The reaction of carbonyl cyanide phenylhydrazones with thiols

Carbonyl cyanide phenylhydrazone and its ring-substituted analogs react with thiols (thioglycolic acid, 2-mercaptoethanol, dithiothreitol) and amino-thiols (cysteine, glutathione) to give corresponding N-(substituted phynyl)-N′-(alkythiodicyano)-methylhydrazine derivatives. These addition products decompose to the original components in alkaline solution. On the other hand, in the presence of an excess of thiols in aqueous buffered systems the addition reactions are practically quantitative with respect to phenylhydrazones, follow pseudo-first-order kinetics and can be investigated spectrophotometrically. These reactions are of the bimolecular Ad_N type where the non-dissociated form of carbonyl cyanide phenylhydrazones function as an electrophilic component, while the RS^? ion plays the role of nucleophilic component in the case of thiols (the attack of the azomethine group).The reactivity of carbonyl cyanide phenylhydrazones with respect to thiols increases in the order carbonyl cyanide phenylhydrazone < carbonyl cyanide m-chlorophenylhydrazone < carbonyl cyanide p-trifluoromethoxyphenylhydrazone which corresponds to the order of decreasing values of the pK_a constants. On the other hand, the reactivity of thiols increases with their basicity.The reactivity of carbonyl cyanide phynylhydrazone with thiols is comparable with the reactivity of phynyl isothiocyanate and N-ethylmaleimide. It was demostrated that carbonyl cyanide phenylhydrazone is an efficient inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12).The results obtained are discussed in relation to the biological activity of carbonyl cyanide phenylhydrazones.     

Effects of Carbonyl Cyanide Phenylhydrazones on Two Mitochondrial Ion Channel Activities

The respiratory uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) affect the activities of two mitochondrial ion channels from mouse liver. At micromolar concentrations, the phenylhydrazones block the voltage-dependent 100-pS channel, mCS, and induce the multiple-conductance-level channel, MCC. The binding site(s) involved in perturbation of channel activities are probably distinct from the sites involved in uncoupling of oxidative phosphorylation which occurs at nanomolar concentrations of the phenylhydrazones. The effects of FCCP and CCCP on the mitochondrial ion channels could be partially reversed by washing with fresh media and were always reversed by perfusion with dithiothreitol. These results indicate that the effects of the phenylhydrazones on mitochondrial ion channels may be related to the ability of these compounds to act as sulfhydryl reagents and not to their protonophoric and uncoupling activity.     

Uncoupling effect of protonophoric and nonprotonophoric analogs of carbonyl cyanide phenylhydrazone on mitochondrial oxidative phosphorylation

Analogs of carbonyl cyanide phenylhydrazone providing no reaction with nucleophilic groups and lacking acidobasic properties, respectively, were synthesized for study of mechanism of uncoupling effect on oxidative phosphorylation. Their retention, influence on proton transport, abilities to SH--groups modify and to stimulate respiration in rat liver mitochondria, together with their physico-chemical properties, namely lipophilicity, acidobasicity and reactivity were characterized. The substitution of acidic hydrogen of the imino group resulted in the loss of both acidobasicity and uncoupling effect on oxidative phosphorylation. A decreased reactivity resulted from the substitutions of nitrile groups with the uncoupling activity remaining preserved.     

Mobilization of iron from endocytic vesicles. The effects of acidification and reduction

The factors necessary to dissociate iron from transferrin in endocytic vesicles and to mobilize the iron across the vesicle membrane were studied in a preparation of endocytic vesicles markedly enriched in transferrin-transferrin receptor complexes isolated from rabbit reticulocytes. Vesicles were prepared with essentially fully saturated transferrin by incubating the reticulocytes with the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prior to incubation with 59Fe, 125I-transferrin with or without fluorescein isothiocyanate labeling. Initiation of acidification by the addition of ATP was sufficient to achieve dissociation of 59Fe from transferrin with a rate constant of 0.054 +/- 0.06 s-1. Mobilization of 59Fe out of the vesicles required, besides ATP, the addition of a reductant with 1 mM ascorbate, allowing approximately 60% mobilization at 10 min with a rate constant of 0.0038 +/- 0.0006 s-1. An NADH:ferricyanide reductase activity could be demonstrated in the vesicles with an activity of 7.1 x 10(-9) mol of NADH reduced per min/mg of vesicle protein. Both dissociation and mobilization were inhibited by N-ethylmaleimide, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and monensin. Mobilization, but not dissociation, was inhibited by the permeant Fe(II) chelator alpha,alpha'-dipyridyl. The Fe(III) chelators deferoxamine, diethylenetriaminepentaacetic acid, and apotransferrin did not promote mobilization of dissociated iron in the absence of a reductant. This study establishes the basis for the cellular incorporation of iron through the endocytic pathway in which the endocytic vesicle membrane utilizes, in a sequential way, an acidification system, an iron reduction system, and an Fe(II) transporter system.     

