Characterisation of a homogeneous plant aminoaldehyde dehydrogenase.

According to our knowledge, this is the first purification method developed, enabling isolation of a homogeneous aminoaldehyde dehydrogenase (AMADH) from etiolated pea seedlings. The procedure involved initial purification with precipitants followed by three low pressure chromatographic steps. Partially purified enzyme was further subjected to fast protein liquid chromatography on a Mono Q column and to affinity-interaction chromatography on 5'-AMP Sepharose. Purity of the final enzyme preparation was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and chromatofocusing. Pea AMADH exists as a tetramer of 230 kDa in the native state, a molecular mass of one subunit was determined as 57 kDa. The enzyme was found to be an acidic protein with pI 5.4. AMADH showed a broad substrate specificity utilising various aminoaldehydes (C3-C6) as substrates. The best substrate of pea AMADH was 3-aminopropionaldehyde, the enzyme also efficiently oxidised 4-aminobutyraldehyde and omega-guanidinoanalogues of the aminoaldehydes. Pea AMADH was inhibited by SH reagents, several elementary aldehydes and metal-binding agents. Although AMADH did not oxidise betaine aldehyde at all, the N-terminal amino acid sequence of the enzyme shows a high degree of homology with those of plant betaine aldehyde dehydrogenases (BADHs) of spinach, sugar beet and amaranth. Several conserved amino acids were found in comparison with BADH from cod liver of known crystal structure.

Two amine oxidases from Aspergillus niger AKU 3302 contain topa quinone as the cofactor: unusual cofactor link to the glutamyl residue occurs only at one of the enzymes.

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 x 75 kDa) and AO-II (80 kDa), were examined to determine the cofactor structure. Inactivated with p-nitrophenylhydrazine, they showed absorption and fluorescence spectra similar to those published for other copper amine oxidases and to topa hydantoin p-nitrophenylhydrazone. After digestion by thermolysin and pronase, cofactor peptides were purified by HPLC and sequenced. For thermolytic peptides, a typical topa consensus sequence, Asn-X-Glu-Tyr, was obtained for AO-II, although in case of AO-I it overlapped with Val-Val-Ile-Glu-Pro-Tyr-Gly. For pronase peptides of AO-I, only the latter sequence was obtained. NMR and mass spectroscopy confirmed the residue X as topa p-nitrophenylhydrazone in AO-II and revealed the presence of a residue Z attached to the Glu in the peptide Val-Val-Ile-Glu(Z)-Pro of AO-I. This residue was separated from the peptide by hydrolysis and identified as a product derived from topa quinone. The data, together with amino-acid sequence of AO-I, confer strong evidence for topa quinone as the cofactor, bound in the typical consensus sequence. Raman spectra of the p-nitrophenylhydrazone derivative of AO-I and its pronase peptide showed essentially the same peaks matching to a model compound for topa p-nitrophenylhydrazone. However, there may exist an unusual ester link between the topa-404 and Glu-145 in the native enzyme.

Redox hydrogel-based amperometric bienzyme electrodes for fish freshness monitoring

This work presents the design and optimization of amperometric biosensors for the determination of biogenic amines (e.g., histamine, putrescine, cadaverine, tyramine, cystamine, agmatine, spermidine), commonly present in food products, and their application for monitoring of freshness in fish samples. The biosensors were used as the working electrodes of a three-electrode electrochemical cell of wall-jet type, operated at -50 mV vs Ag/AgCl, in a flow injection system. Two different bienzyme electrode designs were considered, one based on the two enzymes [a newly isolated and purified amine oxidase (AO) and horseradish peroxidase (HRP)] simply adsorbed onto graphite electrodes, and one when they were cross-linked to an Os-based redox polymer. The redox hydrogel-based biosensors showed better biosensors characteristics, i.e., sensitivity of 0.194 A M-1 cm-2 for putrescine and 0.073 A M-1 cm-2 for histamine, and detection limits (calculated as three times the signal-to-noise ratio) of 0.17 microM for putrescine and 0.33 microM for histamine. The optimized redox hydrogel-based biosensors were evaluated in terms of stability and selectivity, and were used for the determination of total amine content in fish samples kept for 10 days in different conditions.

