kinetics and specificity of guinea pig liver aldehyde ... · oxidase and bovine milk xanthine...

Kinetics and specificity of guinea pig liver aldehydeoxidase and bovine milk xanthine oxidase towardssubstituted benzaldehydes

Georgios I. Panoutsopoulos1,2� and Christine Beedham1

1Department of Pharmaceutical Chemistry, School of Pharmacy, University of Bradford,Bradford, U.K., and 2Department of Experimental Pharmacology, Medical School, AthensUniversity, Athens, Greece

Received: 09 July, 2003; revised: 26 February, 2004; accepted: 25 March, 2004

Key words: aldehyde oxidase, benzaldehydes, dopamine, isovanillin, protocatechuic aldehyde, xanthineoxidase

Molybdenum-containing enzymes, aldehyde oxidase and xanthine oxidase, are im-portant in the oxidation of N-heterocyclic xenobiotics. However, the role of these en-zymes in the oxidation of drug-derived aldehydes has not been established.The present investigation describes the interaction of eleven structurally related

benzaldehydes with guinea pig liver aldehyde oxidase and bovine milk xanthineoxidase, since they have similar substrate specificity to human molybdenumhydroxylases. The compounds under test included mono-hydroxy and mono-methoxybenzaldehydes as well as 3,4-dihydroxy-, 3-hydroxy-4-methoxy-, 4-hydroxy-3-metho-xy-, and 3,4-dimethoxy-benzaldehydes. In addition, various amines and catecholswere tested with the molybdenum hydroxylases as inhibitors of benzaldehyde oxida-tion.The kinetic constants have shown that hydroxy-, and methoxy-benzaldehydes are

excellent substrates for aldehyde oxidase (Km values 5×10–6 M to 1×10–5 M) withlower affinities for xanthine oxidase (Km values around 10–4 M). Therefore, alde-hyde oxidase activity may be a significant factor in the oxidation of the aromatic alde-hydes generated from amines and alkyl benzenes during drug metabolism. Com-pounds with a 3-methoxy group showed relatively high Vmax values with aldehydeoxidase, whereas the presence of a 3-hydroxy group resulted in minimal Vmax valuesor no reaction. In addition, amines acted as weak inhibitors, whereas catechols had amore pronounced inhibitory effect on the aldehyde oxidase activity. It is thereforepossible that aldehyde oxidase may be critical in the oxidation of the analogousphenylacetaldehydes derived from dopamine and noradrenaline.

Vol. 51 No. 3/2004

649–663

QUARTERLY

�Address for correspondence: Georgios I. Panoutsopoulos, 5 Tenedou Street, Platia Amerikis, Athens112 57, Greece; tel.: (302 10) 864 9617; fax: (302 10) 746 2554; e-mail: [email protected] [email protected]

Aldehydes are widely distributed in nature.They constitute a group of relatively reactiveorganic compounds which occur as naturalconstituents in a wide variety of foods. Morethan 300 aldehydes have been identified infoods and food components and are widelyused as flavouring agents (Feron et al., 1991;Lindahl, 1992).Most aldehydes are irritants producing tis-

sue damage at the site of application, andmany possess genotoxic activity (Dellarco,1988; Ma & Harris, 1988). Some aldehydesare potentially carcinogenic in several animalspecies, whereas others have limited or nocarcinogenicity (Feron et al., 1991).Although aldehydes are not typically found

in any measurable concentration in cells,many compounds (these include alcohols,amines and hydrocarbons) are ultimately con-verted, via an aldehyde intermediate, to thecorresponding acid.Even though only a few drugs contain alde-

hyde groups in their structures, there aremany drugs with an amino group attached toan unsubstituted methylene carbon whichcan be transformed into aldehydes via the ac-tion of monoamine oxidase. Exposure to exog-enous aldehydes can also occur through theingestion of metabolic precursors such as pri-mary alcohols. Another common source of analdehydic group is the hydroxylation of an ar-omatic methyl group to an alcohol, which sub-sequently undergoes oxidation, such as theoral hypoglycaemic agent tolbutamide, whichis used for the treatment of diabetes mellitus(Washio et al., 1993).Aldehydes are also produced in the body via

oxidative deamination of compounds such asbiogenic amines, catalyzed by the action ofmonoamine oxidase. For example, homo-vanillamine is deaminated to homovanillyl al-dehyde (Beedham et al., 1995b). In addition,nearly all of the common �-amino acids areconverted to reactive aldehydes by the en-zyme myeloperoxidase (Hazen et al., 1998).Biogenic and xenobiotic aldehydes can ei-

ther be oxidized to the corresponding acids,

which may be catalysed by aldehyde dehydro-genase (EC 1.2.1.3, aldehyde:NAD+ oxido-reductase), aldehyde oxidase (EC 1.2.3.1, al-dehyde:oxygen oxidoreductase) and/or xan-thine oxidase (EC 1.2.3.2, xanthine:oxygenoxidoreductase), or reduced by aldehydereductase and/or alcohol dehydrogenase tothe corresponding alcohols (Fig. 1). Althoughsome aldehydes may be reduced to alcohols invivo, the main metabolic routes are conver-sion to acids.