Respiratory inhibitors and uncouplers prevent the aeration-induced increase in mitochondrial anion conductivity.

1. When mitochondria are stirred in air the rate of anion conductivity increases, this effect being enhanced by the addition of respiratory substrate. 2. This effect is reversible if the mitochondria are stored for a period of time under N2. 3. The aeration-induced increase in mitochondrial anion conductivity can also be prevented by the addition of respiratory inhibitors rotenone and antimycin A, as well as by 30 microM-cyanide. 4. A decrease in this aeration-induced anion conductivity can also be observed upon the addition of the uncouplers carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (2 microM) and 2,4-dinitrophenol (100 microM). 5. Simultaneous measurements of mitochondrial anion conductivity and membrane potential show a relationship between the level of membrane potential and anion conductivity. 6. It is suggested that the level of membrane potential is either directly or indirectly responsible for the level of mitochondrial anion conductivity.     

Effect of reducing agents and uncouplers on the electrical potential generated by mitochondrial ATPase activity

Beef heart submitochondrial particles bound to phospholipids impregnated filters generated an electrical potential upon the addition of ATP. The magnitude of the electrical potential reached depended on the phospholipid mixture composition used for filter impregnation, phosphatidylethanolamine being the active component for the electrical potential generation. Uncoupler FCCP (p-trifluoromethoxy carbonyl cyanide phenylhydrazone) inhibited the transmembrane electrical potential generation by diminishing the electrical resistance of the system as a result of its protonophoric action. However, uncouplers 2, 4-dinitrophenol and dicoumarol did not provoke large modifications of the electrical resistance under the conditions of pH and concentration used, and their action varied with the time elapsed after the submitochondrial particles purification, favouring the idea of the uncoupler interaction with a specific site on the membrane. Addition of sodium dithionite resulted in a higher plateau value for the electrical potential consistent with the promoted increase in ATPase activity. The effect of this agent was reversed by the 2,6-dichlorophenol-indophenol added at equivalent concentrations.     

Energy-conserving reactions in phosphorylating electron-transport particles from Nitrobacter winogradskyi. Activation of nitrite oxidation by the electrical component of the protonmotive force.

1. In electron-transport particles (ET particles) prepared from Nitrobacter winogradskyi, the uncoupling agent carbonyl cyanide phenylhydrazone increased the rate of NADH oxidation but decreased the rate of oxidation of NO2-. Its effectiveness in stimulating NADH oxidation closely paralleled its effectiveness in inhibiting NO2- oxidation. 2. In the presence of ADP and phosphate the oxidation of NADH was stimulated, whereas the oxidation of NO2- was inhibited. In the presence of excess of Pi the concentration dependence with respect to ADP was the same for acceleration of NADH oxidation and inhibition of NO2- oxidation. 3. Oligomycin inhibited NADH oxidation and stimulated the oxidation of NO2-. The concentration of oligomycin required to produce half-maximal effect in both systems was the same. 4. The apparent Km for NO2- was not affected by ADP together with Pi, by uncoupling agent or by oligomycin. 5. With NADH as substrate, classical respiratory control was observed. With NO2- as substrate the respiratory-control ratio was less than unity. 6. A reversible uptake of H+ accompanied the oxidation of NO2- by ET particles. 7. In the presence of NH4Cl or cyclohexylamine hydrochloride, H+ uptake was abolished and increased rates of NO2- oxidation were observed. When valinomycin was present in the reaction medium, low concentrations of NH4Cl inhibited NO2- oxidation. 8. Pretreatment of ET particles with oligomycin enhanced the stimulation of NO2- oxidation induced by NH4Cl or by cyclohexylamine hydrochloride. Pretreatment with the uncoupler carbonyl cyanide phenylhydrazone prevented these stimulations. 9. In the presence of dianemycin together with K+, the uptake of H+ was abolished and the rate of NO2- oxidation was increased. In contrast, in the presence of valinomycin together with K+, the uptake of H+ was increased, and the rate of NO2- oxidation decreased. 10. Sodium tetraphenylboron was found to be an inhibitor of NO2- oxidation, but caused a stimulation of NADH oxidation which was dependent on the presence of NH4Cl or cyclohexylamine hydrochloride. 11. It is concluded that the enhanced rate of NO2- oxidation observed in the absence of energy-dissipating processes clearly relates to some state before the involvement of adenine nucleotides, and it is suggested that the oxidation of NO2- generates a protonmotive force, the electrical component of which controls the rate of NO2- oxidation.     

