Vol. 66

The Biochemistry of Aromatic Amines3. ENZYMIC HYDROXYLATION BY RAT-LIVER MICROSOMES*

By J. BOOTH AND E. BOYLANDChester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London, S. W. 3

(Received 8 October 1956)

Several hydroxylating systems showing differentintracellular distributions but requiring a reducedphosphopyridine nucleotide, or the nucleotide andan enzyme system capable of reducing it, have beendescribed. Mueller & Miller (1948) reported thata rat-liver homogenate, fortified with diphospho-pyridine nucleotide (DPN+), nicotinamide, mag-nesium ions and hexose diphosphate, demethylatesand hydroxylates 4-dimethylaminoazobenzene togive 4'-hydroxy derivatives. Kensler, Miller &Hyatt (1956) showed that the hydroxylation couldbe carried out by rat-liver microsomes in thepresence of the soluble liver fraction. The systemresponsible for the 1 l,B-hydroxylation of 1 1-deoxy-corticosterone by suspensions of ox-adrenocorticalmitochondria requires reduced triphosphopyridinenucleotide (TPNH) (Hayano & Dorfman, 1954;Grant & Brownie, 1955); the 21-hydroxylation of17oa-hydroxyprogesterone requires the same co-enzyme, but for this reaction to take place both themicrosomes and the soluble fraction are needed(Ryan & Engel, 1956). Aniline and acetanilide arehydroxylated, chiefly in the p-position, by rabbit-liver microsomes and this sytem requires TPNHand oxygen (Brodie et al. 1955; Mitoma & Uden-friend, 1955; Mitoma, Posner, Reitz & Udenfriend,1956), whereas the conversion of 2-acetamido-fluorene into the 7-hydroxy derivative by rat-liverhomogenate takes place in the presence of DPN+,nicotinamide and succinate (Peters & Gutman,1956).Two examples of naturally occurring compounds

which are hydroxylated by similar systems havebeen reported, but in these the microsomes are notinvolved. A soluble enzyme system from rat liverwhich catalyses the conversion of phenylalanineinto tyrosine requires reduced diphosphopyridinenucleotide (DPNH) and oxygen (Udenfriend &Cooper, 1952; Mitoma, 1956), and the conversion ofkynurenine into 3-hydroxykynurenine by livermitochondria of cats and rats requires TPNH (deCastro, Price & Brown, 1956).The isolation of derivatives of 2-amino-1-naph-

thol and 2-amino-6-naphthol from the urine ofanimals dosed with 2-naphthylamine and 2-acet-

* Part 2: Boyland, Manson & Orr (1957).

amidonaphthalene hasdemonstratedthat hydroxyl-ation of the amines occurs in vivo (Wiley, 1938;Dobriner, Hofmann & Rhoads, 1941; Manson &Young, 1950; Boyland & Manson, 1955) and in vitroexperiments have shown that the reaction alsooccurs in the presence of rat-liver slices (Booth,Boyland & Manson, 1955). The present paper reportsan investigation on the enzyme system concernedwith this reaction.

EXPERIMENTAL

Materials. Triphosphopyridine nucleotide (TPN+) as thedihydrate of the sodium salt, TPNH as the sodium salt,glucose 6-phosphate (G 6-P) as the sodium salt or the hepta-hydrate ofthe barium salt and glucose 6-phosphate dehydro-genase were obtained from Sigma Chemical Co. Potassium2-naphthyl sulphamate and potassium 6-hydroxy-2-naph-thyl sulphamate were prepared by the action of chloro-sulphonic acid on 2-naphthylamine and 2-amino-6-naph-thol respectively [Boyland et al. 1957; Boyland & Manson(in preparation)]. Other derivatives of 2-naphthylaminewere obtained as described in a previous paper (Booth et al.1955). Pyrophosphate buffers were prepared from solutionsof Na2H2P2O7 and K4P207 -

