Analysis of methylene blue in human urine by capillary electrophoresis

A capillary electrophoresis method for the determination of the dye methylene blue (tetramethylthionine, MB) in human urine depending on liquid/liquid-extraction and diode array detection has been developed, validated, and applied to samples of healthy individuals, who had been dosed with methylene blue within clinical studies. After extraction with dichloromethane and sodium hexanesulfonate, sample extracts were measured on an extended light path capillary. The dye was detected simultaneously at 292 and 592 nm using methylene violet 3 RAX as internal standard. The limit of quantification was 1.0 microg/ml. The accuracy of the method varied between -15.2 and +0.8% and the precision ranged from 2.0 to 12.0%. The method was linear at least within 1.0 and 60 microg/ml. In contrast to earlier indirect determinations no leuco methylene blue (LMB) was directly detected in urine, whereas in aqueous test solutions containing surplus amounts of ascorbic acid leuco methylene blue was well separated from MB in a single run.     

Ca(2+)-sensitive reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by rat liver mitochondria.

Energized rat liver mitochondria in the presence of EGTA reduced linearly 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at the rate of 7 nmol SH/min per mg protein within more than 1 hour at 20 degrees C. The Km for DTNB, 1.4 mM, was decreased by Mg2+ and spermine to 0.5 and 0.7 mM, respectively. The reaction was suppressed under conditions of decreasing mitochondrial content of NADPH, was blocked by 1,3-bis-(2-chloroethyl)-1-nitrosourea, the inhibitor of disulfide reductases, and was sensitive to external free Ca2+ in the micromolar range. After lysis of mitochondria the reduction of DTNB required the addition of NADPH and EGTA and was inhibited by 1 mM sodium arsenite. These observations suggest that the reduction of DTNB by mitochondria is catalyzed by Ca(2+)-sensitive thioredoxin reductase (EC 1.6.4.5).     

Quantification of cationic anti-malaria agent methylene blue in different human biological matrices using cation exchange chromatography coupled to tandem mass spectrometry

Selective and sensitive methods for the determination of the cationic dye and anti-malarial methylene blue in human liquid whole blood, dried whole blood (paper spot), and plasma depending on protein precipitation and cation exchange chromatography coupled to electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) have been developed, validated according to FDA standards, and applied to samples of healthy individuals and malaria patients within clinical studies. Acidic protein precipitation with acetonitrile and trifluoroacetic acid was used for liquid whole blood and plasma. For the extraction of methylene blue from paper spots aqueous acetonitrile was used. Sample extracts were chromatographed on a mixed mode column (cation exchange/reversed phase, Uptisphere MM1) using an aqueous ammonium acetate/acetonitrile gradient. Methylene blue was quantified with MS/MS in the selected reaction monitoring mode using ESI and methylene violet 3RAX as internal standard. Depending on the sample volume (whole blood and plasma 250 microL, and 100 microL on paper spots) the method was linear at least within 75 and 10,000 ng/mL and the limit of quantification in all matrices was 75 ng/mL. Batch-to-batch accuracies of the whole blood, plasma, and paper spot methods varied between -4.5 and +6.6%, -3.7 and +7.5%, and -5.8 and +11.1%, respectively, with corresponding precision ranging from 3.8 to 11.8% CV. After a single oral dose (500 mg) methylene blue concentrations were detectable for 72 h in plasma. The methods were applied within clinical studies to samples from healthy individuals and malaria patients from Burkina Faso.     

