Reactions of the Wurster's red radical cation with hemoglobin and glutathione during the cooxidation of N,N-dimethyl-p-phenylenediamine [correction of phenlenediamine] and oxyhemoglobin in human red cells.

N,N-Dimethyl-p-phenylenediamine (DMPD) reacted directly with oxyhemoglobin under formation of ferrihemoglobin and, presumably, the N,N-dimethyl-p-phenylenediamine radical cation (DMPP.+). The apparent second-order rate constant of this reaction was 1 M-1 s-1 (pH 7.4, 37 degrees C). The reaction rate was diminished by catalase (by 1/3) and by superoxide dismutase (by 1/5). The apparent second-order rate constant of ferrihemoglobin formation by DMPD.+ was 5 x 10(3) M-1 s-1. Since DMPD.+ is disproportionated by 50% at pH 7.4, the quinonediimine could not be excluded as the ultimate ferrihemoglobin forming oxidant. To prove this hypothesis, the disproportionation equilibrium was shifted to the radical side by addition of excess DMPD. Ferrihemoglobin formation was thereby increased, indication that the radical was the responsible oxidant. In contrast to ferrihemoglobin formation, reactions with glutathione occurred predominantly with the quinonediimine. The second-order rate constant of this reaction was 4 x 10(5) M-1 s-1 which approaches the value obtained with p-benzoquinone. In contrast to the corresponding reactions of the N,N,N',N'-tetramethyl-p-phenylenediamine radical cation, the disporportionation reaction of DMPD.+ was very fast, k = 2 x 10(6) M-1 s-1. Formation of glutathione disulfide was negligible and the main reaction products were two isomeric glutathione adducts, 2- and 3-(glutathione-S-yl)-N,N-dimethyl-p-phenylenediamine. In human erythrocytes, DMPD produced many equivalents of ferrihemoglobin, diminished glutathione and produced both thioethers. In contrast to ferrihemoglobin formation, DMPD and glutathione disappearance as well as thioether appearance occured only after a marked lag phase. The calculated steady state concentration of DMPD.+ was only 4 x 10(-6) the DMPD concentration, as long as ferrihemoglobin was low. At increasing ferrihemoglobin higher steady state concentrations of the radical are attained. In fact, preformed ferrihemoglobin in red cells significantly accelerated DMPD and glutathione disappearance. This effect was completely prevented in the presence of ferrihemoglobin-complexing cyanide. The presented experiments once more appoint blood as a metabolically competent organ for the biotransformation of aromatic amines.     

Reactions of the Wurster's blue radical cation with thiols, and some properties of the reaction products

Formation of 1-electron oxidation products of aromatic amines in biological systems have been ascertained. The mechanisms of the toxic actions of the aminyl radicals and their corresponding detoxication reactions are much less established. During the studies of reactions of GSH with the N,N,N',N'-tetramethyl-p-phenylenediamine radical cation (TMPD) (Wurster's blue) two pathways were detected: (1) a slow second order reaction (k = 5 M-1.s-1) which gave the parent amine and (ultimately) GSSG, and (2) a fast, complex reaction which yielded 2-(glutathione-S-yl)-N,N,N',N'-tetramethyl-p-phenylenediamine (2-GS-TMPD). From kinetic reasons, this reaction was suggested to be composed of a rapid disproportionation reaction followed by a reductive 1,4-Michael-addition. This reaction pathway prevailed at GSH concentrations below 1 mM. At higher GSH concentrations formation of the thioether was suppressed. This hypothesis was confirmed when the reaction of the highly labile N,N,N',N'-tetramethyl-p-quinonediiminium dication (TMQDI++) with GSH was followed: In this case, thioether formation outweighed clearly reductive mechanisms, the latter yielding ultimately the amine and GSSG. Similar to N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), 2-GS-TMPD was also capable of producing ferrihemoglobin in a catalytic reaction. Its rate, however, was only 3% that observed with the parent amine. During this reaction the thioether was apparently oxidized to the corresponding quinonediiminium dication, which gave the corresponding quinonemonoimine on acidification.     

Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine,N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine methyl ester, and reductively methylated concanavalin A

Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine (TML) and N epsilon,N epsilon,N alpha,N alpha-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl alpha-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the alpha-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2 approximately equal to 1-10 M-1 s-1, k3 approximately equal to 10(6) M-1 s-1 and ki, the rate constant for nitrogen inversion was about 10(8)-10(9) s-1. Upper limits of 10(12) and 10(3) M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 X 10(4) s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reductively methylated peptides or proteins.     

Characterization of electron donation to cytochrome c-555 in Chromatium vinosum from ferrocyanide, tetramethylphenylenediamine and reduced dimethylquinone. Effects of redox potential, pH and salt concentration

1. The dependences of the reduction of ferricytochrome c-555 in the reaction center-cytochrome c complex on the redox potential and pH were investigated using N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), ferrocyanide, and reduced 2,5-dimethyl-p-quinone as electron donors. 2. In the reduction of cytochrome c-555 by TMPD, the unprotonated form was the exclusive electron donor to the cytochrome with a second-order rate constant of 1.0 X 10(5) M-1.s-1. 3. Ferrocyanide reduced cytochrome c-555 slowly with a rate constant of 7.8 X 10(3) M-1.s-1 at infinite salt concentration. The value of -5.2 X 10(-4) elementary charge/A2 was estimated as the surface charge density in the vicinity of cytochrome c-555 by analyzing the salt effect on the cytochrome reduction using the Gouy-Chapman theory. 4. The characteristics of the dependences of the reduction of cytochrome c-555 by reduced 2,5-dimethyl-p-quinone on the redox potential and pH were well explained by the redox potential and pH dependences of the formation of the semiquinone. In the neutral-to-alkaline pH range the anionic semiquinone was the main electron-donating species with a second-order rate constant of 6.0 X 10(7) m-1.s-1.     

Stopped-flow kinetic study of the peroxidase reactions of mangano-microperoxidase-8

We have investigated the kinetics for the peroxidase-type reaction of mangano microperoxidase 8 (Mn(III)-MP8) by the time-resolved and single-wavelength stopped-flow technique. The formation of intermediate and its subsequent reaction with substrates were studied separately. Oxidation of Mn(III)-MP8 by H2O2 at pH 10.7 yields an intermediate (1) with a rate constant of 2.9 x10(4) M-1 s-1. The formation of 1 exhibits no deuterium solvent isotope effect, favoring the homolytic cleavage of the Mn(III)-MP8 bound hydroperoxide. The rate for the formation of 1 increases sharply as the pH increases and no other intermediate was detected in the entire pH range. Addition of substrate to 1 leads to the regeneration of Mn(III)-MP8. Monitoring the conversion of 1 to Mn(III)-MP8 allows the determination of the substrate reactivity. The substrate reactivity varies by more than two orders of magnitude ranging from 1.04 x 10(6) M-1 s-1 for ascorbic acid to 4.61 x 10(3) M-1s-1 for aniline. It is linearly correlated with the reduction potential for most of the substrates studied, with the easier oxidized species showing greater reactivity. The substrate reactivity drops rapidly as the pH increases. The substrate reactivity at pH 10.7 for the Mn(III)-MP8 system is smaller than that of the corresponding Fe(III)-MP8 system by 2- to 25-fold, depending on the substrate used.     

Stopped-flow investigation of antioxidant activity of estrogens in solution

A kinetic study of the reaction between estrogens (female hormone) and substituted phenoxyl radical has been performed, as a model for the reactions of estrogens with lipid peroxyl radical in biological systems. The rates of reaction of estrogens (estrone 1, estradiol 2, 2-methoxyestrone 3, 3-methoxyestrone 4, and 2-hydroxyestrone 5) with substituted phenoxyl radical in benzene have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants, k2, obtained are 84 M-1.s-1 for 1, 138 M-1.s-1 for 2, 520 M-1.s-1 for 3, less than 10(-4) M-1.s-1 for 4, and 2.6 X 10(5) M-1.s-1 for 5 at 25.0 degrees C. 2-Hydroxyestrone 5 was found to be 2.9-times more active than alpha-tocopherol, which has the highest antioxidant activity among natural tocopherols. The order of magnitude of k2 value (1 less than 2 less than 3 less than alpha-Toc less than 5) is in agreement with that of in vitro tests of their antioxidant activities, as measured by the inhibition of lipid peroxidation. Further, similar measurements have been performed for the reaction between the above estrogens 1-5 and tocopheroxyl 6 in benzene solution. It was found that the estrogens having an OH group at the aromatic ring have an ability to regenerate the tocopheroxyl 6 to tocopherol. Especially, the 2-hydroxyestrone 5 showed about three orders of magnitude higher reactivity than ascorbic acid.     