Inhibition of copper amine oxidases by pyridine-derived aldoximes and ketoximes.

The reactions of pea diamine oxidase (PSAO) and 2-phenylethylamine oxidase from Arthrobacter globiformis (AGAO) with pyridine-derived oximes were studied. Pyridine carbaldoximes and alkyl pyridyl ketoximes act as strong non-competitive inhibitors of the enzymes. The inhibition constants K(i) of these compounds vary between 10(-4) and 10(-5) M, for AGAO and some of the studied oximes were found even micromolar K(i) values. The presence of pyridine moiety in the studied compounds has remarkable influence on the inhibition potency. Elementary oximes lacking the heterocyclic ring, i.e., aliphatic (acetone oxime), alicyclic (cyclohexanone oxime) and aromatic (benzaldoxime), are considerably weaker non-competitive inhibitors (K(i) similar to 10(-3) or 10(-2) M). The position of the pyridine ring substitution by -C(R)=NOH group does not play a significant role for the inhibition potency of the studied oxime compounds. If the pyridine nitrogen is quaternised (in hydroxyiminomethyl-1-methylpyridinium iodides), the compound looses its inhibitory properties. Extended length of alkyl substituents on the ketoxime group of alkyl pyridyl ketoximes increases the K(i) value. The enzyme-bound copper represents one of possible target sites for pyridine-derived oxime inhibitors. The addition of an alkyl pyridyl ketoxime or a pyridine carbaldoxime to a native PSAO sample perturbs the absorption spectrum of the enzyme (by an absorption increase in the region 300-400 nm) that is not observed in the spectrum of reacted PSAO apoenzyme. However, an additional formation of hydrogen bonds with amino acid side-chains at the active site should be considered, namely for 3- and 4-substituted pyridine derivatives.

Inhibition of diamine oxidases and polyamine oxidases by diamine-based compounds.

This review reports on inhibitors of copper-containing amine oxidases and flavoprotein polyamine oxidases, which are structurally based on diamines. In the introduction, basic characteristics and classification of amine oxidases are described together with the significance of their synthetic inhibitors. The following text is divided into several chapters, which deal with diaminoketones, aza-diamines, unsaturated diamine analogs and diamines with heterocyclic substituents. Then it continues with diamine- and agmatine-based inhibitors of polyamine oxidases. Each chapter gives detailed information on the inhibition mode, potency and structural relationships. The conclusion points out possible roles of mechanism-based inhibitors of amine oxidases in physiological and medicinal research.

1,4-Diamino-2-butyne as the mechanism-based pea diamine oxidase inhibitor.

1,4-Diamino-2-butyne is a mechanism-based inhibitor of diamine oxidase (EC 1.4.3.6) from pea cotyledons. It shows saturation kinetics Km = 1 mM like a substrate, but its interaction leads to time-dependent loss of enzyme activity which is not restored by gel filtration. The substrate 1,4-diaminobutane and the competitive inhibitor 1,4-diamino-2-butanone protect the enzyme against inactivation. Changes in the enzyme electronic spectra with 1,4-diamino-2-butyne were found. The mechanism of the interaction involves an intermediate aminoallenic compound, which is formed with covalent bound pyrrole in the reaction of the nucleophile with the enzyme. The presence of a pyrrole in the inactivated enzyme was confirmed by reaction with Ehrlich's reagent. The kinetic data obtained in this study indicate that 1,4-diamino-2-butyne is a mechanism-based inactivator with number of turnovers, r = 17 and characteristic constants K' = 0.32 mM and k(in) = 4.89 min-1.

Competition of homologous substrates, putrescine and cadaverine, in the reaction catalyzed by pea diamine oxidase.

