o-Quinones formed in plant extracts. Their reactions with amino acids and peptides

1. The reactions of amino acids and peptides with the o-quinones produced by the enzymic oxidation of chlorogenic acid and caffeic acid have been studied manometrically and spectrophotometrically. 2. Amino acids, except lysine and cysteine, react primarily through their alpha-amino groups to give red or brown products. These reactions, which compete with the polymerization of the quinones, are followed by secondary reactions that may absorb oxygen and give products with other colours. 3. The in-amino group of lysine reacts with the o-quinones in a similar way. The thiol group of cysteine reacts with the quinones, without absorbing oxygen, giving colourless products. 4. Peptides containing cysteine react with the o-quinones through their thiol group. 5. Other peptides, such as glycyl-leucine and leucylglycine, react primarily through their alpha-amino group and the overall reaction resembles that of the N-terminal amino acid except that it is quicker. 6. With some peptides, the secondary reactions differ from those that occur between the o-quinones and the N-terminal amino acids. The colours produced from carnosine resemble those produced from histidine rather than those from beta-alanine, and the reactions of prolylalanine with o-quinones are more complex than those of proline.     

Production of Diagnostic Pigment by Phenoloxidase Activity of Cryptococcus neoformans

Cryptococcus neoformans produces brown pigmented colonies when grown on agar media made from an extract of potatoes and carrots, broad beans (Vicia faba), or Guizotia abyssinica seeds. Since other yeasts do not produce the pigment, these media are useful as differential isolation media for C. neoformans. Similar specific pigment was produced by C. neoformans on chemically defined agar media which contained six different substrates of phenoloxidase (o-diphenol: oxygen oxidoreductase EC 1.10.3.1) an enzyme which catalyses the oxidation of o-diphenols to melanin. Substrates were incorporated singly into the media and included L-3, 4-dihydroxyphenylalanine (L-DOPA), chlorogenic acid, protocatechuic acid, catechol, norepinephrine, and 3-hydroxytyramine hydrochloride (dopamine). No pigment was produced on media without substrate. Phenoloxidase activity in (NH(4))(2)SO(4) precipitates of C. neoformans cell-free extract was assayed by measuring increases in absorbance at 480 nm produced in solutions of L-DOPA. This reaction showed oxygen uptake and was effectively inhibited by copper chelators, but not by catalase. The enzyme also oxidized the five other substrates which induced pigment formation. Electron micrographs of cells incubated in L-DOPA showed deposition of the pigment in the cell wall.     

Phenylacetyl-CoA:acceptor oxidoreductase, a membrane-bound molybdenum-iron-sulfur enzyme involved in anaerobic metabolism of phenylalanine in the denitrifying bacterium Thauera aromatica.

Phenylacetic acids are common intermediates in the microbial metabolism of various aromatic substrates including phenylalanine. In the denitrifying bacterium Thauera aromatica phenylacetate is oxidized, under anoxic conditions, to the common intermediate benzoyl-CoA via the intermediates phenylacetyl-CoA and phenylglyoxylate (benzoylformate). The enzyme that catalyzes the four-electron oxidation of phenylacetyl-CoA has been purified from this bacterium and studied. The enzyme preparation catalyzes the reaction phenylacetyl-CoA + 2 quinone + 2 H2O --> phenylglyoxylate + 2 quinone H2 + CoASH. Phenylacetyl-CoA:acceptor oxidoreductase is a membrane-bound molybdenum-iron-sulfur protein. The purest preparations contained three subunits of 93, 27, and 26 kDa. Ubiquinone is most likely to act as the electron acceptor, and the oxygen atom introduced into the product is derived from water. The protein preparations contained 0.66 mol Mo, 30 mol Fe, and 25 mol acid-labile sulfur per mol of native enzyme, assuming a native molecular mass of 280 kDa. Phenylglyoxylyl-CoA, but not mandelyl-CoA, was observed as a free intermediate. All enzyme preparations also catalyzed the subsequent hydrolytic release of coenzyme A from phenylglyoxylyl-CoA but not from phenylacetyl-CoA. The enzyme is reversibly inactivated by a low concentration of cyanide, but is remarkably stable with respect to oxygen. This new member of the molybdoproteins represents the first example of an enzyme which catalyzes the alpha-oxidation of a CoA-activated carboxylic acid without utilizing molecular oxygen.     