Adduct formation between sulfite and the flavin of phototrophic bacterial flavocytochromes c. Kinetics of sequential bleach, recolor, and rebleach of flavin as a function of pH.

The kinetics of sulfite adduct formation with the bound flavin in flavocytochromes c from the purple phototrophic bacterium Chromatium vinosum and the green phototrophic bacterium Chlorobium thiosulfatophilum have been investigated as a function of pH. Both species of flavocytochrome c rapidly react with sulfite to form a flavin sulfite adduct (k = 10(3)-10(5) M-1 s-1) which is bleached at 450-475 nm and has associated charge-transfer absorbance at 660 nm. The rate constant for adduct formation in flavocytochrome c is 2-4 orders of magnitude faster than for model flavins of comparable redox potential and is likely to be due to a basic residue near the N-1 position of the flavin, which not only raises the redox potential but also stabilizes the negatively charged adduct. There is a pK for adduct formation at 6.5, which suggests that the order of magnitude larger rate constant at pH 5 as compared to pH 10 in flavocytochrome c is due the influence of another positive charge, possibly a protonated histidine residue. The adduct is indefinitely stable at pH 5 but decomposes (the flavin recolors) in a first-order process accelerating above pH 6 (at pH 10, k = 0.1 s-1). The pK for recoloring is 8.5, which is suggestive of a cysteine sulfhydryl. On the basis of the observed pK and available chemical information, we believe that recoloring is due to a secondary effect of the reaction of sulfite with a protein cystine disulfide, which is adjacent to the flavin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and mechanism of anionic ligand binding to carbonic anhydrase

The kinetics of complex formation between Co(II)-carbonic anhydrase B and the anions cyanate, thiocyanate and cyanide has been studied at different pH values employing temperature-jump relaxation spectrometry. Formation of the 1:1 complex occurs via binding of the deprotonated state of the anion to an acidic state of the enzyme. The determined formation rate constants range from 10(8) to 3 X 10(9) M-1 s-1 and are two to three orders of magnitude higher than the value estimated for a ligand coordination to the central Co2+, based on a solvate substitution mechanism. These kinetic results strongly indicate that the deprotonated anion binds to an unoccupied coordination position of the protein-bound heavy metal ion in the form of an addition reaction. Upon binding of the anion, the coordination number of the Co2+ in the acidic state of the enzyme is increased from four to five. In the case of cyanide, a 2:1 anion complex is also formed. The formation rate constant is 5 X 10(5) M-1 s-1 which provides good evidence that this binding process is controlled by a solvate substitution mechanism.     

Dimerization kinetics of the IgE-class antibodies by divalent haptens. I. The Fab-hapten interactions.