Liver preparations. In experiments to determine theintracellular location of the enzyme, male rat-liver homo-genates in 0-25M-sucrose were fractionated bythe method ofSchneider (1948) and the sedimented material was resus-pended in 0-2M-pyrophosphate buffer, pH 7-2.To obtain an active 'microsome preparation', chilled

livers of male rats were homogenized with an MSE Ato-MixBlender (Measuring and Scientific Equipment Co. Ltd.) for0*5 min. or with a Potter & Elvehjem (1936) homogenizer in4 vol. of 0 05M-pyrophosphate buffer, pH 7-2. The homo-genates were centrifuged for 10 min. at 600g to remove celldebris and nuclei and the supernatant was centrifuged for10 min. at 22000 g in a Spinco Model L preparative ultra-centrifuge with rotor no. 40, since this force gave maximumactivity in the supernatant under these conditions. Thesupernatant (100 ml.) was brought to pH 5-4 by the additionof 10% (v/v) acetic acid (approximately 6 ml.), allowed tostand for 0.5 hr. and centrifuged at 10500g for 30 min. Thesupernatant was discarded, and the precipitate, consistingof microsomes and some liver dehydrogenases, was sus-pended in 100 ml. of pyrophosphate buffer, pH 7-2, anddialysed against 5 1. of the same buffer solution for approxi-mately 18 hr. All operations were carried out between 0°and 80 and the centrifugal forces refer to the centre of thetube employed. The preparations correspond to 200 mg. of

73

J. BOOTH AND E. BOYLANDliver/mi. and the protein content, determined by a modifica-tion of the biuret method (Fincham, 1954), was 9 mg. ofprotein/mi.

Estimation of hydroxylating activity with 2-acetamido-naphthalene as substrate. The activity was determined byestimating the rate of formation of 2-acetamido-6-naphtholfrom 2-acetamidonaphthalene. Incubations were carriedout in air in flasks which were shaken for 30min. at38°. Thestandard reaction mixture consisted of 3mM-G 6-P, 0*2mM-TPK, 35 mM-nicotinamide, 2 mM-2-acetamidonaphthaleneand1 ml. of microsome preparation in a total volume of3 ml. of pyrophosphate buffer (0.2M), pH7-2. The 2-acet-amidonaphthalene was added in ethylene glycol mono-

methyl ether (01 ml.), and the reaction was stopped by theaddition of 10% (w/v) Na2CO3 (1 ml.). The reaction mixturewas poured into aglass-stoppered tube, the flask washed outwith n-butanol (3 ml.) and the washings were added to thetube. The addition of diazotized sulphanilic acid (0 5 ml.),freshly prepared by the addition of 0*5% NaNO2 (1.6 ml.) to0.2% sulphanilic acid in0-1N-HCl (10 ml.), followed byshaking and centrifuging to separate the layers, produced anorange colour in the n-butanol phase; this phase (2-5 ml.)was removed to a 5 ml. graduated tube containing 2N-NaOH (2 ml.). After shaking and allowing the layers toseparate, the n-butanol layer was yellow owing to thepresence of 2-naphthylamine which had been formed bydeacetylation of the substrate, and the red colour producedby the 2-acetamido-6-naphthol was in the alkaline layer.This was removed (2 ml.) and added to another tube con-

taining ethanol (0.5 ml.) to prevent turbidity, and theoptical density was read at 520m,u on a Unicam SP. 500spectrophotometer. A standard curve was prepared by theestimation of known amounts of 2-acetamido-6-naphtholadded to a tissue preparation under the same conditions andthe relationship between optical-density readings andjmoles of compound added was linear up to 04,umole.Flasks in which the tissue preparation had been boiled were

run as blanks in each series of experiments.In experiments to determine the intracellular distribution

of the enzyme it was necessary to precipitate the protein byheating in a water bath at 700 for 2 min. After centrifuging,the precipitated protein was shaken with n-butanol (3 ml.)which was then added to the supernatant and the colourdeveloped as before.