Effect of hydrogen peroxide on d-amino acid oxidase from Rhodotorula gracilis

D-amino acid oxidase from Rhodotorula gracilis is a FAD-containing enzyme that belongs to the oxidase class that is characterized by the ability of the reduced flavin to react quickly with oxygen, yielding hydrogen peroxide and the oxidized cofactor. Hydrogen peroxide, necessary for the production of glutaryl-7-ACA from cephalosporin C had a deleterious effect on the enzyme. H(2)O(2) induced the oxidation of tryptophan and cysteine residues of the protein that could be involved in the dimerization process, required for the attainment of a fully competent enzyme. H(2)O(2) had also a kinetic effect on the reaction catalyzed by D-amino acid oxidase. It was a pure noncompetitive inhibitor; the corresponding inhibition constants were K(is) = 0.52 mM and K(ii) = 0.70 mM.     

On the acceptor specificity of glycollate oxidase of Nicotiana tabacum

Summary The effect of compounds on the activity of ammonium sulphate preparations of glycollate oxidase from Nicotiana tabacum cv. John Williams' Broadleaf and the aurea mutant Su/su is reported. Coupling to DCPIP as terminal oxidant under anaerobic conditions gave greater rates of glycollate oxidation than when measured as O₂ uptake in the presence of cyanide. The enzyme also linked to DCPIP in the presence of O₂, showing that it is a facultative aerobic dehydrogenase. Catalytic amounts of PMS stimulated enzyme-dependent oxygen uptake and DCPIP reduction under aerobic and anaerobic conditions. This further suggests that an intermediate carrier, or alternate acceptor, depending on concentration, exists before O₂ in vivo. Naturally occurring quinoid compounds may fulfill such a role, as evidenced by the enhancement of aerobic DCPIP reduction upon addition of catalytic amounts of caffeic and chlorogenic acid. The observation that PMS, caffeic and chlorogenic acid, biopterin, 6-hydroxy-2-amino-4-hydroxypteridine and a quinone extract of N. tabacum quenched the inhibitory effect of blue light on tobacco glycollate oxidase, is in accordance with the possible function of such compounds in glycollate oxidation.Abbreviation DCPIP 2,6-dichlorophenolindophenol - FMN flavin mononucleotide - PMS phenazine methosulphate     

Oxidation of peptidyl lysine by copper complexes of pyrroloquinoline quinone and other quinones. A model for oxidative pathochemistry.

Various o- and p-quinones were assessed as oxidants of peptidyl lysine in elastin and collagen substrates in the presence and absence of divalent copper as paradigms of protein-lysine 6-oxidase (lysyl oxidase) which contains both quinone and copper cofactors. Pyrroloquinoline quinone was among the most active in the absence and the most active of the o- and p-quinones tested in the presence of copper. The optimal rate of elastin oxidation occurred at a 2:1 PQQ/Cu(II) ratio while Cu(II) itself oxidized elastin relatively slightly. Elastin oxidation by 2:1 PQQ/Cu(II) required aerobic conditions consistent with oxygen-dependent turnover of this catalytic pair. Dimethylsulfoxide and catalase individually or in combination inhibited elastin oxidation by PQQ/Cu(II) by approx. 50%, suggesting that oxygen free radical species participate in the reaction. Amino-acid analysis of elastin and collagen substrates oxidized by 2:1 PQQ/Cu and then reduced with borohydride revealed that alpha-aminoadipic-delta-semialdehyde and lesser amounts of covalent cross-linkages were generated by this oxidant. In contrast, lysine oxidase produced aldehydes and significantly greater quantities of cross-linkage products, consistent with the known specificity of the enzyme. These data, thus, indicate the potential for free quinones, such as PQQ, particularly when stimulated by appropriate metal ions, to act as adventitious oxidants of lysine side-chains in proteins.     

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.     