The molybdenum hydroxylases aldehydeoxidase and xanthine oxidase catalyse the oxi-dation of a wide range of N-heterocycles andaldehydes (Krenitsky et al., 1972; Beedham,1985; Beedham et al., 1995a). Most of the re-ports in the literature have utilizedheterocycles as substrates rather than alde-hydes and many studies have been carriedout with rabbit liver aldehyde oxidase, whichhas different properties than human liver al-dehyde oxidase (Johns, 1967; Beedham,1987). Aldehyde oxidase activity is predomi-nant in the liver (Sasaki et al., 1983; Beedhamet al., 1987), whereas xanthine oxidase activ-ity is present at high levels in the lactating

650 G.I. Panoutsopoulos and C. Beedham 2004

Figure 1. The pathways of aldehyde metabolism.

mammary gland (Bruder et al., 1982) and pri-marily in cow’s milk but not in human milk(Modi et al., 1959). Although these enzymesare similar in structure and molecular proper-ties, they differ in substrate specificity(Krenitsky et al., 1972). While the substratespecificity towards aromatic heterocycles hasbeen extensively investigated, very few sys-tematic studies on the enzymatic oxidation ofaldehydes are found in the literature (Booth,1938; Gordon et al., 1940; Pelsy & Klibanov,1983).The present study examines the interaction

of eleven structurally related benzaldehydeswith guinea pig liver aldehyde oxidase and bo-vine milk xanthine oxidase, as these enzymeshave similar substrate specificity to humanmolybdenum hydroxylases (Beedham et al.,1987a; Critchley, 1989). The compounds un-der test included mono-hydroxy-, and mono-methoxy-benzaldehydes, as well as 3,4-dihy-droxy-, 3-hydroxy-4-methoxy-, 4-hydroxy-3-me-thoxy-, and 3,4-dimethoxy-benzaldehydes. Inaddition, a number of amines and catecholswere tested with guinea pig liver aldehydeoxidase as inhibitors of benzaldehyde oxida-tion.

MATERIALS AND METHODS

Animals. The animals used in this studywere Dunkin-Hartley guinea pigs (450–950 g). They were fed with FD1 pellets supple-mented with ascorbic acid and received haythree times weekly. All animals were allowedfood and water ad libitum and maintained in astrictly controlled temperature; 18 ± 1�C. Hu-midity was kept at 50% and the lighting cyclewas 07.00–19.00 h light and 19.00–07.00 hdark.Chemicals. 4-Hydroxy-3-methoxybenzal-

dehyde (vanillin), 3,4-dihydroxybenzaldehyde(protocatechuic aldehyde), 3-hydroxy-4-me-thoxybenzaldehyde (isovanillin), 3-methoxy-benzaldehyde (3-anisaldehyde), 2-phenylethyl-amine hydrochloride, 3,4-dimethoxy-2-phenyl-

ethylamine (homoveratrylamine), 3,4-dihy-droxyphenylethylamine (dopamine), 3,4-dihy-droxyphenylethanolamine (noradrenaline),L-3,4-dihydroxyphenylalanine (L-DOPA) and3,4-dihydroxyphenylacetic acid (DOPAC)were supplied from Sigma Chemical Com-pany Ltd. Benzaldehyde, 2-hydroxybenzal-dehyde (salicylaldehyde), 3,4-dimethoxybenz-aldehyde, 4-hydroxybenzaldehyde, 2-methoxy-benzaldehyde (2-anisaldehyde), 4-methoxy-benzaldehyde (4-anisaldehyde), phthalazine,4-hydroxy-3-methoxy-2-phenylethylamine(homovanillamine), and phenanthridine werepurchased from Aldrich Chemical Co. Ltd.3-Hydroxybenzaldehyde was from RalphEmanuel, 1,2-dihydroxybenzene (catechol)from BDH Chemicals Ltd, xanthine (pure)from Koch-Light Laboratories Ltd,Coomassie Blue G-250 from Pierce, and2-methylphthalaziniun iodide was synthe-sized according to procedures of Smith &Otremba (1962).Enzymes. Xanthine oxidase (grade I) was

obtained from Sigma Chemical Company Ltd:from bovine buttermilk (EC 1.2.3.22), 0.47units/g protein, 38 mg protein/ml (biuret).Preparation of partially purified aldehyde

oxidase from guinea pig liver. Guinea pigswere killed by cervical dislocation and theirlivers were placed in 3–4 volumes of ice-coldisotonic potassium chloride solution (1.15%KCl, w/v). They were then homogenized onice in a homogenizer fitted with a Teflon pes-tle.The homogenate was heated at 55–57�C for

11 min and then centrifuged at 15000 � g for45 min at 4�C. The resulting clear redsupernatant was filtered through glass wooland then solid ammonium sulphate wasadded to give 50% saturated solution (35.3g/100 ml at 4�C).The resulting suspension was centrifuged at

6000 � g for 20 min at 4�C and the precipi-tate re-dissolved in 5 ml of 1 mM EDTA peranimal liver. The enzyme was stored in liquidnitrogen in the form of small beads untilneeded.