Zinc Chloride Methylene Blue, Ii. Preparation of Giesma and Wright Type Low Azure B Blood Stains from Dichromate Oxidized Commercial Dye

TO determine the amount of K₂Cr₂O₇ required to produce optimal Giemsa type staining, six 1 g amounts (corrected for dye content) of zinc methylene blue were oxidized with graded quantities of K₂Cr₂O₇ to produce 4, 8, 12, 16, 20 and 24% conversion of methylene blue to azure B. These were heated with a blank control 15 minutes at 100 C in 60-65 ml 0.4 N HCI. cooled, and adjusted to 50 ml to give 20 mg original dye/ml. Aliquots were then diluted to 1% and stains were made by the “Wet Giemsa” technic (Lillie and Donaldson 1979) using 6 ml 1% polychrome methylene blue, 4 ml 1% cosin (corrected for dye content), 2 ml 0.1 M pH 6.3 phosphate buffer, 5 ml acetone, and 23 ml distilled water. The main is added last and methanol fixed blood films are stained immediately for 20-40 min.

For methylene blue supplied by MCB 12-H-29, optimal stains were obtained with preparations containing 20 and 24% conversion of methylene blue to azure B. With methylene blue supplied by Aldrich (080787), 16% conversion of methylene blue to azure B was optimal. Eosinates prepared from a low azure B/methylene blue preparation selected in this way give good stains when used as a Wright stain in 0.3% methanol solution. However, when the 600 mg eosinate solution in glycerol methanol is supplemented with 160 mg of the same azure B/methylene blue chloride the mixture fails to perform well. The HCI precipitation of the chloride apparently produces the zinc methylene blue chloride salt which is poorly soluble in alcohol. It appears necessary to have a zinc-free azure B/methylene blue chloride to supplement the probably zinc-free eosinate used in the Giemsa mixture.     

DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography.

A new method of affinity chromatography using blue dextran-Sepharose 4B resin was established to purify NADP+-dependent isocitrate dehydrogenase [EC 1.1.1.42] from Bacillus stearothermophilus in high yield. The purified preparation was found to be homogeneous on disc gel electrophoresis. The SH groups of the enzyme were modified with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to determine the number of SH groups per molecule and their contribution to the enzyme activity. One SH group was titrated with DTNB per subunit (the native enzyme consisted of two subunits) and after complete denaturation with 4 M guanidine-HCl the number of titratable SH groups remained unchanged. ORD and CD measurements showed that the alpha-helical conformation of the polypeptide backbone was unaffected by DTNB modification, though the near ultraviolet CD spectrum was evidently altered. The fluorescence derived from tryptophanyl residue(s) was quenched by the modification to 30% of the native level, which may indicate the presence of SH in the vicinity of tryptophanyl residue(s). A remarkable decrease of the enzyme activity was detected upon modification with DTNB, but there was some discrepancy between the rate of inactivation and that of modification of SH groups. The presence of substrate and Mg2+ gave partial protection against modification of the SH groups by DTNB. Complete protection of the native enzyme activity against heating at 65 degrees was observed in the presence of substrate and Mg2+, but the thermostability of the enzyme was markedly reduced by modification of the SH groups.     

Studies on the reactivity of the essential sulfhydryl groups as a conformational probe for the fatty acid synthetase of chicken liver. Inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid) and intersubunit cross-linking of the inactivated enzyme