The determination of diamine oxidase activity with the ninhydrin reagent was used for monitoring of simultaneous oxidation of two homologous substrates, putrescine and cadaverine, which give different colour products (519 and 417 nm). We measured the reaction rates of oxidation of both substrates in different proportion and compared them with the total reaction rate determined by the guaiacol method. The substrates show competition with inhibition constants of putrescine against cadaverine of 0.14 mmol.l-1 and cadaverine against putrescine of 6.4 mumol.l-1.

Comparison of coupling subsites and inhibition effects of piperidine alkaloids and aminoketones on plant amine oxidases.

In the present work we compare the binding subsites of inhibitors from a series of alkaloids and aminoketones on pea and sainfoin diamine oxidase (EC 1.4.3.6; DAO) by the graphical method. As standard competitive inhibitors we have chosen oxoanalogs of the substrates, namely, 1,4-diamino-2-butanone and 1,5-diamino-3-pentanone, which were compared with the alkaloids (+)-sedamine, (-)-norallosedamine, (-)-norsedamine, L-lobeline, cinchonine and aromatic analogs of aliphatic aminoketones such as 1-amino-3-phenyl-3-propanone and 1-amino-3-phenyl-2-propanone. In the case of pea DAO all inhibitors compete for the same subsites with 1,4-diamino-2-butanone and 1,5-diamino-3-pentanone (alpha = infinity). In the case of sainfoin enzyme they are bound to other subsites and the interaction constants (0 < alpha < 1) point to a positive attraction between these two types of inhibitors. With sainfoin DAO, 1-amino-3-phenyl-3-propanone is bound into the same subsite as 1,4-diamino-2-butanone. Cinchonine and 1-amino-3-phenyl-3-propanone are bound to two different subsites and the value of the interaction constant (1 < alpha < infinity) shows repulsion between the inhibitors.

Proteolytic activity assay based on enhanced peroxidase effect of hydrolyzed cytochrome c.

A method using cytochrome c as the substrate of proteinases trypsin, chymotrypsin and subtilisin based on peroxidase effect of the formed haem octapeptide, which is much higher than that of cytochrome c, is described. The octapeptide is degraded by excess hydrogen peroxide and the competition between oxidation of guaiacol catalyzed by the octapeptide and octapeptide degradation leads under experimental conditions to rapid production of a stable colour. Absorption at 476 nm is proportional to proteolytic activity. The method was standardized using the Anson test for proteolytic activity with haemoglobin as a substrate.

Some amines as inhibitors of pea diamine oxidase.

The inhibition of pea diamine oxidase (DAO; EC 1.4.3.6) by some amines and chelating agents has been studied. Ethylenediamine (competitive, Ki = 5.2 mmol.l-1), cis-1,2-diaminocyclohexane (noncompetitive, Ki = 2.9 mmol.l-1) and trans-1,2-diaminocyclohexane (competitive, Ki = 3.6 mmol.l-1) are weak inhibitors whereas diethylenetriamine (noncompetitive, Ki = 7.2 mumol.l-1), triethylenetetraamine (partial noncompetitive, Ki = 0.57 mumol.l-1) are potent inhibitors. The chelating agents 1,10-phenanthroline (Ki = 0.031 mmol.l-1), and 2,2'-bipyridyl (Ki = 0.058 mmol.l-1) are noncompetitive inhibitors. The pH-inhibition profile for the inhibitors indicates that the protonated forms are the active species.

Determination of the dissociation constants of pea diamine oxidase.

The activity of diamine oxidase [EC 1.4.3.6] (DAO) isolated from pea cotyledons was measured in Britton-Robinson buffers at pH range 5.0-9.6 by spectrophotometric method with E-1,4-diamino-2-butene as substrate. The enzyme has the highest activity at pH = 7.7 and in pH greater than 8.0 it is irreversible denaturated with time. The dissociation constants of the enzyme and enzyme-substrate complex were calculated by Dixon's method from plots of log Vmax, log KM and log Vmax/KM against pH. The pKEA = 6.5 suggests that histidine is in active site of DAO.