Vol. 51 Oxidation of benzaldehydes by aldehyde and xanthine oxidases 651

Spectrophotometric determination of enzymeactivity. All kinetic measurements and UVspectra were determined using a Pye-UnicamSP8-250 UV/Visible Spectrophotometer fit-ted with a Pye-Unicam cell temperature con-troller at 37�C.Aldehyde oxidase activity was routinely esti-

mated using potassiun ferricyanide (1 mM) asthe electron acceptor, at 420 nm, using aheterocyclic substrate such as phthalazine.Xanthine oxidase activity was checked withxanthine, at 295 nm, and molecular oxygen asthe electron acceptor.Protein content estimation in aldehyde

oxidase. The protein content in aldehydeoxidase was determined using CoomassieBlue dye reagent and bovine serum albuminas the standard, measured at 595 nm, accord-ing to the method of Bradford (1976).Determination of kinetic constants for alde-

hyde oxidase. Kinetic constants were deter-mined using aldehyde oxidase which was pre-pared from three animal livers. All assayswere carried out in 67 mM Sörensen’s phos-phate buffer, pH 7.0, containing 0.1 mMEDTA, varying concentrations of substrate,potassium ferricyanide (1 mM), in a final vol-ume of 3 ml and sufficient enzyme to producemeasurable rates over the range of substrateconcentrations under study.Michaelis–Menten constants were deter-

mined by measuring the initial oxidationrates from a minimum ten different substrateconcentrations over the first 2–5 min, follow-ing spectrophotometrically the decrease inabsorbance at 420 nm. All aldehydes were ini-tially tested for non-enzymatic oxidationand/or interaction with potassium ferri-cyanide.Since many of the benzaldehydes interacted

with potassium ferricyanide, the oxidationrates of these substrates were monitored di-rectly. This was achieved by determining theUV spectra of aldehydes and their acid prod-ucts, which were scanned individually in therange between 200–400 nm. Absorbancemaxima were chosen in the UV spectrum of

the aldehydes, at which wavelength the corre-sponding acid had minimal absorbance at asimilar concentration. The molar absorptionof each aldehyde was calculated at thespecified wavelength.The wavelengths chosen for measurement

of the initial oxidation rates for each alde-hyde, using molecular oxygen as the electronacceptor, and the corresponding molar ab-sorption are presented in Table 1.Therefore, the oxidation rates of benzalde-

hydes were measured at the chosen wave-length under similar conditions as with potas-sium ferricyanide assay. All initial rates mea-sured were found to be linear over the first2–3 min of reaction.Assays were validated by incubating each al-

dehyde with aldehyde oxidase and scanningthe resultant UV spectrum from 200–400 nmat various time intervals until acid formationwas maximal. In each case the final UV spec-tra corresponded to that of the acid. This is il-lustrated in Fig. 2 for 3-methoxybenzalde-hyde.The Michaelis–Menten constant (Km) and

maximum velocity (Vmax) were determinedfrom the ‘Lineweaver-Burk’ plot of 1/V versus1/[S]. The line of the best fit through thepoints was calculated using simple linear re-gression by the least squares method. The ‘co-efficient of determination’ was used to ex-press the goodness of fit of the line. The sub-strate efficiency (Ks), was calculated from theformula: Ks=Vmax/Km.Determination of kinetic constants for

xanthine oxidase. The rates of aldehyde oxida-tion by xanthine oxidase were measured withmolecular oxygen as electron acceptor and atthe same wavelengths as for aldehydeoxidase.Determination of the mode of inhibition and

inhibitor constants for molybdenum hydroxy-lase inhibitors. The effect of inhibitors uponthe oxidation rate of a substrate was also in-vestigated spectrophotometrically. Com-pounds which showed no reaction as sub-strates by aldehyde oxidase were tested as in-


hibitors of benzaldehyde or 2-hydroxybenz-aldehyde oxidation. The rate of oxidation ofat least ten varying concentrations of2-hydroxybenzaldehyde was monitored in the

presence or absence of two concentrations ofan inhibitor, [I].The type of inhibition was determined by ex-

amination of double reciprocal plots of bothnon-inhibited and inhibited reactions and theinhibitor constant (Ki) was calculated by theuse of information derived from these plots.