Fatty acid synthetase of chicken liver is rapidly and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at a rate (k2 = 132 mM-1 S-1 in 3 mM EDTA, 1% (v/v) glycerol, pH 7.0, at 25 degrees C) up to 2200 times higher than the reaction of this reagent with simple thiol compounds. The inactivation is caused by the reaction of the phosphopantetheine SH group, since it is protected competitively by either acetyl- or malonyl-CoA, and since the inactivated enzyme is unreactive with the phosphopantetheine label chloroacetyl-CoA but reactive with the cysteine reagent 1,3-dibromopropanone. Moreover, chloroacetyl-CoA prevents the modification of the rapidly reacting essential SH group by DTNB. The number of SH groups involved in inactivation was determined by correlating activity loss with the extent of reaction and by stopped-flow analysis of substrate (or chloroacetyl-CoA) protection. Values between 0.91 and 1.15 SH groups/dimer were obtained, indicating the presence of substoichiometric amounts of the prosthetic group in the fatty acid synthetase preparations used in this study. Inactivation of the synthetase by DTNB is strongly inhibited by increasing salt concentration and protected noncompetitively by NADP+ and NADPH. Treatment of the enzyme inactivated at low salt by salt, NADP+, or NADPH also effectively reduced cross-linking between enzyme subunits. The parallel effects of these treatments on the reaction with DTNB and subsequent dimerization are consistent with a minimum model of two discreet conformation states for fatty acid synthetase. In the low salt conformer, the phosphopantetheine and cysteine SH groups are juxtaposed, and the DTNB reaction (k2 approximately 132 mM-1 S-1) and dimerization are both facilitated. Transition to the high salt conformer by the above treatments is accompanied by an approximately 20-fold reduction of reactivity with DTNB (k2 = 6.8 mM-1 S-1) and reduced dimerization, due to spatial separation of the SH groups. During palmitate synthesis, the enzyme may oscillate between these conformation states to permit the reaction of intermediates at different active sites. Results obtained by studies on the effect of pH on DTNB inactivation implicate a pK of 5.9-6.1 for the essential SH group independent of salt concentration. This value is 1.5-1.8 pH units lower than the pK of 7.6-7.7 for CoA and may explain the 23-fold increase of the rate constant from a value of 0.3 mM-1 S-1 for CoA to that of the high salt conformer.     

The purification of methylene blue and azure B by solvent extraction and crystallization.

Detailed schemes are described for the preparation of purified methylene blue and azure B from commercial samples of methylene blue. Purified methylene blue is obtained by extracting a solution of the commercial product in an aqueous buffer (pH 9.5) with carbon tetrachloride. Methylene blue remains in the aqueous layer but contaminating dyes pass into the carbon tetrachloride. Metal salt contaminants are removed when the dye is crystallized by the addition of hydrochloric acid at a final concentration of 0.25 N. Purified azure B is obtained by extracting a solution of commercial methylene blue in dilute aqueous sodium hydroxide (pH 11-11.5) with carbon tetrachloride. In this pH range, methylene blue is unstable and yields azure B. The latter passes into the carbon tetrachloride layer as it is formed. Metal salt contaminants remain in the aqueous layer. A concentrated solution oa azure B is obtained by extracting the carbon tetrachloride layer with 4.5 X 10(-4)N hydrobromic acid. The dye is then crystallized by increasing the hydrobromic acid concentration to 0.23 N. Thin-layer chromatography of the purified dyes shows that contamination with related thiazine dyes is absent or negligible. Ash analyses reveal that metal salt contamination is also negligible (sulphated ash less than 0.2%).     

Differential Cytophysiological Diagnosis of Cancerous and Normal Tissues

A method is offered for he differential diagnosis of cancer cells. It depends on the use of methylene blue decolorized with sodium thiosulfate (denoted here HLM, i.e. “hyposulfite methylene blue”); this is prepared by dissolving 800 mg. sodium thiosulfate in 10 ml. of 0.1% aqueous methylene blue and adding 3-5 drops of dilute (1:3) HCl. Frozen sections are treated with this reagent for 2-3 minutes, rinsed with a large amount of distilled water, then stained 2-3 minutes with 0.05% aqueous acid fuchsin. Staining should be performed in a darkened room. If all due precautions are observed, normal tissue appears blue, malignant tissue red.     

Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver

An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.     

Methylene Violet (Bernthsen), by Zinc-Alkali-Chlorate Hydrolysis of Methylene Blue