Tyrosinase catalyzes an unusual oxidative decarboxylation of 3,4-dihydroxymandelate

Tyrosinase usually catalyzes the conversion of monophenols to o-diphenols and oxidation of diphenols to the corresponding quinones. However, when 3,4-dihydroxymandelic acid was provided as the substrate, it catalyzed an unusual oxidative decarboxylation reaction generating 3,4-dihydroxybenzaldehyde as the sole product. The identity of the product was confirmed by high-performance liquid chromatography (HPLC) as well as ultraviolet and infrared spectral studies. None of the following enzymes tested catalyzed the new reaction: galactose oxidase, ceruloplasmin, superoxide dismutase, ascorbate oxidase, dopamine beta-hydroxylase, and peroxidase. Phenol oxidase inhibitors such as phenylthiourea, potassium cyanide, and sodium azide inhibited the reaction drastically, suggesting the participation of the active site copper of the enzyme in the catalysis. Mimosine, a well-known competitive inhibitor of tyrosinase, competitively inhibited the new reaction also. 4-Hydroxymandelic acid and 3-methoxy-4-hydroxymandelic acid neither served as substrates nor inhibited the reaction. Putative intermediates such as 3,4-dihydroxybenzyl alcohol and (3,4-dihydroxybenzoyl)formic acid did not accumulate during the reaction. Oxidation to a quinone methide derivative rather than conventional quinone accounts for this unusual oxidative decarboxylation reaction. Earlier from this laboratory, we reported the conversion of 4-alkylcatechols to quinone methides catalyzed by a cuticular phenol oxidase [Sugumaran, M., & Lipke, H. (1983) FEBS Lett. 155, 65-68]. Present studies demonstrate that mushroom tyrosinase will also catalyze quinone methide production with the same active site copper if a suitable substrate such as 3,4-dihydroxymandelic acid is provided.     

The oxidation of cytochrome c peroxidase by hydrogen peroxide. Characterization of products.

H2O2 reacts with cytochrome c peroxidase in a variety of ways. The initial reaction produces cytochrome c peroxidase Compound I. If more than a 10-fold excess of H2O2 is added to the enzyme, a portion of the H2O2 will react with Compound I to produce molecular oxygen. The remainder oxidizes the heme group and various amino acid residues in the protein. If less than a 10-fold excess of H2O2 is added to the enzyme, essentially all the H2O2 is utilized by oxidation of amino acid residues in the protein. The oxidation of the amino acid residues by H2O2 substantially modifies the reactivity of cytochrome c peroxidase. The modification of reactivity could be the direct result of amino acid oxidation or an indirect result caused by a perturbation of the protein structure at the active site. The products oxidized at pH 8 lose their ability to react with H2O2. The products oxidized at pH4 react with H2O2 but their reactivity toward Fe(CN)4-6 is substantially reduced.     

Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system

Anomalies both kinetic and equilibrium in nature are described for the inhibition of cytochrome c oxidase activity by sulphide in the isolated enzyme and in submitochondrial particles. These anomalies are related to the involvement of more than 1 mol of sulphide in the blockage of one cytochrome aa3 centre. Sulphide reduces resting cytochrome a3, a reaction that results in oxygen uptake and the loss of a sulphide molecule. Sulphide can also reduce cytochromes c and a; in the former case, a part of the one-equivalent oxidation product, presumed to be the SH radical, reacts with oxygen. Such oxygen uptake is also seen under aerobic conditions when ferricyanide reacts with sulphide. Three phases are identified in the inhibitory interaction of sulphide with the cytochrome c oxidase enzyme itself: an initial rapid reaction involving sulphide oxidation, oxygen uptake, and conversion of cytochrome aa3 into the low-spin "oxyferri" form; a subsequent step in which sulphide reduces cytochrome a; and the final inhibitory step in which a third molecule of sulphide binds the a3 iron centre in the cytochrome a2+ a3 3+ (oxy) species to give cytochrome a2+ a3 3+ H2S. the initial events parallel some of the events in the interaction of the cytochrome c-cytochrome aa3 system with monothiols; the final inhibitory event resembles that with cyanide.     

Phenolic Components of Brown Scales of Onion Bulbs Produce Hydrogen Peroxide by Autooxidation

2 O₂ when the brown scales were suspended in water. Brown components isolated from the brown scales also transformed molecular oxygen into H₂O₂. During the autooxidation process, absorbance in the visible region was increased. On acid hydrolysis of the brown fraction, 2,4,6-trihydroxyphenylglyoxylic acid, 3,4-dihydroxybenzoic acid and the quinone form of benzoic acid were detected. In addition, glucose was detected as a sugar. 3,4-Dihydroxybenzoic acid was preferentially oxidized during autooxidation of the brown fraction. One of the oxidation products was the quinone form. Stable electron spin resonance (ESR) signals were detected in the brown fraction. New ESR signals appeared on oxidation of the brown fraction by hexacyanoferrate (III). One of the newly formed radicals seemed to have a 3,4-dihydroxyphenyl group. Based on these results, possible structures, mechanism of H₂O₂ formation and biological significance of the brown components are discussed. Received 11 April 2001/ Accepted in revised form 3 August 2001     