RESULTS AND DISCUSSION

Kinetic constants for the oxidation of sub-stituted benzaldehydes by guinea pig liveraldehyde oxidase

The Km, Vmax and Ks values calculated foraldehyde oxidase with the heterocyclic sub-strate phthalazine, using ferricyanide as anelectron acceptor, were found to be 0.089mM, 0.44 �mol/min per mg protein and 5.0ml/min per mg protein, respectively. Thesekinetic constants are similar to those re-ported previously for phthalazine (Beedhamet al., 1990; Critchley et al., 1992).All kinetic plots showed substrate inhibition

at concentrations of around 10 � Km value.Substrate inhibition was also noticed in theoxidation of 2-hydroxybenzaldehyde (50 �


SubstrateWavelength(nm)

Molar absorption, �

(M–1 cm–1)

Benzaldehyde2-Hydroxybenzaldehyde3-Hydroxybenzaldehyde4-Hydroxybenzaldehyde2-Methoxybenzaldehyde3-Methoxybenzaldehyde4-Methoxybenzaldehyde3,4-Dihydroxybenzaldehyde3-Hydroxy-4-methoxybenzaldehyde4-Hydroxy-3-methoxybenzaldehyde3,4-Dimethoxybenzaldehyde

249255255290256256284330230310310

1008397289813

129171104210313171308499650088549333

Table 1. Wavelengths and molar absorption used for the measurement of the enzymatic oxidationof substituted benzaldehydes.

UV spectra of aldehydes and their acid products were scanned individually in the range of 200–400 nm. The absorbance max-ima were chosen in the UV spectrum of the aldehydes, at which wavelength the corresponding acid had minimal absorbance ata similar concentration.

Figure 2. UV spectra of oxidation of 3-methoxy-benzaldehyde by aldehyde oxidase at consecutivetime intervals.

3-Methoxybenzaldehyde (0.024 mM) was incubatedwith 0.015 ml aldehyde oxidase (18.6 mg/ml) in 3 ml67 mM Sörensen’s phosphate buffer, pH 7.0, contain-ing 0.1 mM EDTA, at consecutive time intervals (1, ab-sence of enzyme; 2, 0 min; 3, 5 min; 4, 10 min; 5, 15min; 6, 20 min).

Km value) with rabbit liver aldehyde oxidase(Rajagopalan & Handler, 1964) and 4-hy-droxy-3-methoxybenzaldehyde at concentra-tions greater than 5 mM with aldehydeoxidase from Streptomyces viridosporus T7A(Wilkof et al., 1984). However, this phenome-non is not only restricted to aldehydes, butalso to uncharged substrates such asphthalazine (Coughlan, 1980) and quinolinewhich exhibit marked substrate inhibition(Rajagopalan & Handler, 1964). Excess sub-strate may cause an over reduction of Mo(III)which results in a slower reoxidation toMo(VI) (Coughlan, 1980). In contrast, quater-nary substrates such as N-methylquinoliniumand N-ethylquinolinium do not show sub-strate inhibition with aldehyde oxidase (Tay-lor et al., 1984).Most of the substituted benzaldehydes were

found to be excellent substrates of hepaticguinea pig aldehyde oxidase (Table 2). Thelowest Km values were obtained with 2-me-thoxy-, 3-hydroxy-, and 4-hydroxy-benzaldehy-des and the best substrate for guinea pig alde-

hyde oxidase, in terms of substrate efficiency(Ks), is 2-methoxybenzaldehyde. Relativelylow Vmax values were obtained with 3-hy-droxy-, and 4-hydroxy-benzaldehydes.Benzaldehyde oxidation has also been stud-

ied with human liver aldehyde oxidase(Johns, 1967) under similar conditions tothose employed in the present study. Johnsfound the Km for the human liver enzyme tobe of the same order as the Km value reportedhere for guinea pig liver aldehyde oxidase.This is further evidence of the similarity insubstrate specificity between guinea pig andhuman liver aldehyde oxidase (Krenitsky etal., 1986; Beedham et al., 1987a). Althoughabsolute oxidation rates were not reported byJohns (1967), benzaldehyde had higher reac-tion velocities than the aliphatic aldehydestested as substrates for the human liver alde-hyde oxidase.4-Hydroxy-3-methoxybenzaldehyde was found

to be an excellent substrate for guinea pigliver aldehyde oxidase with Km value (Ta-ble 2) similar to that reported by Wilkof et al.


SubstituentR2 R3 R4

Km*(mM)

Vmax(�mol/min per mg)

Ks=(Vmax/Km)(ml/min per mg)

H H HOH H HH OH HH H OHOCH3 H HH OCH3 HH H OCH3H OH OHH OH OCH3H OCH3 OHH OCH3 OCH3

0.0190.020.0050.0080.0040.020.013I**I**0.0290.019

0.340.300.050.120.330.380.28

0.450.38

18151015731922

1721

Table 2. Kinetic constants for the oxidation of substituted benzaldehydes by guinea pig liver alde-hyde oxidase.