Though Bernthsen's methylene violet (MV) is a common constituent of polychrome methylene blue, the hydrolytic oxydation of methylene blue to yield azure-free MV has been considered a difficult chemical reaction since the time of Bernthsen, who used Ag₂O in the hydrolysis. MV is qualitatively distinguished from azures by Bernthsen's criteria and the author's new tests: (1) light-excited isomeric change, (2) reactivity to acidity, (3) reaction with KCIO, and (4) reaction with Na₂SO₃ of azures in CHCI₃, while MV gives none. But MV shows (5) indicator properties at pH 4, while azures do not. For practical hydrolysis, treat methylene blue (10 parts by weight) and KCIO₃ (1 part) with 1-2 N NaOH to convert methylene blue to a mixture of MV and azures. Then dilute the solution, add a Zn salt and NaHCO₃ in excess of the amount needed to convert the NaOH to Na₂CO₃. Boil the solution gently for 1-2 hr. The end point of the reaction is found by pipetting a drop of reactant into 3% acetic acid in a test tube, adding CHC1₃ and extracting. The acetic layer should then be almost colorless while the CHC1₃ is colored intensely cherry red. After cooling, the precipitated dye is filtered and dried. This procedure gives good yields of a dye which meets the criteria given by Bernhsen. The peak of the absorption curve in solution, pH 4-11, is at 624 mμ (Bernthsen 625 mμ) and in acid solution, pH 0-4, 588 mμ (Biological Stains, 1953; 580 μ). The dye contains so little azures, that purification of the MV fraction obtained from the reaction mixture is unnecessary when it is used in the Wright-type Romanowsky stain. The remarkable staining effect of MV is its power to bring out red azurophil granules of monocytes and lymphocytes when used with eosinated thiazins in Wright's stain.     

Modification of the Cl−-activated arginine aminopeptidases from rat liver and human erythrocytes: A comparative study

The amino acid residues important for the catalytic activity of the Cl^?-activated arginine aminopeptidases from human erythrocytes and rat liver were studied using enzyme modification. The general inhibition characteristics were similar with both enzymes. Inactivation with 5,5′-dithiobis-(2-nitrobenzoic acid) revealed one essential SH-group per active enzyme unit in both aminopeptidases. l-Arginyl-l-phenylalanine and N-l-arginyl-2-naphthylamide protected the enzymes against inactivation by DTNB, the former substrate being more effective. The rat liver enzyme was more sensitive to DTNB than the erythrocyte enzyme. Titration with DTNB revealed only fast reacting SH-groups in rat liver APB (mean 7.8). The erythrocyte enzyme, however, revealed SH-groups which reacted fast with low concentrations of DTNB, while high concentrations of DTNB or SDS treatment were needed to reveal all enzyme SH-groups (mean 8.0). The presence of at least one essential imidazole group in the erythrocyte enzyme was indicated by photooxidation in the presence of methylene blue, as previously found with the rat liver enzyme (5., 22.). The pH dependence curves of both enzymes also supported the presence of SH- and imidazole groups at or near the active site. Thus, the functional groups identified were the same for both enzymes. Neither enzyme had essential COOH or arginyl groups and they did not contain any zinc. The absence of Zn suggests that the reaction mechanism recently presented by other authors, based on the presence of Zn in the active center, does not apply to the Cl^?-activated arginine aminopeptidases. Accordingly, this enzyme group cannot be classified to metallopeptidases.     

Zinc Chloride Methylene Blue. I. Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples

Zinc chloride methylene blue appeared on the market almost contemporaneously with the zinc-free medicinal form. The former has rarely been reported as being used in blood stains. Recent suspension of manufacture of medicinal methylene blue by it. principal American producer has excited interest in the use of the zinc chloride form for the preparation of blood stains. According to Lillie (1944a,b) the azure B content of zinc chloride methylene blue may have varied from 5 to 30% in the samples studied. Taking the Merck Index (1968, 1976) figures for the spectroscopic absorption maximum (λmax) of 667.8 and 668 nm as standard, recent samples of zinc chloride methylene blue are calculated to contain 6-8% azure B. These figures are baaed on 1) the shift of λmax after exhaustive pH 9.5 chloroform extraction, 2) evaluation of the actual ratio of the observed TiCl₂ dye content to the theoretical for pure zinc chloride methylene blue, 3) comparison of spectroscopic and staining effects of graded hot dichromate oxidation products with those of highly purified azure B-methylene blue mixtures of known proportions.

