauryl maltoside, or by low concentrations of 2-mercaptoethanol. The enzyme is protected by high concentrations of 2-mercaptoethanol or by the substrate, thiosulfate. The fluorescence of 1,8-anilinonaphthalene sulfonate reports the appearance of hydrophobic sites between 30–40°C. Light scattering kinetics at 40°C shows three phases: an initial lag, a relatively rapid increase, and then a more gradual increase. The light scattering decreases under several conditions: at increased protein concentration; at high concentrations of 2-mercaptoethanol; with lauryl maltoside; or with thiosulfate. Aggregated enzyme is inactive, although enzyme can inactivate without significant aggregation. Gluteraldehyde cross-linking shows that rhodanese can form dimers, and that higher molecular weight species are formed at 40°C but not at 23°;C. Precipitates formed at 40°C contain monomers with disulfide bonds, dimers, and multimers. We propose that thermally perturbed rhodanese has increased hydrophobic exposure, and it can either: (a) aggregate after a rate-limiting inactivation; or (b) reversibly dimerize and protect itself from inactivation and the formation of large aggregates.     

Conformational changes accompany the oxidative inactivation of rhodanese by a variety of reagents

Rhodanese is oxidatively inactivated by several reagents, some of which are not normally considered oxidants. Rhodanese, in a form not containing persulfide sulfur (E), was inactivated by phenylglyoxal under conditions where disulfides are formed. There was the concomitant increase in the fluorescence of the apolar probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). At 0.2 mg/ml protein, there was no turbidity, while at 1 mg/ml, turbidity formed after an induction period of 23 min. Phenylglyoxal-inactivated E was extensively digested by endoproteinase glutamate C (V8 protease) to give two discrete high molecular weight fragments (Mr = 29,500 and 16,000). Enzymatically active E or ES, the form of rhodanese containing transferred sulfur (Mr = 33,000) was totally refractory to V8 protease and gave only small fluorescent enhancement of bisANS. Phenylglyoxal inactivated ES (reaction at arginine) gave very little fluorescence enhancement of bisANS and was not digested by V8. Hydrogen peroxide rapidly inactivated E (t1/2 less than 2 min) giving a slow increase in bisANS fluorescence (t1/2 greater than 10 min) identical to that observed with phenylglyoxal. The turbidity also increased after an induction period of approximately 30 min. Inactivation of E by hydrogen peroxide gave the same digestion pattern as that observed with phenylglyoxal inactivation. The turbidity was associated with the formation of disulfide-bonded structures that formed with the stoichiometry of E, 2E, 4E, 6E, 8E, etc. relative to the native enzyme, E. E was inactivated with several other reagents that lead to oxidatively inactivated rhodanese including NADH, dithiothreitol, mercaptoethanol, and m-dinitrobenzene. Enzyme inactivated with dithiothreitol or NADH gave an identical digestion pattern as above. In addition, with the exception of NADH which could not be used due to optical interference, each of the reagents gave rise to increased fluorescence of bisANS after inactivation. The results are consistent with a model in which the oxidized rhodanese resulting from diverse treatments is in a new conformation that has extensive exposed apolar surfaces and can form both noncovalent and disulfide-bonded aggregates.     

Active site modifications quench intrinsic fluorescence of rhodanese by different mechanisms

Beef liver rhodanese can be modified covalently at the active site (Cys-247) either reversibly or irreversibly by sulfur, selenium, iodoacetate, and hydrogen peroxide. Each derivative shows an intrinsic fluorescence lower than that of the free enzyme. The reaction of rhodanese with iodoacetate or hydrogen peroxide is time-dependent and accompanied by enzyme inactivation, by the loss of one or two sulfhydryl groups, respectively, by quenching and bathochromic shift of fluorescence, and by an absorbance perturbation in the near UV. The latter findings are indicative for a displacement of some tryptophyl side chains from hydrophobic to hydrophilic environment. The fluorescence decays of the various rhodanese derivatives can be fitted by a double-exponential function with two lifetimes: a shorter one of 1-1.7 ns and a longer one of 2.8-4.6 ns. The S-loaded and Se-loaded rhodanese samples have proportionally shorter lifetimes and lower quantum yields. No such proportionality was observed for the iodoacetate-treated and for the hydrogen peroxide treated enzyme. These findings indicate that two different quenching mechanisms are operating in rhodanese derivatives, a long-range energy transfer from tryptophan to persulfide (or sulfoselenide) group and a static quenching accompanying a conformational change of the protein after modification of the active site.     

Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase

Guaiacol peroxidase from spinach catalyzes the oxidation of p-aminophenol to produce the aminophenoxy radical as the primary product which is converted further into a stable oxidation product with an absorption peak at 470 nm. The p-aminophenol radicals oxidize ascorbate (AsA) to produce monodehydroascorbate radicals. Kinetic analysis indicates that p-aminophenol radicals also oxidize monodehydroascorbate to dehydroascorbate. Incubation of AsA peroxidase from tea leaves and hydrogen peroxide with p-aminophenol, p-cresol, hydroxyurea, or hydroxylamine results in the inactivation of the enzyme. No inactivation of the enzyme was found upon incubation of the enzyme with these compounds either in the absence of hydrogen peroxide or with the stable oxidized products of these compounds. The enzyme was protected from inactivation by the inclusion of AsA in the incubation mixture. The radicals of p-aminophenol and hydroxyurea were produced by AsA peroxidase as detected by their ESR signals. These signals disappeared upon the addition of AsA, and the signal characteristic of monodehydroascorbate was found. Thus, AsA peroxidase is inactivated by the radicals of p-aminophenol, p-cresol, hydroxyurea, and hydroxylamine which are produced by the peroxidase reaction, and it is protected from inactivation by AsA via the scavenging of the radicals. Thus, these compounds are the suicide inhibitors for AsA peroxidase. Isozyme II of AsA peroxidase, which is localized in chloroplasts, is more sensitive to these compounds than isozyme I. In contrast to AsA peroxidase, guaiacol peroxidase was not affected by these various compounds, even though each was oxidized by it and the corresponding radicals were produced.     

Active rhodanese lacking nonessential sulfhydryl groups contains an unstable C-terminal domain and can be bound,inactivated, and reactivated by GroEL

Mutation of all nonessential cysteine residues in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). This less stable mutant allowed testing of two hypotheses; (a) the two domains of rhodanese are differentially stable, and (b) the chaperonin GroEL can bind better to less stable proteins. Reduced temperatures during expression and purification were required to limit inclusion bodies and obtain usable quantities of soluble C3S. C3S and WT have the same secondary structures by circular dichroism. C3S, in the absence of the substrate thiosulfate, is cleaved by trypsin to give a stable 21-kDa species. With thiosulfate, C3S is resistant to proteolysis. In contrast, wild type rhodanese is not proteolyzed significantly under any of the experimental conditions used here. Mass spectrometric analysis of bands from SDS gels of digested C3S indicated that the C-terminal domain of C3S was preferentially digested. Active C3S can exist in a state(s) recognized by GroEL, and it displays additional accessibility of tryptophans to acrylamide quenching. Unlike WT, the sulfur-loaded mutant form (C3S-ES) shows slow inactivation in the presence of GroEL. Both WT and C3S lacking transferred sulfur (WT-E and C3S-E) become inactivated. Inactivation is not due to irreversible covalent modification, since GroEL can reactivate both C3S-E and WT-E in the presence of GroES and ATP. C3S-E can be reactivated to 100%, the highest reactivation observed for any form of rhodanese. These results suggest that inactivation of C3S-E or WT-E is due to formation of an altered, labile conformation accessible from the native state. This conformation cannot as easily be achieved in the presence of the substrate, thiosulfate.     