Identification of hydroxylated metabolites of varioussubstrates. The microsome preparation was incubated for2 hr. in the reaction mixture described for the quantitativeexperiments, the various substrates being added in ethyleneglycol monomethyl ether (0.1 ml.). The addition of the less-soluble substrates resulted in some of them being pre-

cipitated, and in these experiments they remained partly insuspension throughout the incubation. With 2-naphthyl-amine it was not possible to detect 2-amino-1-naphthol, butwhen a phosphate buffer fortified with adenosine triphos-phate (ATP), MgCl2 and K2SO4 to facilitate sulphate con-

jugation (Bernstein & McGilvery, 1952) was employed,2-amino-1-naphthylsulphuric acid could be identified.

Identification of metabolites by paper chromatography. Atthe end of the incubation period the reaction mixture was

poured into a glass-stoppered tube and shaken with ethylacetate (1 ml.). After centrifuging to separate the layers theethyl acetate phase was applied to VVhatman no. 1 chro-matography paper and descending chromatograms were run

for approximately 18 hr. in the solvent system n-butanol-

n-propanol-water(2:11:1, by vol.). Authentic specimens ofthe hydroxy compounds were run alongside the metabolitesin each case. The following sprays were employed foridentification by colour reactions: (a) hexylresorcinol-N-HCI followed by NaNO2 (05%) and then by hexylresorcinol(0-5 % in 2N-NaOH); (b) 2:6-dichloroquinonechloroimide(0-5%inethanol) followed byNa2CO3(1%);(c) 1-naphthol-N-HCI followed by NaNO2 (0.5%) and then by 1-naphthol(0 5% in2N-NaOH); (d) diazotized sulphanilic acid [1-6 ml.of NaNO (0.5%) added to 10 ml. of sulphanilic acid (0.2%inN-HCI)] followed by Na2CO3 (10%); (e) NNCD reagent(saturated 2-chloro-4-nitrobenzenediazonium naphthalene-2-sulphonate in 01 N-HCI) followed by Na2CO3 (10%);(f) p-dimethylaminobenzaldehyde (0.5% in ethanol con-

taining 1 ml. of conc. HCl/100 ml.); (g) diazotized p-nitro-aniline [1-6 ml. of NaNO2 (0.5%) added to 10 ml. of p-

nitroaniline (0-2% inN-HCI)] followed by Na2C03 (10%).Identification of metabolites by colour tests on the reaction

mixture. (a) 2-Acetamido-7-hydroxyfluorene and 2-amino-7-hydroxyfluorene were identified by the 'nitrite test' beforeand after hydrolysis withN-HCI for 1 hr. in a boiling-waterbath (Damron & Dyer, 1953). (b) Phenol was identified byadding a saturated solution of NNCD reagent in 0-1 N-HCI(1 ml.), 5N-NaOH (0 5 ml.) and n-butanol (3 ml.) to thereaction mixture and shaking. The red colour produced,both by the reaction mixture and by an authentic specimenof phenol, was extracted into the n-butanol phase and hada maximum absorption at 530m,u. The formation of phenolfrom benzene was confirmed with 2:6-dichloroquinone-chloroimide. The reaction mixture was shaken with n-

butanol (3 ml.) and centrifuged. Afterbreakingtheemulsionwith a glass rod andre-centrifuging, the n-butanol phasewasremoved. After the addition of 0.5% 2:6-dichloroquinone-chloroimide in ethanol (0.1 ml.) and 0 1M-borate (2 ml.) thetube was allowed to stand for 0.5 hr. with occasional shaking.The blue colour in the n-butanol produced by the reactionmixture and by an authentic specimen of phenol had an

absorption maximum at 640 mjs. A blank in which thetissue preparation has been boiled was run at the same timein both cases. (c) p-Acetamidophenol was detected by theindophenol reaction essentially as described by Brodie &Axelrod (1948).