Reaction of rat liver phenylalanine hydroxylase with fatty acid hydroperoxides. Characterization and mechanism

Rat liver phenylalanine hydroxylase must be in a reduced form to be catalytically active (Marota, J.J. A., and Shiman, R. (1984) Biochemistry 23, 1303-1311). In this communication we show that a fatty acid hydroperoxide, 13-hydroperoxylinoleic acid (LOOH), can efficiently oxidize the reduced enzyme. In the process, the hydroperoxide is decomposed, oxygen consumed, and hydrogen peroxide formed. Enzyme reduction by the tetrahydropterin cofactor and reoxidation by LOOH can occur as two single steps or, when the enzyme concentration is low compared to that of the substrates, as part of a catalytic cycle. In this latter case, phenylalanine hydroxylase is a hydroperoxide-dependent tetrahydropterin oxidase. The reaction requires 1.0 mol of O2, 1.0 mol of tetrahydropterin, and 0.5 mol of LOOH to yield 1.0 mol of quinonoid dihydropterin, 0.4 mol of H2O2, and fatty acid products. Thus far, the catalytic and single-step reactions appear the same in all properties, consistent with the steady-state reaction following a ping-pong mechanism. Phenylalanine hydroxylase is an excellent catalyst for this reaction: the turnover number with LOOH is slightly greater than with phenylalanine; the Km(app) for LOOH is 11 +/- 4 microM; and the kcat/Km ratio for LOOH is about 25 times greater than for phenylalanine. LOOH and phenylalanine appear to react at different sites on phenylalanine appear to react at different sites on phenylalanine hydroxylase, and the reaction of LOOH is inhibited only slightly by phenylalanine and not at all by 5-deaza-6-methyltetrahydropterin, a competitive inhibitor of phenylalanine hydroxylation. The reaction of LOOH with phenylalanine hydroxylase strongly resembles the nonenzymatic reaction of LOOH with hematin, implying similar mechanisms for the two reactions and implicating the enzyme's non-heme iron as both the site of reaction of LOOH and of electron transfer during oxidation and reduction. The formation of hydrogen peroxide during a reaction of phenylalanine hydroxylase is unusual. Indirect evidence indicates a reduced oxygen species, formed on the enzyme during the reduction step, is (partially) released as H2O2 when the hydroperoxide reacts.     

Stabilization and sclerotization ofRaja erinacea egg capsule proteins

Synopsis Sclerotization of skate egg capsule occurs after secretion of capsule precursors from the shell gland and involves a form of quinone tanning in which catechols are introduced in utero and subsequently oxidized to quinones by catechol oxidase. A latent form of enzyme is incorporated in the capsular matrix during secretion. Oxidase activity increases concomitantly with increasing catechol and quinone contents. Six major proteins ranging in size from 95kDa to 20kDa comprise the skate egg capsule, all of which contain elevated levels of glycine, serine, proline and tyrosine. Hydroxyproline occurs in all but one protein, however, none has an amino acid composition typical of collagen. Solubilization of two proteins from pre-tanned capsule requires reducing agents indicating that an early event leading to matrix stabilization is mediated by disulfide bonds. Stabilization of the other proteins along with the disulfide bonded proteins directly correlates with increasing catechol content, catechol oxidase activity and quinone formation.     

Characterization of quinone tautomerase activity in the hemolymph of Sarcophaga bullata larvae