Detection of time-dependent and oxidatively induced antigens of bovine liver rhodanese with monoclonal antibodies

Rhodanese has been extensively utilized as a model protein for the study of enzyme structure-function relationships. An immunological study of conformational changes occurring in rhodanese as a result of oxidation or thermal inactivation was conducted. Three monoclonal antibodies (MABs) to rhodanese were produced. Each MAB recognized a unique epitope as demonstrated by binding of the MABs to different proteolytic fragments of rhodanese. While none of the MABs significantly bound native, soluble, sulfur-substituted bovine rhodanese, as indicated in indirect enzyme-linked immunosorbent assay experiments, each MAB was immunoadsorbed from solution by soluble rhodanese as a function of the time rhodanese was incubated at 37 degrees C. Thus, as rhodanese was thermally inactivated, conformational changes resulted in the expression of three new epitopes. Catalytic conformers demonstrated different rates of thermally induced antigen expression. Each MAB also recognized epitopes expressed when rhodanese was immobilized on microtiter plates at 37 degrees C. Two conformers resulting from oxidation of rhodanese by hydrogen peroxide were identified immunologically. All MABs recognized rhodanese that was oxidized with peroxide and allowed to undergo a secondary cyanide-dependent reaction which also resulted in a fluorescence shift and increased proteolytic susceptibility. Only one MAB was capable of recognizing an epitope expressed when rhodanese was oxidized with peroxide and then separated from the reactants to prevent induction of the secondary conformational changes.     

Reversible inactivation of maize leaf nitrate reductase

Preincubation of maize leaves crude extracts with NADH resulted in a progressive accumulation of nitrite which mimicked a rapid and lineal activation of nitrate reductase. Nevertheless, in partially purified preparations it was found that preincubation at pH 8.8 with NADH promoted a gradual inactivation of nitrate reductase. At pH 7.5, the enzyme was not inactivated by the presence of NADH alone, but, with the simultaneous presence of a low concentration of cyanide, a fast inactivation took place. The NADH-cyanide-inactivated nitrate reductase remained inactive after removing the excess of NADH and cyanide by filtration through Sephadex G-25. However, it could be readily reactivated by incubation with ferricyanide or by a short exposure to light in the presence of FAD. Prolonged irradiation caused a progressive inactivation of the photoreactivated enzyme.     

Oxidative inactivation of pneumolysin by the myeloperoxidase system and stimulated human neutrophils

Pneumolysin, a hemolytic toxin from Streptococcus pneumoniae, is a member of the group of thiol-activated, oxygen-labile cytolysins produced by various Gram-positive bacteria. The toxin activity of pneumolysin, as determined by lysis of 51Cr-labeled human erythrocytes, was destroyed on exposure to the neutrophil enzyme myeloperoxidase, hydrogen peroxide, and a halide (chloride or iodide). Detoxification required each component of the myeloperoxidase system and was prevented by the addition of agents that inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The entire myeloperoxidase system could be replaced by sodium hypochlorite at micromolar concentrations. Toxin inactivation was a function of time of exposure to the myeloperoxidase system (less than 1 min), the rate of formation of H2O2 (0.05 nmol/min), and the concentration of toxin employed. Toxin that had been inactivated by the myeloperoxidase system was reactivated on incubation with the reducing agent dithiothreitol. Pneumolysin was also inactivated when incubated with human neutrophils (10(5)) in the presence of a halide and phorbol myristate acetate, an activator of neutrophil secretion and oxygen metabolism. Toxin inactivation by stimulated neutrophils was blocked by azide, cyanide, or catalase, but not by superoxide dismutase. Neutrophils from patients with impaired oxygen metabolism (chronic granulomatous disease) or absent myeloperoxidase (hereditary deficiency) failed to inactivate the toxin unless they were supplied with an exogenous source of H2O2 or purified myeloperoxidase, respectively. Thus, inactivation of pneumolysin involved the secretion of myeloperoxidase and H2O2, which combined with extracellular halides to form agents (e.g., hypochlorite) capable of oxidizing the toxin. This example of oxidative inactivation of a cytolytic agent may serve as a model for phagocyte-mediated detoxification of microbial products.