The binding of divalent haptens to IgE-class antibodies leads predominantly to their oligomerization into open and closed dimers. Kinetics of the open dimer formation was investigated by fluorescence titrations of Fab fragments of monoclonal DNP-specific IgE antibodies with divalent haptens having different spacer length (gamma = 14-130 A). Binding was monitored by quenching of intrinsic tryptophan emission of the Fab. Addition of divalent haptens with short spacers (gamma = 14-21 A) to the Fabs at rates larger than a distinct threshold value caused a significant decrease of Fab-binding site occupation in the initial phase of the titration. This finding was interpreted to reflect a nonequilibrium state of hapten-Fab-binding. Such nonequilibrium titrations were analyzed by inserting a kinetic model into a theory of antibody aggregation as presented by Dembo and Golstein (Histamine release due to bivalent penicilloyl haptens. 1978. J. Immunol. 121, 345). Fitting of this model to the fluorescence titrations yielded dissociation rate constants of 7.8 x 10(-3) s-1 and 6 x 10(-3) s-1 for the Fab dimers formed by the flexible divalent haptens N alpha, N epsilon-di(dinitrophenyl)-L-lysine (gamma = 16 A) and bis(N beta-2,4-dinitrophenyl-alanyl)-meso-diamino-succinate (gamma = 21 A). Making the simplifying assumption that a single step binding equilibrium prevails, the corresponding dimer formation rate constants were calculated to be 1.9 x 10(5) M-1 s-1 and 1.1 x 10(4) M-1 s-1, respectively. In contrast, all haptens with spacers longer than 40 A (i.e., bis(N alpha-2,4-dinitrophenyl-tri-D-alanyl)-1,7-diamino-heptane, and di(N epsilon-2,4-dinitrophenyl)-6-aminohexanoate-aspartyl-(prolyl)n-L-l ysyl (n = 24, 27, 33) exhibit a relative fast dimerization rate of the Fab fragments (greater than 7 x 10(6) M-1 s-1). These observations were interpreted as being caused by orientational constraints set by the limited solid angle of the reaction between the macromolecular reactants. Thus, ligands having better access to the binding site would react faster.     

A pulse radiolysis investigation of the reactions of myeloperoxidase with superoxide and hydrogen peroxide

Using pulse radiolysis, the rate constant for the reaction of ferric myeloperoxidase with O2- to give compound III was measured at pH 7.8, and values of 2.1.10(6) M-1.s-1 for equine ferric myeloperoxidase and 1.1.10(6) M-1.s-1 for human ferric myeloperoxidase were obtained. Under the same conditions, the rate constant for the reaction of human ferric myeloperoxidase with H2O2 to give compound I was 3.1.10(7) M-1.s-1. Our results indicate that although the reaction of ferric myeloperoxidase with O2- is an order of magnitude slower than with H2O2, the former reaction is sufficiently rapid to influence myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.     

Reactions of 4-dimethylaminophenol with hemoglobin,and autoxidation of 4-dimethylaminophenol

The reactions between 4-dimethylaminophenol and hemoglobin were studied with 4-dimethylaminophenol ¹⁴C-labelled either in the methyl groups or in C₁ of the ring.In the absence of oxygen 4-dimethylaminophenol was stable in red cell suspensions or hemoglobin solutions. In the presence of oxygen oxyhemoglobin rapidly oxidized 4-dimethylaminophenol. The following reaction products were found in incubates of 4-dimethylaminophenol with red cells or hemoglobin: ferrihemoglobin, formaldehyde, dimethylamine, and hemoglobin with derivatives of 4-dimethylaminophenol covalently bound to its protein moiety.4-Dimethylaminophenol catalytically transferred electrons from ferrohemoglobin to oxygen. It was oxidized by oxyhemoglobin, and oxidized 4-dimethylaminophenol was reduced to 4-dimethylaminophenol by ferrohemoglobin with formation of ferrihemoglobin. Hydrolysis of oxidized 4-dimethylaminophenol, N,N-dimethylquinonimine, and its covalent binding to globin limited the catalytic ferrihemoglobin formation by 4-dimethylaminophenol to an average between 50 and 100 electron transfers per molecule of 4-dimethylaminophenol, when 4-dimethylaminophenol concentration was low and hemoglobin concentration was high. Since 4-dimethylaminophenol reduced ferrihemoglobin to ferrohemoglobin, though more slowly than the catalytic cycle produced it, the increase in ferrihemoglobin content does not indicate the amount of ferrihemoglobin produced.In red cell suspensions at 37° 4-dimethylaminophenol, 0.58 mM, disappeared in 10 min, but dimethylamine continued to be formed, obviously from protein-bound derivative(s) of 4-dimethylaminophenol.The rate of autoxidation of 4-dimethylaminophenol was found to be much lower that the rate of oxidation of 4-dimethylaminophenol by oxyhemoglobin. After autoxidation of 4-dimethylaminophenol several products were isolated and identified which were not detected in incubates of 4-dimethylaminophenol with oxyhemoglobin, namely hydroquinone, 4-methylaminophenol, 4-aminophenol, 2-dimethylamino-1, 4-benzoquinone, a purple and a yellow dye.Nuclear magnetic resonance (NMR), mass spectroscopy, and synthesis from 1,4-benzoquinone and 4-methylaminophenol proved the purple dye to be 2-(N- methyl-N-(p-hydroxyphenyl)-amino-1,4-benzoquinone.The structure of the yellow dye, which is produced also by oxidation of the purple dye with hydrogen peroxide, was not proved unequivocally. IR, NMR spectra and the product of hydrogenation with Pd-charcoal and acetylation showed the compound to be an epoxide of 2-(N-methyl-N-(p-hydroxyphenyl)-amino)-benzoquinone.     