The hemolymph of Sarcophaga bullata larvae was activated with either zymosan or proteolytic enzymes such as chymotrypsin or subtilisin and assayed for phenoloxidase activity by two different assays. While oxygen uptake studies readily attested to the wide specificty of activated phenoloxidase, visible spectral studies failed to confirm the accumulation of quinone products in the case of 4-alkyl substituted catechols such as N-acetyldopamine and N-β-alanyldopamine. Sepharose 6B column chromatography of the activated hemolymph resolved phenoloxidase activity into two fractions, designated as A and B. Peak A possessed typical o-diphenoloxidase (o-diphenol, oxygen oxidoreductase EC 1.10.3.1) activity, while peak B oxidized physiologically important catecholamine derivatives such as N-acetyldopamine, N-acetylnorepinephrine, and N-β-alanyldopamine into N-acetylnorepinephrine, N-acetylarterenone, and N-β-alanylnorepinephrine, respectively, and converted 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol into 3,4-dihydroxymandelic acid, 3,4-dihydroxybenzaldehyde, and 2-hydroxy-3′,4′-dihydroxyacetophenone, respectively. These transformations are consistent with the conversion of phenoloxidase-generated quinones to quinone methides and subsequent non-enzymatic transformations of quinone methides. Accordingly, Peak B contained both o-diphenoloxidase activity and quinone tautomerase activity. Sepharose 6B column chromatography of unactivated hemolymph resulted in the separation of quinone tautomerase from prophenoloxidase. The tautomerase rapidly converted both chemically made and mushroom tyrosinase-generated quinones to quinone methides. Thus the failure to observe the accumulation of quinones with N-acyl derivatives of dopamine and related compounds in the whole hemolymph is due to the rapid conversion of these long lived toxic quinones to short lived quinone methides. The latter, being unstable, undergo rapid non-enzymatic transformations to form side-chain-oxygenated products that are non-toxic. The possible roles of quinone isomerase and its reaction products—quinone methides—as essential components of sclerotization of cuticle and defense reaction of Sarcophaga bullata are discussed.     

Thiols oxidation and covalent binding of BSA by cyclolignanic quinones are enhanced by the magnesium cation

A novel cyclolignanic quinone, 7-acetyl-3',4'-didemethoxy-3',4'-dioxopodophyllotoxin (CLQ), inhibits topoisomerase II (TOPO II) activity. The extent of this inhibition was greater than that produced by the etoposide quinone (EQ) or etoposide. Glutathione (GSH) reduces EQ and CLQ to their corresponding semiquinones under anaerobic conditions. The latter were detected by EPR spectroscopy in the presence of MgCl₂ but not in its absence. Semiquinone EPR spectra change with quinone/GSH mol ratio, suggesting covalent binding of GSH to the quinones. Quinone-GSH covalent adducts were isolated and identified by ESI-MS. These orthoquinones also react with nucleophilic groups from BSA to bind covalently under anaerobic conditions. BSA thiol consumption and covalent binding by these quinones are enhanced by MgCl₂. Complex formation between the parent quinones and Mg⁺² was also observed. Density functional calculations predict the observed blue-shifts in the absorption spectra peaks and large decreases in the partial negative charge of electrophilic carbons at the quinone ring when the quinones are complexed to Mg⁺². These observations suggest a possible role of Mg⁺² chelation by these quinones in increasing TOPO II thiol and/or amino/imino reactivity with these orthoquinones.     

Active site-directed irreversible inhibition of glutathione S-transferases by the glutathione conjugate of tetrachloro-1,4-benzoquinone

Purified glutathione S-transferase from rat liver cytosol are irreversibly inhibited by the glutathione conjugate of tetrachloro-1,4-benzoquinone, 2-S-glutathionyl-3,5,6-trichloro-1,4-benzoquinone. The inhibition is due to covalent binding in or near the active site, resulting in modification of a single amino acid residue/subunit, presumably a cysteine residue. The amount of inhibition is related to the molar ratio of the inhibitor and the enzyme and is independent of the enzyme concentration. A 70-80% inhibition is obtained on incubating the enzyme with a 5-fold molar excess of the conjugate. Complete 100% inhibition is never reached. The derivative bound to the enzyme still possesses a quinone structure and is able to react with thiol-containing compounds. Reduction of the enzyme-bound quinone abolishes its reactivity but does not decrease the inhibition. At 0 degrees C, the glutathione conjugate of tetrachloro-1,4-benzoquinone inhibits the glutathione S-transferases at a much higher rate than the corresponding beta-mercaptoethanol conjugate, indicating a distinct targetting effect of the glutathione moiety. However, the parent compound, tetrachloro-1,4-benzoquinone, also has a considerable affinity for the enzymes. Although it does not react as fast as the glutathione conjugate, it reacts with the same amino acid residue. Protection from inhibition by the substrate analog S-hexylglutathione also indicates an active site-directed modification. Small but significant differences exist between the different rat liver transferase isoenzymes; using a 20-fold molar excess the inhibition ranges from 78 to 98% for the conjugate, and from 72 to 93% for the quinone, with isoenzyme 1-1 being the most and isoenzyme 2-2 the least inhibited forms.     