Estimation of the mitochondrial calcium pool in rat brain synaptosomes using Rhod-2 AM fluorescent dye

The literature data on the role of synaptic mitochondria in the regulation of the cytosolic calcium level are contradictory. In the present paper calcium storage by mitochondria in rat brain synaptosomes using the fluorescent dye Rhod-2 has been investigated. The addition of 60 mM KCl increases Rhod-2 fluorescence. This effect is completely abolished by replacing K⁺ with Na⁺ or withdrawing Ca²⁺ from the incubation medium. A proton ionophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, and a mixture of rotenone/oligomycin mitochondrial toxins cause a two-fold decrease in Rhod-2 fluorescence. Thapsigargin, an inhibitor of endoplasmic reticulum ATPase (1 μM), but not bafilomycin, an inhibitor of ATPase in synaptic vesicles (500 nM) also leads to a mitochondrial calcium influx. The addition of calcium to synaptosomes with the retained plasma membrane potential increased Rhod-2 fluorescence; however, this effect is insensitive to carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. We have shown that mitochondria can serve as a calcium store in synaptosomes only in the case of a high cytosolic concentration of calcium.     

Effect of carbonyl compounds on red blood cells deformability

The effect of Maillard reaction on red blood cells (RBC) deformability was investigated. Exposure of RBC to carbonyl compounds (dl-glyceraldehyde, glyoxal, glycolaldehyde, 3-deoxyglucosone, and d-glucose) leading to Maillard reaction caused a marked decrease in RBC deformability even at 1 mM level. The decrease rate depended on the kind of carbonyl compounds, in which both dl-glyceraldehyde and glyoxal significantly decreased the RBC deformability (p < 0.05). In addition, the decrease rate also differed among volunteers tested, indicating that the sensitivity against carbonyl compounds varies among them. In order to elucidate the mechanism of the decrease in RBC deformability, RBC was exposed to carbonyl compounds in the presence of aminoguanidine which is the inhibitor of AGE formation in Maillard reactions. Aminoguanidine inhibited the decrease in RBC deformability by dl-glyceraldehyde and glyoxal. When Hb which has a high reactivity with carbonyl compounds was incubated with those carbonyl compounds, dl-glyceraldehyde and glyoxal showed the high reactivity with Hb compared with other carbonyl compounds. These results indicate that Maillard reaction between RBC proteins and carbonyl compounds leads to the decrease in RBC deformability. On the other hand, generated by carbonyl compounds involved in lowering the deformability only to a negligible level.     

Zinc binding and its trapping by allosteric transition in glucosamine-6-phosphate deaminase from Escherichia coli

Glucosamine-6-phosphate isomerase deaminase from Escherichia coli, a typical allosteric enzyme, becomes less cooperative and 50% inhibited when treated with zinc. This metal cation behaving as a tight-bound and slow partial inhibitor. Modification of a pair of vicinal reactive thiols with some sulfhydryl reagents mimics this effect. On the other hand, sulfhydryl reactivity disappears in the presence of saturating concentrations of Zn2+, which does not modify the kinetics of S-methylated enzyme, a finding that indicates that vicinal thiols are an essential part of the zinc-binding site. Allosteric activation of the deaminase causes trapping of the metal, which cannot be released by dialysis against a buffer containing EDTA. Cadmium and nickel(II) cations also produce a similar effect.     

Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H/K Exchanger and an OH/Pi Antiporter

Evidence is presented that K⁺ uptake in corn root segments is coupled to an electrogenic H⁺/K⁺ -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH⁻/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K⁺ uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH⁻/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K⁺ uptake. Collapse of the proton gradient across the membrane by (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K⁺ uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K⁺ and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K⁺ uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H⁺/K⁺ -exchanging ATPase to an OH⁻/Pi antiporter in corn root tissue.     

Lipid requirements for coupled cytochrome oxidase vesicles

Cytochrome c oxidase has been reconstituted with two synthetic phospholipids, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine. Vesicles prepared from either of these two lipids alone showed no stimulation of enzyme activity upon addition of carbonyl cyanide (trifluoromethoxy)phenylhydrazone and valinomycin, indicating that they were leaky to small ions. However, when mixtures of the two lipids were used for the reconstitution, tightly coupled vesicles could be obtained. The coupling ratio was dependent upon the ratio of dioleoylphosphatidylcholine to dioleoylphosphatidylethanolamine and also on the lipid-to-protein ratio. Maximal rates of enzyme activity were not significantly different with different lipid mixtures. The results are discussed in terms of both the size distribution of the reconstituted vesicles and the possible requirement for a variety of lipid species to ensure tight sealing at the lipid-protein interface.     

Intracellular and plasma membrane-initiated pathways involved in the [Ca2+]i elevations induced by iodothyronines (T3 and T2) in pituitary GH3 cells

The role of 3,5,3'-triiodo-l-thyronine (T3) and its metabolite 3,5-diiodo-l-thyronine (T2) in modulating the intracellular Ca(2+) concentration ([Ca(2+)](i)) and endogenous nitric oxide (NO) synthesis was evaluated in pituitary GH(3) cells in the absence or presence of extracellular Ca(2+). When applied in Ca(2+)-free solution, T2 and T3 increased [Ca(2+)](i), in a dose-dependent way, and NO levels. Inhibition of neuronal NO synthase by N(G)-nitro-l-arginine methyl ester and l-n(5)-(1-iminoethyl)ornithine hydrochloride significantly reduced the [Ca(2+)](i) increase induced by T2 and T3. However, while depletion of inositol trisphosphate-dependent Ca(2+) stores did not interfere with the T2- and T3-induced [Ca(2+)](i) increases, the inhibition of phosphatidylinositol 3-kinase by LY-294002 and the dominant negative form of Akt mutated at the ATP binding site prevented these effects. Furthermore, the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prevented the increases in both [Ca(2+)](i) and NO elicited by T2 or T3. Interestingly, rotenone blocked the early [Ca(2+)](i) increases elicited by T2 and T3, while antimycin prevented only that elicited by T3. Inhibition of mitochondrial Na(+)/Ca(2+) exchanger by CGP37157 significantly reduced the [Ca(2+)](i) increases induced by T2 and T3. In the presence of extracellular calcium (1.2 mM), under carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, T2 and T3 increased both [Ca(2+)](i) and intracellular Na(+) concentration; nimodipine reduced the [Ca(2+)](i) increases elicited by T2 and T3, but inhibition of NO synthase and blockade of the Na(+)/H(+) pump by 5-(N-ethyl-N-isopropyl)amiloride prevented only that elicited by T3; and CB-DMB, bisindolylmaleimide, and LY-294002 (inhibitors of the Na(+)/Ca(2+) exchanger, PKC, and phosphatidylinositol 3-kinase, respectively) failed to modify the T2- and T3-induced effects. Collectively, the present results suggest that T2 and T3 exert short-term nongenomic effects on intracellular calcium and NO by modulating plasma membrane and mitochondrial pathways that differ between these iodothyronines.     