The kinetic differences between sodium nitrite, amyl nitrite and nitroglycerin oxidation of hemoglobin

The effect of sodium nitrite, amyl nitrite and nitroglycerin (glyceryl trinitrate) on the hemoglobin of adult erythrocytes was examined in vitro. Both amyl nitrite and nitroglycerin reacted immediately with oxyhemoglobin to effect oxidation into methemoglobin while sodium nitrite required an inductionary period (lag phase) prior to the reaction. Kinetic studies of the biomolecular rate law for each of the preceding reaction's reactionary periods (log phases) allowed rate constant calculations to be made. The values are 1.14 x 10(4) M-1 min-1, 7.45 x 10(4) M-1 min-1, and 3.50 x 10(1) M-1 min-1 for sodium nitrite, amyl nitrite and nitroglycerin, respectively. A comparison of the amyl nitrite and nitroglycerin rate constants reveals that amyl nitrite is approximately 2000-fold more toxic to oxyhemoglobin than nitroglycerin. These oxidant's effect on in vitro hemoglobin solutions are comparable since both reactions approximate to rectangular hyperbolae. Sodium nitrite reacts about 300-fold faster with oxyhemoglobin than does nitroglycerin. However, the sodium nitrite reaction proceeds in a sigmoidal fashion which makes a strict comparison between these compounds relative toxicities less clear cut.     

Nicotinamide adenine dinucleotide species in the horseradish peroxidase-oxidase oscillator.

NADH chemistry ancillary to the oscillatory peroxidase-oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 +/- 0.2) x 105 M-1.s-1 and CpII to HRP at 1 x 105 M-1.s-1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 x 106 M-1.s-1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of alpha-NADH, not only the beta anomer as previously assumed. Rate constants of alpha- and beta-NADH reactions with CpI are (7.4 +/- 0.4) x 105 M-1.s-1, and (1.7 +/- 0.2) x 105 M-1.s-1, and with CpII are estimated as 5 x 104 M-1.s-1, and 4 x 104 M-1.s-1. Apparent rate constants of reduction of methylene blue (MB) to leuco-methylene blue (MBH) are 3.8 x 104 M-1.s-1 for NADH and 6.4 x 104 M-1.s-1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 +/- 0.2) x 103 M-1.s-1 All the rates were measured in 0.1 M acetate buffer, pH 5.1.     

The oxidation of Pseudomonas cytochrome c-551 oxidase by potassium ferricyanide.

Stopped-flow kinetics were made of the reaction between ascorbate-reduced Pseudomonas cytochrome oxidase and potassium ferricyanide under both N2 and CO atmospheres. Under N2 three kinetic processes were observed, two being dependent on ferricyanide concentration, with second-order rate constants of 9.6 X 10(4)M-1.s-1 and 1.5 X 10(4)M-1.s-1, whereas the other was concentration-independent, with a first-order rate constant of 0.17 +/- 0.03s-1. Measurements of their kinetic difference spectra have allowed the fastest and second-fastest phases of the reaction to be assigned to direct bimolecular reactions of ferricyanide with the haem c and haem d, moieties of the enzyme respectively. Under CO, the second-order rate constant for the reaction of the haem c was, at 1.3 X 10(5)M-1.s-1, slightly enhanced over the rate in a N2 atmosphere, but the reaction velocity of the haem d1 component was greatly decreased, being apparently limited to that of the rates of CO dissociation from the molecule (0.15s-1 and 0.03s-1). The results are compared with those obtained during a previous study of the reaction of reduced Pseudomonas cytochrome oxidase with oxidized azurin.     