The oxidation rates of the substituted benzaldehydes by aldehyde oxidase were measured spectrophotometrically at 37�C withmolecular oxygen as an acceptor. For details see Materials and Methods. All correlation coefficients (r) > 0.998; *n = 2; **I = in-hibitor.

(1984) for both soluble and immobilised alde-hyde oxidase prepared from S. viridosporusT7A. 4-Hydroxy-3-methoxybenzaldehyde hasalso been used to study species variation of al-dehyde oxidase occurrence in 79 species.Wurzinger & Hartestein (1974) found 4-hy-droxy-3-methoxybenzaldehyde to be a sub-strate of aldehyde oxidase from mollusks,crustaceans and insects. Aldehyde oxidase ac-tivity was not found in birds, but it was ob-served in four other classes of vertebrates:osteichthyes, amphibia, reptilia and mam-malia (Sprague-Dawley rat liver and DBAmouse liver).The Km value obtained for 2-hydroxy-

benzaldehyde with guinea pig hepatic alde-hyde oxidase is markedly lower than the Kmvalue of 0.15 mM calculated by Rajagopalan &Handler (1964) for this compound using rab-bit liver aldehyde oxidase. However, it hasbeen noted previously that the substrate spec-ificity of the lapine liver enzyme is quite dif-ferent to that of guinea pig and human alde-hyde oxidase (Johns, 1967; Beedham et al.,1987a; Beedham et al., 1990).In general, hydroxy-, and methoxy-benzalde-

hydes are more efficient substrates for guineapig liver aldehyde oxidase (Table 2) than mostheterocyclic substrates such as phthalazine(Km= 0.052 mM, Vmax= 0.542 �mol/min permg protein) (Beedham et al., 1990),quinazoline (Km= 0.031 mM, Vmax= 0.639�mol/min per mg protein) (Critchley, 1989)and N-methylquinolinium salts (Km= 0.05mM) (Taylor et al., 1984). This is due tohigher affinities of the enzyme rather thanhigher Vmax values. Km values for hetero-cyclic substrates tend to range from 0.02 mMto 0.01 mM, whereas those for these aromaticaldehydes are between 0.004 mM to 0.03 mM.This may be due to methodological differ-ences, for example the use of different elec-tron acceptors. The oxidation rates of the al-dehydes in this study were measured by usingmolecular oxygen as the electron acceptorwhereas those of heterocyclic substrates withan artificial electron acceptor, ferricyanide.

Reoxidation of aldehyde oxidase occurs at dif-ferent redox centers depending on the elec-tron acceptor used. Oxygen reacts at theflavin center (FAD) of aldehyde oxidase,whereas ferricyanide acts as the oxidizingsubstrate at an iron/sulphur center of the en-zyme (Coughlan & Ni Fhaolain, 1979). How-ever, it was possible to determine the kineticconstants for one of the aldehydes, 4-hy-droxy-3-methoxybenzaldehyde, using thesame enzyme preparation with either molecu-lar oxygen or ferricyanide as electron accep-tors. The Km and Vmax values calculated forthis compound were 0.029 mM and 0.45�mol/min per mg protein, respectively, whenmolecular oxygen was used as the electron ac-ceptor, whereas in the presence of ferri-cyanide the values were 0.035 mM and 0.41�mol/min per mg protein, respectively. Thisindicates that, for this group of compounds,the choice of electron acceptor does not havea significant effect on the kinetic constants.Similarly, uncharged substrates such asphthalazine and quinoline show only smallvariations in Km values with different elec-tron acceptors (Taylor et al., 1984).In contrast to most of the other aldehydes,

3-hydroxy-4-methoxy-, and 3,4-dihydroxy-benzaldehydes did not react with guinea pigliver aldehyde oxidase, although the othertwo di-substituted benzaldehydes, 4-hydroxy-3-methoxy-, and 3,4-dimethoxy-benzalde-hydes, were found to be reasonable sub-strates (Table 2). These compounds werefound to be inhibitors of aldehyde oxidasefrom guinea pig liver (Panoutsopoulos, 1994;Beedham et al., 1995b).

3-Hydroxy-4-methoxy-, and 3,4-dihydroxy-benzaldehydes as inhibitors of aldehydeoxidation by guinea pig liver aldehydeoxidase

3-Hydroxy-4-methoxy-, and 3,4-dihydroxy-benzaldehydes were tested as inhibitors of2-hydroxybenzaldehyde oxidation (Ta-ble 3).