As far as can be found, medicinal methylene blue is almost the exclusive source of cosin polychrome methylene blue blood stains. Lillie (1944c) included a short series comparing 5 zinc chloride methylene blues with a dozen medicinal methylene blue samples; all were oxidized with hot dichromate to produce successful Wright stains. No effort was made to remove the zinc Exhaustive pH 9.5 chloroform extraction of zinc chloride methylene blue (lot MCB 12-H-29) yielded a small amount of red dye which when extracted into 0.1 N HCI gave λmax = 650. The extraction moved the absorption peak of the zinc chloride methylene blue from 667 to 668 nm and the midpoint of the 90% maximum absorption band, 18 nm wide, from 666.5 to 667.5 nm.     

The role of various amino acid residues in the functioning of NADP-specific isocitrate dehydrogenase from bovine adrenal cortex cytoplasm

The modification of SH-groups in the native isocitrate dehydrogenase accessible to 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) is accompanied by the enzyme inactivation. Isocitrate rather than NADP and MnCl2 protects two SH-groups of the enzyme from modification by DTNB and attendant inactivation. The isocitrate dehydrogenase inactivation by DTNB obeys pseudofirst-order reaction kinetics. The number of DTNB-titrated sulphydryl groups does not change after the isocitrate dehydrogenase denaturation by sodium dodecyl sulphate. In the presence of manganese ions isocitrate and to a lesser extent NADP protect isocitrate dehydrogenase from the inactivation induced by 2,3-butanedione, a specific modifier of arginine residues. It has also been shown that the methylene blue-sensitized photoinactivation of the enzyme associated with the photooxidation of histidine residues decreases in the presence of NADP. These data provide evidence for an essential role of the SH-groups, arginine residues and, probably, histidine in the functioning of NADP-dependent isocitrate dehydrogenase from adrenal cortex.     

Disulfide formation within the regulatory light chain of skeletal muscle myosin

Thiol-disulfide exchange reactions between myosin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) lead to the formation of 5-thio-2-nitrobenzoic acid (TNB)-mixed disulfides as well as to protein disulfide bonds. After incubation with DTNB, myosin was treated with an excess of N-ethylmaleimide (NEM) before electrophoretic analysis of the protein subunits in sodium dodecyl sulfate (SDS) without prior reduction by dithiothreitol (DTT). Without NEM treatment, thiol-disulfide rearrangement reactions occurred in the presence of SDS between the residual free thiols and DTNB. In the absence of divalent metal ions at 25 degrees C, DTNB was shown to induce an intrachain disulfide bond between Cys-127 and Cys-156 of the RLC. This intrachain cross-link restricts partially the unfolding of the RLC in SDS and can be followed as a faster migrating species, RLC'. Densitometric evaluation of the electrophoretic gel patterns indicated that the stoichiometric relation of the light chains (including RLC and RLC') remained unchanged. The two cysteine residues of the fast migrating RLC' were no more available for reaction with [14C]NEM, but upon reduction with DTT, the electrophoretic mobility of the RLC' reverted to that of unmodified RLC and of the RLC modified with two TNB groups. Ca2+ or Mg2+ was able to prevent this disulfide formation in the RLC of myosin by 50% at a free ion concentration of 1.1 X 10(-8) and 4.0 X 10(-7) M, respectively, at 25 degrees C and pH 7.6. Intrachain disulfide formation of RLC never occurred in myosin at 0 degree C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of myosin DTNB light chain on the actin-myosin interaction in the presence of ATP.