Mode of interaction of aminooxy compounds with sheep liver serine hydroxymethyltransferase

The interaction of aminooxy compounds such as aminooxyacetate (AAA), L-canaline, and hydroxylamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) was studied by absorption spectra and stopped-flow spectrophotometry and compared with the unique feature of interaction of O-amino-D-serine (OADS) with the enzyme [Baskaran, N., Prakash, V., Appu Rao, A. G., Radhakrishnan, A. N., Savithri, H. S., & Appaji Rao, N. (1989) Biochemistry (preceding paper in this issue)]. The reaction of AAA (0.5 mM) with the Schiff base of the enzyme resulted in the formation of pyridoxal 5'-phosphate (PLP) and was biphasic with rate constants of 191 and 19 s-1. The formation of the PLP-AAA oxime measured by decrease in absorbance at 388 nm on interaction of AAA with the enzyme had a rate constant of 5.2 M-1 s-1. On the other hand, the reaction of L-canaline with the enzyme was slower as measured by the disruption of enzyme-Schiff base than the reaction of OADS and AAA. In contrast, the formation of PLP as an intermediate could not be detected upon the interaction of hydroxylamine with the enzyme. The reaction of D-cycloserine with the enzyme was much slower (1.6 x 10(2) M-1 s-1) than the aminooxy compounds. These observations indicate that the aminooxy compounds that are structural analogues of serine (OADS, AAA, and canaline) formed PLP as an intermediate prior to the formation of oxime, whereas with hydroxylamine such an intermediate could not be detected.     

Generation of superoxide radical, hydrogen peroxide and hydroxyl radical during the autoxidation of N,N,N',N'-tetramethyl-p-phenylenediamine

The autoxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) at neutral pH has been shown to generate superoxide radical and hydrogen peroxide. The rate of formation of these species was increased in the presence of certain iron and copper compounds; in the presence of iron complexed with EDTA, hydroxyl radical was also produced. Hydrogen peroxide was detected in erythrocytes incubated with TMPD and these cells suffered oxidative damage as reflected by methaemoglobin formation and glutathione depletion; the one-electron oxidation product of TMPD, Wurster's Blue, was equally effective in producing such changes in erythrocytes. N-Methylated p-phenylenediamines are known to be mutagenic and myotoxic, and it is suggested that 'active oxygen' species may be involved in the initiation of these harmful effects.     

Kinetic behavior of the monodehydroascorbate radical studied by pulse radiolysis

The reactions of the monodehydroascorbate radical (As.-) with various biological molecules were investigated by pulse radiolysis. As.- reacted with both fully reduced and semiquinone forms of hepatic NADH-cytochrome b5 reductase with second-order rate constants of 4.3 x 10(6) and 3.7 x 10(5) M-1 s-1, respectively, at pH 7.0. In contrast, no reaction of As.- with ferrous cytochrome b5 could be detected by pulse radiolysis, whereas the oxidation of cytochrome b5 by As.- was observed by ascorbate-ascorbate oxidase method. This suggests that the rate constant of As.- with the ferrous cytochrome b5 must be several orders in magnitude smaller than that of the disproportionation of As.-. On the other hand, As.- reduced Fe3+EDTA with a second-order rate constant of 4.0 x 10(6) M-1 s-1 but did not reduce ferric hemoproteins such as metmyoglobin, methemoglobin, and cytochrome b5 by either the pulse radiolysis or the ascorbate-ascorbate oxidase method.     

Interaction of RNA polymerase with promoters from bacteriophage fd.