3-Hydroxy-4-methoxybenzaldehyde (Fig. 3)and 3,4-dihydroxy-benzaldehyde (Fig. 4) wereboth potent competitive inhibitors of2-hydroxybenzaldehyde oxidation by alde-hyde oxidase with Ki values of 6.64 � 10–4

mM (n = 2) and 1.57 � 10–4 mM (n = 2), re-spectively.Johnson et al. (1985) reported an inhibitory

effect of hydralazine on the oxidation of pu-rine by rabbit, guinea pig and baboon liver al-dehyde oxidase. Critchley (1989) tested the ef-

fect of hydralazine administration on guineapig liver aldehyde oxidase. He found that theoxidation rate of phthalazine, phenanthridineand carbazeran was reduced to less than halfthat of control animals when the enzyme wasprepared from hydralazine (10 mg/kg perday) treated guinea pigs. However, whenxanthine was used as substrate there was noreduction in the oxidation rate. These resultssuggest that hydralazine acts as a potent in-hibitor of aldehyde oxidase in vivo and in vi-tro, but does not have any inhibitory effect onxanthine oxidase.

Inhibition of aldehyde oxidase by aminesand catechols

As inhibition of aldehyde oxidase appearedto be related to the presence of 3-hydroxy-, or3,4-dihydroxy-substituents in the benzalde-hydes, a number of amines and catecholswere tested with guinea pig liver aldehydeoxidase (Table 4). However, the samebenzaldehyde concentration could not alwaysbe employed in each case because (a) difficul-ties were encountered with measuring outsmall amounts of liquid and (b) the cumula-tive absorbance of both benzaldehyde andsome of the inhibitors made spectrophotomet-ric readings not possible.Of all the compounds tested, 2-phenylethyl-

amine, at 1 mM, was the weakest inhibitorshowing only a marginal effect on benzal-dehyde oxidation. 4-Hydroxy-3-methoxy-2-phenylethylamine was also found to be rela-tively ineffective as an inhibitor but


Figure 3. Lineweaver-Burk plots for determina-tion of the inhibitor constant for 3-hydroxy-4-me-thoxybenzaldehyde (isovanillin) with guinea pigliver aldehyde oxidase.

The rate of oxidation of at least ten concentrations of2-hydroxybenzaldehyde was monitored in the presenceor absence of two concentrations of the inhibitor, [I],and measured at 37�C with molecular oxygen as elec-tron acceptor at 255 nm.

Inhibitors Inhibitor concentration(mM)

Inhibition of 2-hydroxy-benzaldehydeoxidation* (%)

3-Hydroxy-4-methoxybenzaldehyde3,4-Dihydroxybenzaldehyde

0.10.1

8787

Table 3. The inhibitory effect of 3,4-dihydroxy-, and 3-hydroxy-4-methoxy-benzaldehydes on guineapig liver aldehyde oxidase with 2-hydroxybenzaldehyde as substrate.

*Concentration of 2-hydroxybenzaldehyde = 0.1 mM

3,4-dimethoxy-2-phenylethylamine inhibitedbenzaldehyde oxidation to a great extent.The catecholamines, 3,4-dihydroxy-2-phenyl-

ethylamine (dopamine), 3,4-dihydroxy-phe-nylethanolamine (noradrenaline) andcatechol (1,2-dihydroxybenzene), had thegreatest effect on benzaldehyde oxidation.Thus at equimolar concentrations, nor-adrenaline (0.1 mM) inhibited the oxidationby 90%. As unsubstituted catechol inhibited

benzaldehyde oxidation to a similar extent asthat of dopamine, it would appear that the in-hibition may be caused by the presence of acatechol group, rather than of the ethylamineside chain. This is supported by the results ob-tained with 2-phenylethylamine. L-DOPA ap-peared to be a less effective inhibitor of alde-hyde oxidase than dopamine, catechol andnoradrenaline. 3,4-Dihydroxyphenylaceticacid (DOPAC), one of the major metabolitesof dopamine (Williams et al., 1960; Goodall &

Alton, 1968), was only a weak inhibitor of al-dehyde oxidase.Inhibition studies of 2-hydroxybenzaldehyde

oxidation showed dopamine to be a potent un-competitive inhibitor of aldehyde oxidasewith a Ki value of 0.0071 mM (Fig. 5).Uncompetitive inhibition, as exhibited to-

wards aldehyde oxidase, normally implies re-action of the inhibitor with the enzyme–sub-strate complex [ES] to produce a dead end

product [ESI], the inhibitor preventing theregeneration of the enzyme in a form capableof reacting directly with the substrate.