The influence of the DTNB light chain of myosin on its enzymatic activities was examined by studying the superprecipitation of actomyosin and the actin-activated ATPase of heavy meromyosin (HMM) [EC 3.6.1.3]. Although the Ca2+-, Mg2+-, and EDTA-ATPase activities of control and DTNB myosin were practically the same, the superprecipitation of actomyosin prepared from actin and DTNB myosin occurred more slowly than that of control myosin. The apparent binding constant obtained from double-reciprocal plots of actin-activated ATPase of DTNB HMM was lower than that of control HMM. Recombination of DTNB myosin and HMM with DTNB light chains restored the original properties of myosin and HMM. The removal of DTNB light chain from myosin had no effect on the formation of the rigor complex between actin and myosin. These results suggest that the DTNB light chain participates in the interaction of myosin with actin in the presence of ATP.     

Mitochondrial sulfhydryl group modification by adriamycin aglycones

Induction of Ca2+ release from isolated, preloaded rat heart mitochondria by low concentrations (less than 5 micrM) of adriamycin aglycones, has recently been reported [(1988) Biochem. Pharmacol. 37, 803]. Ca2+ release occurs via a generalized, Ca2+-dependent increase in the permeability of the inner mitochondrial membrane to small molecules. The process is antagonized by dithiothreitol, suggesting thiol involvement. This communication demonstrates modification of mitochondrial sulfhydryl groups, detected as decreased 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) reactivity, by adriamycin aglycones. Ca2+ release and sulfhydryl modification are shown to depend similarly on aglycone concentration and on the C-7 substituent of the anthracycline ring. In addition, DTNB elicits Ca2+ release. It can therefore be proposed that adriamycin aglycones alter mitochondrial membrane permeability by altering mitochondrial thiol status.     

Accumulation of methylene blue by erythrocytes and determination of its maximum sensitivity to cell damage

The accumulation of methylene blue in native and damaged erythrocytes treated by cetyltrimethylammonium bromide at different initial concentrations (c0) of methylene blue and different volume ratios between external solution and cells (Vs/Vc) was studied. It was shown that at low methylene blue concentrations (c0 < 200 microM), the sorption of the dye by cells made the main contribution to its accumulation. As a result, the internal concentration of methylene blue exceeded manifold its external concentration and their ratio Q was 4-6 for native and 6-9 for damaged cells. As Vs/Vc decreased and especially as c0 increased, the diffusion of methylene blue inward the cells increased concentration gradient, and Q sharply fell to 0.9-1.0. The optimal values of c0 and Vs/Vc that provide the maximum sensitivity of Q to cell damage were determined. The advantages of using Q over other parameters of methylene blue accumulation were shown.     

Modifications of Sulfhydryl Groups on Phytochrome and Their Influence on Physicochemical Differences between the Red- and Far-Red-Absorbing Forms

Phytochrome extracted from shoots of dark-grown rye (Secale cereale cv Rymin) and oat (Avena sativa cv Garry) as the far-red-form (Pfr) and/or under conditions conducive to oxidation exhibited a blue shift in the visible absorption maximum of its red-light-absorbing form (Pr) relative to that measured in vivo. This spectral alteration could not be reversed but could be prevented by inclusion of 10 millimolar diethyldithiocarbamate and 140 millimolar 2-mercaptoethanol in homogenization buffers. Similar blue shifts were induced in purified rye phytochrome by addition of the sulfhydryl-modifying reagent, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). In spectrally normal phytochrome (i.e., no detectable blue shift), Pfr had three to four more sulfhydryls available for rapid reaction with DTNB than did Pr. This difference was maintained over a 2.5-hour time course. Phytochrome purified under conditions resulting in a blue-shifted Pr absorption maximum exhibited a decreased short-term reactivity of Pfr to DTNB. Comparison of the binding and elution of altered and unaltered phytochrome from agarose-immobilized Cibacron blue 3GA confirmed that the Pfr form of spectrally normal phytochrome had a greater affinity for the dye than did the Pr form but that spectral alteration of phytochrome was accompanied by a loss of this difference as evidenced by an increased binding of Pr to the dye. It was concluded that phytochrome has highly reactive sulfhydryl residues located on the portion of the protein that undergoes conformational changes on interconversion of Pr and Pfr and that these residues require rigorous protection in order to extract the native form of the protein from plant tissue.     