Replicative form DNA of bacteriophage fd, which had been fragmented with the restriction endonuclease II from Hemophilus parainfluenzae (endo R- HpaII), was reacted with Escherichia coli RNA polymerase; the resulting stable preinitiation complexes were analysed using the filter binding assay followed by gel electrophoresis. At 120mM KCL the first-order rate constants for complex decay were determined to be 10(-2)-10(-6)s-1. The second-order rate constants for complex formation were found to be about 10(6) -10(7) M-1 s-1. From these values association constants for the individual promoters were calculated to be 2 x 10(-8) -2 x 10(-11) M-1. The rate of formation and the stability of promoter complexes was enhanced in superhelical DNA. No evidence was found for stable promoter-specific closed complexes consisting of enzyme and helical DNA. This and the kinetic data suggest that the unwinding of base pairs is already important early in promoter selection, and not only for the formation of the final open complex. The initiation of RNA synthesis form the preinitiation complex was faster than complex dissociation and complex formation for all promoters. Consequently, the initiation efficiency of a promoter is determined by the rate of complex formation, and not by its 'affinity' for the enzyme. No correlation was found between the relative order of the fd promoters for the binding and the dissociation reaction. This is explained by different structural determinants, for the two reactions, which are located in different parts of the promoter DNA.     

Kinetics of slow, tight-binding inhibitors of angiotensin converting enzyme

Five phosphorus-containing inhibitors of angiotensin converting enzyme were found to exhibit slow, tight-binding kinetics by using furanacryloyl-L-phenylalanylglycylglycine as substrate at pH 7.50 and T = 25 degrees C. Two of the inhibitors, (O-ethylphospho)-Ala-Pro (2) and (O-isopropylphospho)-Ala-Pro (3), are found to follow at minimum a two-step mechanism of binding (mechanism B) to the enzyme. This mechanism consists of an initial fast formation of a weaker enzyme-inhibitor complex (Ki = 130 nM for 2 and 180 nM for 3) followed by a slow reversible isomerization to a tighter complex with measurable forward (K3) and reverse (k4) rate constants (k3 = 4.5 X 10(-2) s-1 for 2 and 5.4 X 10(-2) s-1 for 3; k4 = 9.2 X 10(-3) s-1 for 2 and 3.5 X 10(-3) s-1 for 3). For the remaining three inhibitors, phospho-Ala-Pro (1), (O-benzyl-phospho)-Ala-Pro (4), and (P-phenethylphosphono)-Ala-Pro (5), a one-step binding mechanism (mechanism A) is observed under the conditions of the experiment. The second-order rate constants k1 (M-1 s-1) for the binding of these inhibitors to converting enzyme are found to have values more than 3 orders of magnitude lower than the diffusion-controlled limit for a bimolecular reaction involving the enzyme, viz., 3.9 X 10(5) for 1, 2.2 X 10(5) for 4, and 4.8 X 10(5) for 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome oxidase from Pseudomonas aeruginosa. IV. Reaction with oxygen and carbon monoxide.

The reaction between a cytochrome oxidase from Pseudomonas aeruginosa and oxygen has been studied by a rapid mixing technique. The data indicate that the heme d1 moiety of the ascorbate-reduced enzyme is oxidized faster than the heme c component. The oxidation of heme d1 is accurately second order with respect to oxygen and has a rate constant of 5.7 - 10(4) M-1 - s-1 at 20 degrees C. The oxidation of the heme c has a first order rate constant of about 8 s-1 at infinite concentration of O2. The results indicate that the rate-limiting step is the internal transfer of electrons from heme c to heme d1. These more rapid reactions are followed by more complicated but smaller abcorbance changes whose origin is still not clear. The reaction of ascorbate-reduced oxidase with CO has also been studied and is second order with a rate constant of 1.8 - 10(4) M-1 - s-1. The initial reaction with CO is followed by a slower reaction of significantly less magnitude. The equilibrium constant for the reaction with CO, calculated as a dissociation constant from titrimetric experiments with dithionite-reduced oxidase, is about 2.3 - 10(-6) M. From these data a rate constant of 0.041 s-1 can be calculated for the dissociation of CO from the enzyme.