Inhibition of aldehyde oxidase by substi-tuted catechols

In order to determine whether the inhibitionobserved with 3-hydroxy-4-methoxybenzal-dehyde and 3,4-dihydroxybenzaldehyde,noradrenaline and dopamine was a general


Inhibitors Inhibitor concentration(mM)

Inhibition of benzaldehydeoxidation (%)

2-Phenylethylamine*10.1

1010

4-Hydroxy-3-methoxy-2-phenylethylamine*10.1

5521

3,4-Dimethoxy-2-phenylethylamine*10.1

10048

Dopamine**10.1

10059

Noradrenaline**10.1

10090

Catechol**10.1

9864

L-Dopa**10.1

9439

DOPAC***10.05

2716

Table 4. The inhibitory effect of catechols and amines on guinea pig liver aldehyde oxidase withbenzaldehyde as substrate.

*Benzaldehyde concentration = 0.04 mM; **Benzaldehyde concentration = 0.1 mM; ***Benzaldehyde concentration = 0.8 mM.


Figure 5. Lineweaver-Burk plots for determina-tion of the inhibitor constant for dopamine withguinea pig liver aldehyde oxidase.

The rate of oxidation of at least ten concentrations of2-hydroxybenzaldehyde was monitored in the presenceor absence of two concentrations of the inhibitor, [I],and measured at 37�C with molecular oxygen as elec-tron acceptor at 255 nm.

Inhibitors Inhibitor concen-tration (mM)

Inhibition of substrate oxidation (%)Phenanthridine* N-methylphthalazinium**

3-Hydroxy-4-methoxybenzal-dehyde

0.10.010.001

87 8517 340 20

3,4-Dihydroxybenzaldehyde0.10.010.001

96 §50 763 64

Noradrenaline

10.10.010.001

91 §51 9517 3210 6

Dopamine0.10.010.001

80 9319 320 6

Table 5. The inhibitory effect of substituted catechols on guinea pig liver aldehyde oxidase withphenanthridine and N-methylphthalazinium chloride as substrates.

*Phenanthridine concentration = 0.05 mM (�max = 322 nm); **N-Methylphthalazinium concentration = 0.05 mM (�max = 320nm); molecular oxygen was used as an acceptor. §, Combined absorbance too high.

Figure 4. Lineweaver-Burk plots for determina-tion of the inhibitor constant for 3,4-dihydroxy-benzaldehyde (protocatechuic aldehyde) withguinea pig liver aldehyde oxidase.

The rate of oxidation of at least ten concentrations of2-hydroxy-benzaldehyde was monitored in the presenceor absence of two concentrations of the inhibitor, [I],and measured at 37�C with O2 as electron acceptor at255 nm.

property of these compounds, or peculiar tothe oxidation of aldehydes, these compoundswere also tested against two N-heterocyclicsubstrates, namely phenanthridine andN-methylphthalazinium chloride.These two heterocyclic substrates were cho-

sen for three reasons. Firstly, both phenan-thridine (Stubley & Stell, 1980; Beedham,1987) and N-methylphthalazinium (Critchley,1989; Beedham et al., 1990) are relatively spe-cific substrates for aldehyde oxidase. Sec-ondly, phenanthridine is a typical unchargedheterocycle, whereas N-methylphthalaziniumis a charged quaternary compound and thesemay bind at different sites. Thirdly, their use

facilitates the experimental conditions, sincesubstrate and product have different UV spec-tra making spectrophotometric measure-ments under oxygen possible.3-Hydroxy-4-methoxybenzaldehyde (0.1

mM) had a similar inhibitory effect on the oxi-dation of phenanthridine, N-methylphthal-azinium (Table 5) and 2-hydroxybenzaldehyde

(Table 3). 3,4-Dihydroxybenzaldehyde inhib-ited aldehyde oxidase with phenanthridine assubstrate and, particularly, N-methylphthala-zinium (Table 5) to a greater extent than thatof 2-hydroxybenzaldehyde (Table 3).The percentage inhibition of N-methyl-

phthalazinium and benzaldehyde oxidationsby noradrenaline were of similar magnitude,whereas the effect upon phenanthridine oxi-dation was less marked. Dopamine appearedto be an even more effective inhibitor ofphenanthridine and N-methylphthalaziniumoxidation than of benzaldehyde.These results showed that 3-hydroxy-4-me-

thoxybenzaldehyde, 3,4-dihydroxybenzalde-

hyde, noradrenaline and dopamine not onlyinhibit aldehyde oxidase, but also the oxida-tion of charged and uncharged heterocycles.This assumption is also supported by the inhi-bition of phthalazine and 4-hydroxy-3-metho-xybenzaldehyde (vanillin) oxidation by alde-hyde oxidase, monitored by HPLC (Pano-utsopoulos, 1994).


SubstituentR2 R3 R4

Km*(mM)

Vmax(�mol/min per mg)

Ks= (Vmax/Km)(ml/min per mg)

H H HOH H H

H OH HH H OH

OCH3 H HH OCH3 HH H OCH3H OH OHH OH OCH3H OCH3 OHH OCH3 OCH3

0.30.190.180.170.190.170.170.0390.180.120.68

0.170.670.953.290.100.590.170.400.950.270.22

0.53.55.419.80.53.41.010.05.31.30.3

Table 6. Kinetic constants for the oxidation of substituted benzaldehydes by bovine milk xanthineoxidase.