Inhibition of nitrogen fixation by nitrite inAnabaena variabilis

Addition of nitrite to rapidly growing, nitrogen-fixing filaments ofAnabaena variabilis caused an immediate drop in nitrogenase activity. This was followed by a transient induction of nitrite reductase, recovery of nitrogen fixation and cyanobacterial growth. The experiments with isolated heterocysts and a partially purified nitrogenase preparation from heterocysts showed that nitrite primarily exerted its inhibitory effect by inactivating nitrogenase irreversibly, rather than interfering with photosynthetic energy conservation.Abbreviations ATCC American type culture collection - Chl chlorophyll - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - Tes 2-{[2 hydroxy-1,1-bis(hydroxymethyl)ethyl] amino} ethane sulfonic acid     

Fluorescence spectroscopic detection of mitochondrial flavoprotein redox oscillations and transient reduction of the NADPH oxidase-associated flavoprotein in leukocytes

Steady-state and time-resolved fluorescence spectroscopy and fluorescence microscopy of leukocyte flavoproteins have been performed. Both living human peripheral blood monocytes and neutrophils have been utilized as experimental models, as the former relies much more heavily on mitochondrial metabolism for energy production than the latter. We confirm previous studies indicating that cellular flavoproteins absorb at 460 nm and emit at 530 nm, very similar to that of the FAD moiety. Furthermore, the emission properties of intracellular flavoproteins were altered by the metabolic inhibitors rotenone, antimycin A, azide, cyanide, DNP (2,4-dinitrophenol), and FCCP [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone]. Kinetic studies revealed flavoprotein emission oscillations in both monocytes and neutrophils. The flavoprotein intensity oscillations correlated with the physiological status of the cell and the nature of membrane receptor ligation. Microscopy revealed the presence of flavoprotein fluorescence in association with the plasma membrane, intracellular granules and distributed throughout the cytoplasm, presumably within mitochondria. Metabolic inhibitors such as cyanide suggest that the plasma membrane and granular components are cyanide-insensitive and therefore are likely associated with the flavoprotein component of the NADPH oxidase, which is located in these two compartments. This interpretation was found to be consistent with structural localization of the NADPH oxidase using an antibody molecule specific for this protein. Using peripheral blood neutrophils, which display less active mitochondria, and time-resolved emission spectroscopy, we show that the NADPH oxidase-associated flavoprotein undergoes a periodic transient reduction of about 54±2 ms in living cells. This finding is consistent with prior studies indicating that propagating substrate (NADPH) waves periodically promote electron transport across the NADPH oxidase.     

Inhibition of the mitochondrial respiratory chain by alkylhydroxynaphthoquines: Reversal on discharge of the energized state

Inhibition of mitochondrial respiration by alkylhydroxynaphthoquinones may be reversed by addition of a variety of uncouplers including substituted phenols, carbonyl cyanide phenylhydrazones, divalent cations and univalent cations in the presence of ionophoretic antibiotics. A likely explanation for such reversibility is the requirement that the anionic inhibitor be transported to a site of action within the mitochondrion. Support for this view includes (1) failure to obtain reversal of inhibition with submitochondrial particles, (2) release of inhibition by a competing anion, succinate, (3) augmentation of inhibition when a divalent cation is taken up, (4) the chemical diversity of uncouplers that release inhibition and (5) inhibiton by uncoupling compounds of the uptake of labeled alkylhydroxynaphthoquinones. It is suggested that a similar explanation may apply to two other inhibitors of the cytochrome b–c region, antimycin and alkylhydroxyquinoline-N-oxides.     

Control of respiration in proteoliposomes containing cytochrome aa3 II. Inhibition by carbon monoxide and azide

1. Carbon monoxide (CO) acts competitively towards oxygen when the latter is taken up in respiration by cytochrome aa₃-containing proteoliposomes, both in the presence of p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin (deenergized state) and in their absence (energized state). At high levels of CO, the double reciprocal plots ($\text{1}\text{v}$ vs. $\text{1}\text{[}\text{O}{}_2\text{]}$) in the energized and deenergized states are parallel, i.e. energization acts “anti-competitively” towards oxygen, and the “respiratory control ratio” decreases as the oxygen concentration decreases.2. Azide acts non-competitively towards cytochrome c when the latter is oxidized by cytochrome aa₃-containing proteoliposomes both in the energized and deenergized (plus p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin) conditions. At low azide concentrations the apparent K_i for azide is unaffected by energization, but at high azide levels the K_i increases in energized liposomes, i.e. the “respiratory control ratio” decreases as the azide concentration increases.3. It is concluded that the inhibitor experiments are consistent with but do not prove the concept that the oxidase molecules in a single vesicle are responding to a single “energization state” or set of electrochemical gradients. This and other models are discussed.     