RESULTS

Distribution and requirements ofthe enzymesystem

When the rat-liver homogenate was fractionatedaccording to the method of Schneider (1948) it wasfound that the hydroxylating activity was presentin the microsome fraction (Table 1). Equal activitywas obtained in the presence of either chemicallyprepared TPNH, TPN+ and the soluble liverfraction or TPN, G 6-P and G 6-P dehydrogenase.In the microsome preparations used in the quanti-tative experiments both the microsomes and thedehydrogenases of the liver are present, only thematerial soluble at pH 5-4 having been discarded.Therefore with this preparation no activity was

obtained unless a substrate for one of the dehydro-genases such as G 6-P, glucose or to a less extent

malate, was also added. The replacement of TPNH

'95774

75ENZYMIC HYDROXYLATION

Table 1. Intracellular di8tribution and requirement8 of enzyme 8y8tem re8pon8iblefor the conver8ion of 2-acetamidonaphthalene into 2-acetamido-6-naphthol

Flasks contain 2 mM-2-

Liver preparation(1 ml.)

Whole homogenateNucleiNucleiMitochondriaMitochondriaMicrosomesSoluble fractionMicrosomes + soluble fractionMicrosomes + soluble fractionMicrosomesMicrosomesMicrosomesMicrosome preparationMicrosome preparationMicrosome preparationMicrosome preparationMicrosome preparationMicrosome preparationMicrosome preparation

-acetamidtonapntnalene

Phosphopyridinenucleotide(0.2 mm)TPN+TPN+TPNHTPN+TPNHTPN+TPN+TPN+TPN+TPNHTPN+DPNHTPN+TPN+TPN+TPN+TPN+TPNHDPNH

in a total volume of 3 ml. of pyrophosphate buffer (0-2m), pH 7-2.

Substrate for G 6-Pdehydrogenase dehydrogenase

(3 mm) (0 1 %)

G 6-P

G 6-PG 6-PGlucoseMalate

1-21

101

0-4-

02

6 7 vpH

Fig. 1. Effect of pH on the rate of hydroxylation of 2-acetamidonaphthalene. Reaction mixtures (3 ml.) con-

tained 1 ml.. of microsome preparation, 3 mM-G 6-P,0.2 mM-TPN, 35 mM-nicotinamide, 2 mM-2-acetamido-naphthalene and 0 2M-pyrophosphate buffer, and were

incubated for 30 min. at 380.

by DPNH resulted in a loss ofapproximately half ofthe activity and complete loss of activity resultedwhen air was replaced by nitrogen.

Fornawtion of 2-acetamido-6-naphtholfrom 2-acetamidonaphthalene

Rate of reaction and effect of pH. The rate ofreaction was constant over 30min. and was

directly proportional to the volume of microsomepreparationaddedbetween 0-2 and 1 ml. The estima-tion of activity at various pH values in the range

5-8-8-2 and with 1 ml. of microsome preparationincubated for 30 min., showed optimum activity at

pH 7-2, the rate of formation of 2-acetamido-6-naphthol being 1 jamole/g. of liver/hr. (Fig. 1). Theactivity of the microsome preparation was de-pendent on either TPNH or a TPNH-generatingsystem such as TPN+, G 6-P and G 6-P dehydro-genase. Sufficient liver-G 6-P dehydrogenase was

precipitated by the treatment with acetic acid,since the microsome preparation showed the same

activity as a suspension ofmicrosomes in the solubleliver fraction, and addition of G 6-P or G 6-P de-hydrogenase did not increase the activity or affectthe pH-activity curve.