The oxidation rates of substituted benzaldehydes by xanthine oxidase were measured spectrophotometrically at 37�C with mo-lecular oxygen as an acceptor. For details see Materials and Methods. All correlation coefficients (r) > 0.998; *n = 2.

Kinetic constants for the oxidation of sub-stituted benzaldehydes with bovine milkxanthine oxidase

In this study, all the compounds examinedwere found to be substrates for bovine milkxanthine oxidase. Km values for these benzal-dehydes were at least ten-fold higher thanwith aldehyde oxidase, but with the exceptionof 3,4-dihydroxybenzaldehyde, they did notvary significantly (Table 6). The lowest Kmvalue was obtained with 3,4-dihydroxy-benzaldehyde but this was still 5–10 foldhigher than that of xanthine (Km=0.004–0.008 mM) (Krenitsky et al., 1986;Spector et al., 1986).Oxidation rates varied to a greater extent

between the compounds examined with thehighest Vmax value being that of 4-hydroxy-benzaldehyde which was thus the best sub-strate in term of substrate efficiency (Ks).The monosubstituted hydroxy-benzaldehydesgave higher Vmax values than the analogousmonosubstituted methoxy-derivatives.Whilst 3,4-dihydroxy-, and 3-hydroxy-4-me-

thoxy-benzaldehydes are potent inhibitors ofaldehyde oxidase in vitro, they do not appearto exert a similar inhibitory effect uponxanthine oxidase. Therefore, it would appearthat the presence of a 3-hydroxy group did nothinder the oxidation reaction catalysed by bo-vine milk xanthine oxidase.Krenitsky et al. (1986) found benzaldehyde

to be a relatively poor substrate for humanliver xanthine oxidase with a low affinity forthe enzyme (Km = 0.93 mM). This value was

in a similar range to the one reported in thepresent studies using bovine milk xanthineoxidase.Oxidation of 2-hydroxybenzaldehyde has

also been reported by Morpeth (1983) (Km= 1mM) for bovine milk xanthine oxidase. ThisKm value is five-fold higher than that re-ported in this study. However, this discrep-ancy is probably due to the different experi-mental conditions used. In addition, Morpeth(1983) found the Km for 2,5-dihydroxybenzal-dehyde with bovine milk xanthine oxidase tobe 0.068 mM, whereas Xia et al. (1999) foundthis substrate to have a Km value of 0.004 mMat pH 7.0.Purines, which are excellent substrates for

xanthine oxidase, have Km values in the orderof 10–4 to 10–3 mM (Krenitsky et al., 1986).Oxidation rates of substituted purines arealso significantly higher than those of benzal-dehydes making the latter group of com-pounds relatively weak substrates. Althoughsome of these compounds were transformedas rapidly as xanthine their high Km valuesmake them relatively inefficient substrates ofxanthine oxidase.

Inhibitors of xanthine oxidase

Noradrenaline and dopamine were alsotested as inhibitors of xanthine oxidase. Incontrast to the results obtained with aldehydeoxidase, they were found to be very weak in-hibitors of benzaldehyde oxidation catalysedby xanthine oxidase (Table 7).


Inhibitors Inhibitor concentration (mM) Inhibition of benzaldehyde oxidation*(%)

Noradrenaline10.1

3015

Dopamine10.1

2323

Table 7. The inhibitory effect of amines on benzaldehyde oxidation by bovine milk xanthineoxidase.

*Concentration of benzaldehyde = 0.1 mM

CONCLUSIONS

The two enzymes used for the spectrophoto-metric determination of the kinetic constantswere guinea pig liver aldehyde oxidase andbovine milk xanthine oxidase. These two en-zymes were chosen as they have similar sub-strate specificity to human liver aldehyde(Beedham et al., 1987a; Beedham et al., 1990)and xanthine (Krenitsky et al., 1986) oxidas-es.The kinetic constants have indicated that

the hydroxy-, and methoxy-benzaldehydes areexcellent substrates for aldehyde oxidase butless efficient substrates for xanthine oxidase.Although the oxidation rates for some of thecompounds with xanthine oxidase are quitecomparable to those for xanthine, the highKm values make these compounds relativelypoor substrates for the enzyme.From these studies it appears, therefore,

that the activity of aldehyde oxidase may be asignificant factor in the oxidation of aromaticaldehydes either generated from biogenicamines, or present in foods. They occur asnatural (flavouring) constituents in a varietyof foods and food components and are alsowidely used as food additives (Coughlan,1980; Feron et al., 1991).

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