A role of iron ions in the SOS DNA repair response induced by nitric oxide in Escherichia coli

An induction of the SOS DNA repair response by physiological nitric oxide donors (dinitrosyl iron complexes (DNIC) with thiols and S-nitrosothiols (RSNO)) was studied in E. coli cells. DNIC with thiols were the most effective SOS-inducers. Being more toxic, RSNO mediated a similar response at 10-100 microM, but they were inactive at concentrations above 0.5 mM. Pretreatment of the cells with chelating agents, o-phenanthroline and picolinic acid, prevented induction of the SOS response by all NO-donors used and led to a decrease in the DNIC-type EPR signal that appeared after incubation of the cells with DNIC or S-nitrosoglutathione (GSNO). Analysis of these effects revealed a dual role of iron ions in reactivity and toxicity of the NO-donating agents. On one hand, they could stabilize GSNO in the form of less toxic DNIC, and, on the other hand, they took part in the formation of the SOS-inducing signal by NO-donating agents.     

A study of dependence of protein synthesis in mitochondria on the transmembrane potential

Summary 1. Incorporation of [H³]leucine into the TCA insoluble fraction of rat liver mitochondria incubatedin vitro is inhibited by uncouplers of oxidative phosphorylation. The inhibition is not correlated with the activation of mitochondrial ATPase. 2. Dependence of mitochondrial protein synthesis on the transmembrane potential is manifested in a wide range of K⁺ and Mg⁺⁺ concentrations in the incubation media. 3. The inhibitory action of uncouplers shows a lag period equal to 5–7 minutes, this lag period however is not observed when the uncoupler is added to puromycin-treated mitochondria. 4. Dependence of mitochondrial protein synthesis on the transmembrane potential, which represents a property characteristic for the inner mitochondrial membrane suggests that mitochondrial ribosomes act in close contact with the mitochondrial membrane system.Abbreviations MPS mitochondrial protein synthesis - CAP chloramphenical - CCP 2,4,6-chlorocarbonyl cyanide phenylhydrazone - FCCP p-trifluoromethoxy carbonyl cyanide phenylhydrazone - PEP phosphoenolpyruvate - Pi inorganic phosphate     

5-Hydroxy-1,4-naphthoquinone (juglone) and 2-hydroxy-1,4-naphthoquinone (lawsone) influence on jack bean urease activity: Elucidation of the difference in inhibition activity

The aim of this study was elucidation of the difference in inhibition influence of 5-hydroxy-1,4-naphthoquinone (juglone) and 2-hydroxy-1,4-naphthoquinone (lawsone) on jack bean urease activity. It was found that juglone acted as a strong, time and concentration dependent inactivator of urease. On the contrary, lawsone showed an inconsiderable inhibition influence. The reactivation of juglone modified urease showed the participation of reversible and irreversible contribution in the inactivation. In the presence of an excess of DTT, urease inactivated by juglone regained 70% of its activity. The reversible inactivation was attributed to oxidation of the essential urease thiols by reactive oxygen species (ROS) realizing during reduction of juglone to seminaphthoquinone. Presence of hydrogen peroxide in the incubation system was proved by direct determination and by application of catalase. The irreversible contribution in the inhibition was assumed as an arylation of urease thiol groups by juglone. The insignificant urease inhibition by lawsone was concluded as an effect of a low hydrogen peroxide generation and lawsone resistance for reaction with protein thiols. It was found that lawsone well reacted with l-cysteine, poorly with glutathione and hardly with urease thiols. The observed sequence was arranged according the rule the more complex thiol the less susceptible for reaction with lawsone. On the other hand, juglone displayed an excellent reactivity towards both thiols and urease. Thus, this indicated a significance of a steric hindrance which appeared when the hydroxyl group changing position from 5 in juglone (5-hydroxy-1,4-naphthoquinone) to 2 in lawsone (2-hydroxy-1,4-naphthoquinone).