Effect of concentration8 of the reactant8. The rate ofhydroxylation was determined in a series of experi-ments in which the concentration of one of thereactants was varied while the others were present in

concentrations givingmaximum activity. The effectof variations in TPN concentration is shown inFig. 2, a half-maximal rate being obtained with a

concentration of 0-018 mM-TPN.The results of experiments in which the activity

was measured in the presence of various concentra-tions of G 6-P (Fig. 3) show a half-maximal ratewith 0-25 mM-G 6-P.When the substrate concentration was varied,

maximal activity occurred at a concentration of2 mM; at higher substrate concentrations thereaction rate was diminished (Fig. 3). Examinationby paper chromatography showed that some of thesubstrate was deacetylated to 2-naphthylamineand this compound may have inhibited the oxida-tion. However, estimations of 2-naphthylamine atthe end of the incubations showed that the concen-

tration was 0 07 mm, whereas a concentration of

Vol. 66

GasAirAirAirAirAirAirAirAirN2AirAirAirAirAirAirAirAirAirAir

2-Acetamido-6-naphthol(Imoles/g.of liver/hr.)

1.00000001-101-21-30-601*21-21-00*31-10-5

J. BOOTH AND E. BOYLAND0-25 mM-2-naphthylamine caused only 32% in-hibition, suggesting that it was the substrate itselfwhich caused a decrease in activity at concentra-tions above 2 mM.

Hydroxylation of other aromatic compound8

The hydroxylated metabolites identified aftertreating other aromatic compounds with the micro-some preparation in the presence of TPN+, nico-

1-'.

0E 1

L.

o >

6 0

Eco4)v

Concn. of TPN (mm)

Fig. 2. Effect ofTPN+ concentration onthe rate ofhydroxyl-ation of 2-acetamidonaphthalene. Reaction mixtures(3 ml.) contained 1 ml. of microsome preparation, 3 mM-G 6-P, 35 mM-nicotinamide, 2 mM-2-acetamidonaph-thalene and 0-2 M-pyrophosphate buffer, pH 7'2, and wereincubated for 30 min. at 380.

tinamide and G 6-P are listed in Table 2. Theseexperiments were purely qualitative and the condi-tions which produce maximum hydroxylation of2-acetamidonaphthalene were employed.When the three acetylated amines acetanilide,

2-acetamidonaphthalene and 2-acetamidofluorenewere used as substrates, deacetylation occurred ineach case, the free amine being identified on thepaper chromatograms. No hydroxy derivatives ofthe deacetylated products, however, were identifiedexcept 2-amino-7-hydroxyfluorene from 2-acet-amidofluorene, but it is uncertain whether thedeacetylation or the hydroxylation occurs first. Noacetylated hydroxy compounds were found inexperiments in which the free amines were used assubstrates.The hydroxy compounds formed from the acetyl-

ated amines acetanilide, 2-acetamidonaphthaleneand 2-acetamidofluorene were p-acetamidophenol,2-acetamido-6-naphthol and 2-acetamido-7-hydr-oxyfluorene respectively, the hydroxylation havingtaken place at the carbon atom which is furthestfrom the amino group. On the other hand, the freeamines aniline and 2-naphthylamine were oxidizedto the o-hydroxy derivatives as well. 2-Naphthylsulphamate appeared to behave in the same way as2-acetamidonaphthalene in that hydroxylationoccurred in the 6-position.The metabolites identified from naphthalene

were 1-naphthol and 1:2-dihydronaphthalene-1:2-diol, but 2-naphthol was not seen, and phenol wasthe only product identified from benzene.

E 1 0

.06

4 04 -

.1 2 3 4Concn. of 2-acetamidonaphthalene

or G 6-P (mM)

Fig. 3. Effect of G 6-P and substrate concentration onthe rate of hydroxylation of 2-acetamidonaphthalene.Reaction mixture (3 ml.) contained 1 ml. of microsomepreparation, 0-2 mM-TPN, 35 mM-nicotinamide and0-2 M-pyrophosphate buffer, pH 7-2, and was incubated for30 min. at 380. 0, Hydroxylation with variations inG 6-P concentrations in the presence of 2 mM-2-acet-amidonaphthalene; *, hydroxylation with variations in2-acetamidonaphthalene concentration in the presence of