Studies on regulatory functions of malic enzymes. VII. Structural and functional characteristics of sulfhydryl groups in NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme from Escherichia coli W contains 7 cysteinyl residues per enzyme subunit. The reactivity of sulfhydryl (SH) groups of the enzyme was examined using several SH reagents, including 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). 1. Two SH groups in the native enzyme subunit reacted with DTNB (or NEM) with different reaction rates, accompanied by a complete loss of the enzyme activity. The second-order modification rate constant of the "fast SH group" with DTNB coincided with the second-order inactivation rate constant of the enzyme by the reagent, suggesting that modification of the "fast SH group" is responsible for the inactivation. When the enzyme was denatured in 4 M guanidine HCl, all the SH groups reacted with the two reagents. 2. Althoug the inactivation rate constant was increased by the addition of Mg2+, an essential cofactor in the enzyme reaction, the modification rate constant of the "fast SH group" was unaffected. The relationship between the number of SH groups modified with DTNB or NEM and the residual enzyme activity in the absence of Mg2+ was linear, whereas that in the presence of Mg2+ was concave-upwards. These results suggest that the Mg2+-dependent increase in the inactivation rate constant is not the result of an increase in the rate constant of the "fast FH group" modification. 3. The absorption spectrum of the enzyme in the ultraviolet region was changed by addition of Mg2+. The dissociation constant of the Mg2+-enzyme complex obtained from the Mg2+- dependent increment of the difference absorption coincided with that obtained from the Mg2+- dependent enhancement of NEM inactivation. 4. Both the inactivation rate constant and the modification rate constant of the "fast SH group" were decreased by the addition of NADP+. The protective effect of NADP+ was increased by the addition of Mg2+. Based on the above results, the effects of Mg2+ on the SH-group modification are discussed from the viewpoint of conformational alteration of the enzyme.     

The roles of superoxide anion and methylene blue in the reductive activation of indoleamine 2,3-dioxygenase by ascorbic acid or by xanthine oxidase-hypoxanthine

To clarify the roles of superoxide anion (O2.-) and methylene blue in the reductive activation of the heme protein indoleamine 2,3-dioxygenase, effects of xanthine oxidase-hypoxanthine used at various oxidase concentration levels as an O2.- source and an electron donor on the catalytic activity of the dioxygenase have been examined in the presence and absence of either methylene blue or superoxide dismutase using L- and D-tryptophan as substrates. In the absence of methylene blue, initial rates of the product N-formylkynurenine formation are enhanced in parallel with the xanthine oxidase level up to approximately 100 and approximately 50% of the apparent maximal activity (approximately 2 s-1) for L- and D-Trp, respectively. Superoxide dismutase effectively inhibits the reactions by 80-98% for both isomers. Additions of methylene blue (25 microM) help to maintain the linearity of the product formation that would be rapidly lost a few minutes after the start of the reaction without the dye, especially for L-Trp. Additions of methylene blue also enhance the activity to the maximal level for D-Trp. In the presence of methylene blue, the inhibitory effects of superoxide dismutase are considerably decreased with the increase in xanthine oxidase concentration, and at near maximal dioxygenase activity levels superoxide dismutase is totally without effect. In separate anaerobic experiments leuco-methylene blue, generated either by photoreduction or by ascorbate reduction, is shown to be able to reduce the ferric dioxygenase up to 25-40%. Substrate Trp and heme ligands (CO, n-butyl isocyanide) help to shift a ferric form----ferrous form equilibrium to the right. Thus, under aerobic conditions leuco-methylene blue might similarly be able to reduce the dioxygenase in the presence of an electron donor with the aid of substrate and O2. These results strongly suggest that indoleamine 2,3-dioxygenase can be activated through different pathways either by O2.- or by an electron donor-methylene blue system. For the latter case, the dye is acting as an electron mediator from the donor to the ferric dioxygenase.