Determination of trimecaine metabolites in blood plasma by capillary isotachophoresis.

A method is proposed for the determination of trimecaine (diethylglycylmesidide) and its de-ethylated metabolites (monoethylglycylmesidide and glycylmesidide) in blood plasma by capillary isotachophoresis. The deproteinated plasma is extracted into chloroform after alkalinization and the total solids in the organic layer are dissolved in acidified 25% 2-propanol. Subsequent isotachophoretic analysis is performed in an operational system consisting of potassium acetate buffer (pH 4.75) as the leading and beta-alanine as the terminating electrolyte. The order of the zones corresponds to the molecular weights of the separated compounds. The recovery of all substances of interest is 55% and the limit of determination is 0.05 micrograms of each substance in 1 ml of plasma.

Pea (Pisum sativum) diamine oxidase contains pyrroloquinoline quinone as a cofactor.

Diamine oxidase was prepared from pea (Pisum sativum) seedlings by a new purification procedure involving two h.p.l.c. steps. We studied the optical and electrochemical properties of the homogeneous enzyme and also analysed the hydrolysed protein by several methods. The data presented here suggest that the carbonyl cofactor of diamine oxidase is firmly bound pyrroloquinoline quinone.

Time-dependent inhibition of pea cotyledon diamine oxidase by some hydrazides.

The inhibition of diamine oxidase (EC 1.4.3.6) from pea cotyledons (PDAO) by some hydrazides has been studied. It was found that PDAO is inhibited in a time-dependent manner at pH = 7.0 by the hydrazides of acetic, benzoic, nicotinic, isonicotinic, picolinic and 3,4-dihydro-4-oxophtalazine-1-carboxylic acids, by 1-(carboxymethyl)trimethylammonium chloride hydrazide (Girard's reagent T), 1-(carboxymethyl)pyridinium chloride hydrazide (Girard's reagent P) and oxalic acid dihydrazide. The inhibition was partially reversible. Rate constants for enzyme inactivation were in the range 0.29-1.95 min-1. The hydrazides give apparent noncompetitive inhibition at pH = 7.0, but for isonicotinic hydrazide, this changes to competitive inhibition at pH = 8.0. Apparent inhibition constants (K1APP) for the hydrazides with PDAO are in the range 0.005-1.5 mmol l-1.

Probing the active site of pea seedlings amine oxidase with optical antipodes of sedamine alkaloids.

Interactions of pea seedlings amine oxidase (PSAO, EC 1.4.3.6) with sedamine derivatives were studied. All compounds exhibited a competitive inhibition with the inhibition constants in the range 0.03-1.0 mM. The inhibition effect increased in the order allosedamine < sedamine << norallosedamine < norsedamine. The nor-derivatives are about five-fold stronger inhibitors and the allo-isomers are slightly weaker inhibitors than the others. Interestingly, the (-)-diastereomers of the studied sedamines were considerably stronger inhibitors than the (+)-antipodes. Absorption spectroscopy was used to differentiate between two known groups of competitive inhibitors of PSAO. A representative of substrate analogues, 1,5-diamino-3-pentanone, bleached the spectrum of the TPQ cofactor producing a very stable intermediate of the enzyme catalytic cycle that was only slowly converted to the product. On the other hand, the alkaloids did not perturb the spectrum of TPQ so they may interact with some other residue near the active site.

Electrooxidation mechanism of biogenic amines at amine oxidase modified graphite electrode.

Amine oxidase (AO, EC. 1.4.3.6) was previously shown to be a very efficient biological recognition element of amperometric biosensors for monitoring biogenic amines. The enzyme was effectively working in both mono- and bienzyme electrode designs, based on either a direct or a mediated electron-transfer pathway. This work focuses on the elucidation of the electron-transfer mechanism of the monoenzymatic unmediated AO-modified biosensor. The observed unmediated catalytic currents were assumed to be caused by (i) a direct electron-transfer process, (ii) the electrooxidation of the formed product, or (iii) their combination. Experiments supporting these assumptions are discussed in detail.