DISCUSSION

Since Mueller & Miller (1948) found that a rat-liverhomogenate required DPN and an oxidizable sub-strate for the hydroxylation of 4-dimethylamino-azobenzene inthe 4'-position, the enzymic hydroxyl-ation of several compounds has been reported, allthe reactions requiring a reduced phosphopyridinenucleotide and oxygen.The enzyme system responsible for the conversion

of 2-acetamidonaphthalene into 2-acetamido-6-naphthol is present in the microsomes of rat liverand requires TPNH and oxygen. Apart from thebuffer system employed the conditions are similarto those described by Mitoma et al. (1956) for thehydroxylation of acetanilide by rabbit-liver micro-somes. Although these workers reported a maxi-mum activity at pH 8-2 for the hydroxylation ofacetanilide, whereas the pH optimum for 2-acet-amidonaphthalene is 7-2, it would seem that thesame enzyme system is involved, and qualitativeexperiments with some of the substrates used byMitoma et al. (1956) such as aniline, acetanilide andnaphthalene yielded essentially the same meta-bolites.

I957

3 mM-G 6-P.

76

1.

ENZYMIC HYDROXYLATION

The mechanism of the hydroxylation reaction isstill not understood, since it is difficult to explain therequirement forTPNH in an oxidative reaction. Asaromatic compounds can be hydroxylated non-enzymically in the presence of H202 and Fe2+ ions(Loebl, Stein & Weiss, 1949) the possibility thatH202 was involved has been investigated. ThusMitoma (1956) found that H202-generating systemssuch as D-amino acid oxidase, uricase or xanthineoxidase and their respective substrates could not

replace the DPN+ or either of the liver fractionsneeded for the hydroxylation of phenylalanine;similarly the TPNH required for the hydroxylationof acetanilide could not be replaced by H202generated by glucose oxidase or D-amino acidoxidase (Mitoma et al. 1956). Furthermore, neitherof these reactions was inhibited by the addition ofcatalase, indicating that H202 was not involved.With 1802 and H2180 it has been demonstrated thatmolecular oxygen, but not water, is utilized by the

Table 2. Compounds hydroxylated by the rat-liver microsomre preparationin the presence of TPN, nicotinamide and G 6-P

Colours were developed by spraying paper chromatograms with the reagents described in Methods unless stated other-wise. Substrates were added in ethylene glycol monomethyl ether (0-1 ml.) to give the final concentration stated, butsome of the less soluble compounds were present partly in suspension.

SubstrateAniline (20 mm)

Acetanilide (2 mM)

2-Naphthylamine (2 mM)

2-Acetamidonaphthalene(2mM)

2-Naphthylsulphamate(20 mm)

2-Aminofluorene (2mM)

2-Acetamidofluorene (2 mM)

Benzene (20 mM)

Naphthalene (20 mm)

Hydroxylated metaboliteo-Aminophenol

p-Aminophenol

p-Acetamidophenol

2-Amino-6-naphthol

2-Amino-l-naphthol (identifiedby formation of 2-amino-i-naphthyl sulphuric acid)

2-Acetamido-6-naphthol

6-Hydroxy-2-naphthylsulphamate

2-Amino-7-hydroxyfluorene

2-Amino-7-hydroxyfluorene

2-Acetamido-7-hydroxyfluorene

Phenol

1-Naphthol

1:2-Dihydronaphthalene-1:2-diol

Method of identificationHexylresorcinol2:6-DichloroquinonechloroimideHexylresorcinol1-Naphthol1-Naphthol after hydrolysis ofreaction mixture

Indophenol test on hydrolysedreaction mixture

HexylresorcinolDiazotized sulphanilic acidNNCD reagent (acid)NNCD reagent (alkaline)Hexylresorcinolp-Dimethylaminobenzaldehyde

Diazotized sulphanilic acid2:6-Dichloroquinonechloroimide

Hexylresorcinol after hydrolysisp-Dimethylaminobenzaldehydeafter hydrolysisI-Naphthol after hydrolysis

p-DimethylaminobenzaldehydeNitrite test on reaction mixture

p-DimethylaminobenzaldehydeNitrite test on reaction mixtureDiazotized p-nitroanilineNitrite test on hydrolysedreaction mixture