Modification of Dopamine Transporter Function: Effect of Reactive Oxygen Species and Dopamine

Abstract: Dopamine can oxidize to form reactive oxygen species and quinones, and we have previously shown that dopamine quinones bind covalently to cysteinyl residues on striatal proteins. The dopamine transporter is one of the proteins at risk for this modification, because it has a high affinity for dopamine and contains several cysteinyl residues. Therefore, we tested whether dopamine transport in rat striatal synaptosomes could be affected by generators of reactive oxygen species, including dopamine. Uptake of [³H]dopamine (250 n M ) was inhibited by ascorbate (0.85 m M ; −44%), and this inhibition was prevented by the iron chelator diethylenetriaminepentaacetic acid (1 m M ), suggesting that ascorbate was acting as a prooxidant in the presence of iron. Preincubation with xanthine (500 µ M ) and xanthine oxidase (50 mU/ml) also reduced [³H]dopamine uptake (−76%). Preincubation with dopamine (100 µ M ) caused a 60% inhibition of subsequent [³H]dopamine uptake. This dopamine-induced inhibition was attenuated by diethylenetriaminepentaacetic acid (1 m M ), which can prevent iron-catalyzed oxidation of dopamine during the preincubation, but was unaffected by the monoamine oxidase inhibitor pargyline (10 µ M ). None of these incubations caused a loss of membrane integrity as indicated by lactate dehydrogenase release. These findings suggest that reactive oxygen species and possibly dopamine quinones can modify dopamine transport function.     

Thiols oxidation and covalent binding of BSA by cyclolignanic quinones are enhanced by the magnesium cation

A novel cyclolignanic quinone, 7-acetyl-3′,4′-didemethoxy-3′,4′-dioxopodophyllotoxin (CLQ), inhibits topoisomerase II (TOPO II) activity. The extent of this inhibition was greater than that produced by the etoposide quinone (EQ) or etoposide. Glutathione (GSH) reduces EQ and CLQ to their corresponding semiquinones under anaerobic conditions. The latter were detected by EPR spectroscopy in the presence of MgCl₂ but not in its absence. Semiquinone EPR spectra change with quinone/GSH mol ratio, suggesting covalent binding of GSH to the quinones. Quinone-GSH covalent adducts were isolated and identified by ESI-MS. These orthoquinones also react with nucleophilic groups from BSA to bind covalently under anaerobic conditions. BSA thiol consumption and covalent binding by these quinones are enhanced by MgCl₂. Complex formation between the parent quinones and Mg⁺² was also observed. Density functional calculations predict the observed blue-shifts in the absorption spectra peaks and large decreases in the partial negative charge of electrophilic carbons at the quinone ring when the quinones are complexed to Mg⁺². These observations suggest a possible role of Mg⁺² chelation by these quinones in increasing TOPO II thiol and/or amino/imino reactivity with these orthoquinones.     

The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.     

The oxidation of lysine and oxalysine by Mytilus edulis: Identification of the products formed in the presence and the absence of catalase

1. O-(2-Aminoethyl)serine (oxalysine) was shown to be a substrate of the l-amino acid oxidase of the digestive gland of the common mussel, Mytilus edulis. 2. Three atoms of oxygen were consumed per mole of oxalysine oxidized in the presence of catalase; l-lysine under the same conditions consumed only one atom. 3. The products of oxidation of oxalysine in the presence and the absence of catalase were: ethanolamine, N-oxalylethanolamine and 3-morpholone (the oxygen analogue of 2-piperidone). After acid hydrolysis 70% of the oxalysine oxidized was recovered as ethanolamine. 4. In the absence of catalase 2-aminoethoxyacetic acid was also detected. 5. The products identified account quantitatively for the oxalysine oxidized and for the oxygen uptake. 6. N-Oxalylethanolamine and 2-aminoethoxyacetic acid have been synthesized. 7. Treatment of extracts of the digestive gland at pH3·0 completely inactivated the catalase, leaving the l-amino acid oxidase unaffected. 8. The major product of the oxidation of lysine in the absence of catalase was 2-piperidone.