A study on the reactions of plant copper amine oxidase with C3 and C4 aliphatic diamines.

The paper reports a study on the reactions of grass pea (Lathyrus sativus) amine oxidase (GPAO) with several aliphatic diamines. The influence of the chain length and of unsaturations in the molecules was examined. Kinetic measurements confirmed that trans-, i.e., (E)-2-butene-1,4-diamine (TDABE) and cis-, i.e., (Z)-2-butene-1,4-diamine (CDABE) could be classified as good substrates. Propane-1,3-diamine (DAP) and propene-1,3-diamine (DAPE) were only weakly oxidized, whereas 1,3-diamino-2-propanol (DAPL) was not utilized as a substrate. Contrary to the inactivator 2-butyne-1,4-diamine (DABI), DAPE was shown to be only a competitive inhibitor. DAP itself did not inhibit the catalytic activity. Irreversible inhibition of the activity occurred only after the incubation of GPAO with DABI; other diamines were without this effect. Differential pulse polarography and chromatofocusing confirmed that the aminoaldehyde product of DABI oxidation binds to the enzyme. Activity assay of pea aminoaldehyde dehydrogenase enabled us to detect the products of the oxidation of TDABE, CDABE, and DAP by GPAO. As the product of DAP oxidation, 3-amino-propanal (APAL) was detected by mass spectrometry and confirmed to be a potent noncompetitive inhibitor of GPAO. The absorption changes that occurred in the course of the reaction of GPAO with the diamines were investigated using rapid-scanning spectrophotometry. DABI, TDABE, CDABE, DAP, and DAPE reacted with GPAO providing characteristic maxima of the Cu(I)-semiquinolamine species that is formed in the catalytic cycle. The results presented here confirm that with the exception of DAPL, all the studied diamines could be classified as GPAO substrates, but only DABI can be considered as a mechanism-based inhibitor.

Cytokinins as inhibitors of plant amine oxidase.

The interaction of pea seedling amine oxidase with cytokinins was examined to probe a possible connection between cytokinin oxidase and amine oxidase by determining whether cytokinins are substrates or inhibitors of the latter. Kinetic measurements suggest that cytokinins are weak competitive inhibitors of amine oxidase while their behaviour as substrates was not observed. The absence of enzymatic activity with cytokinins as substrates denies the identity or even any similarity of these two enzymes which was previously considered [Hare, P.D. and van Staden, J. (1994) J. Physiol. Plant., 91, 128]. From the values of the inhibition constants obtained it seems unlikely that cytokinins take part in the regulation of amine oxidase activity in vivo. Their inhibitory effect on amine oxidase may be similar to that of some alkaloids studied earlier.

Comparison of kinetic properties between plant and fungal amine oxidases.

Kinetic properties of novel amine oxidases isolated from a mold Aspergillus niger AKU 3302 were compared to those of typical plant amine oxidase from pea seedling (EC 1.4.3.6). Pea amine oxidase showed highest affinity with diamines, such as putrescine and cadaverine, while fungal enzymes oxidized preferably n-hexylamine and tyramine. All enzymes were inhibited by carbonyl reagents, copper chelating agents, some substrate analogs and alkaloids, but there were quite significant differences in the sensitivity and inhibition modes. Aminoguanidine, which strongly inhibited pea amine oxidases showed only little effect on fungal enzymes. Substrate analogs such as 1.5-diamino-3-pentanone and 1-amino-3-phenyl-3-propanone, which were potent competitive inhibitors of pea amine oxidases, inhibited fungal enzymes much more weakly and non competitively. Also various alkaloids behaving as competitive inhibitors of pea amine oxidase inhibited the fungal enzymes non competitively. Very surprising was the potent inhibition of fungal enzymes by artificial substrates of pea amine oxidases, E- and Z-1,4-diamino-2-butene. The relationships between the different inhibition modes and possible binding at the active site are discussed.

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.

An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested. Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations. Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum. Possible existence of a second redox centre is discussed.