2:6-Dichloroquinonechloroimidetest on the reaction mixtureNNCD reagent test on thereaction mixture

Diazotized sulphanilic acidDiazotized p-nitroaniline2:6-DichloroquinonechloroimideDiazotized sulphanilic acid afterhydrolysis

Diazotized p-nitroaniline afterhydrolysis2:6-Dichloroquinonechloroimideafter hydrolysis

BP Colour0-82 Mauve- Grey-green0-71 Red- Violet

- Violet

Blue

0-85

0-56

RedMauveMauveBlueMauveYellow

0-89 RedBlue

0-21 RedOrange

Puce

0-76 Orange- Yellow

0-76 Orange- Yellow0-79 Blue

Yellow

- Blue

Red

0-97

0-86

RedBlueBlueRed

Blue

Blue

77Vol. 66

78 J. BOOTH AND E. BOYLAND I957adrenal gland in the 11-f-hydroxylation of steroids(Hayano, Lmdberg, Dorfman, Hancock & Doering,1955). Further evidence for the utilization ofmolecular oxygen was provided by the finding thatthe oxygenation of the hydroxyl group which isintroduced into salicylate by the model hydroxylaseferrous ion, ethylene diaminetetra-acetate, oxygenand ascorbate (Brodie, Axelrod, Shore & Uden-friend, 1954) arose from molecular oxygen (Mason &Onoprienko, 1956), and Mason, Fowlks & Paterson(1955) have shown that molecular oxygen contri-butes directly in phenolase-catalysed hydroxyl-ations of some aromatic compounds. It has in factbeen suggested (Velick, 1956) that phenolase mayprovide a model for the TPNH and oxygen-requiring systems, since if molecular oxygen andoxidizable metallo-enzymes were involved theTPNH would serve to regenerate the reduced andactive enzyme. Both the hydroxylation of phenyl-alanine and acetanilide are inhibited by the ferrousion-complexing agent, oca'-dipyridyl (Mitoma, 1956;Mitoma et al. 1956), whereas the 1 1#-hydroxylationof 1 1-deoxycorticosterone is strongly inhibited bydiethyl dithiocarbamate (Sweat et al. 1956),suggesting that the enzymic activity in this case isassociated with a copper complex.Although naphthalene is converted into both 1-

naphthol and 1:2-dihydronaphthalene-1:2-diol bythis system, it seems likely that two differentenzyme systems having the same location andrequiring the same coenzvme are responsible forthese two different types of reactions. 1:2-Di-hydronaphthalenel:2-diol is readily dehydrated to1 -naphthol by mineral acid, but when it is incubatedwith the enzyme system no l-naphthol can bedetected, so that 1:2-dihydronaphthalene-1:2-diol isunlikely to be an intermediate between naphthaleneand 1-naphthol. A further study of these tworeactions may throw some light on the hydroxyl-ation mechanism.

SUMMARY

1. The enzyme system responsible for the con-version of 2-acetamidonaphthalene into 2-acet-amido-6-naphthol is associated with the micro-somes of rat liver and requires reduced triphospho-pyridine nucleotide (TPNH) and oxygen.

2. The TPNH can be replaced by triphospho-pyridine nucleotide, glucose 6-phosphate (G 6-P)and G 6-P dehydrogenase, and conditions affectingthe rate of hydroxylation of 2-acetamidonaphtha-lene by this sytem have been studied.

3. The system is not specific for 2-acetamido-naphthalene but will hydroxylate other aromaticamines, N-substituted amines and also benzene andnaphthalene.

This work has been supported by grants to the ChesterBeatty Research Institute (Institute of Cancer Research,Royal Cancer Hospital) from the British Empire CancerCampaign, the Jane Coffin Childs Memorial Fund forMedical Research, the Anna Fuller Fund, and the NationalCancer Institute of the National Institutes of Health, U.S.Public Health Service.

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