p-phenylenediamine and its various N-alkyl derivatives are also oxidized by this enzyme system (35). It has generally been assumed that any substance which ...
The
Oxidation
of o-Aminophenols by Cytochrome and Cytochrome Oxidase
c
I. ENZYMATIC OXIDATIONS AND BINDING OF OXIDATION PRODUCTS TO BOVINE SERUM ALBUMIN* H. T. NAGASAWA
AND
H. R.
GUTMANN
From the Radioisotope Service, Veterans Administration Hospital, and the Department of Physiological Chemistry, University of Minnesota, Minneapolis, Minnesota (Received for publication, December 5, 1958) The mechanism by which a chemical agent binds to proteins is of considerable interest, particularly since a large number of compounds with carcinogenic activity have been found to bind Infirmly to cellular proteins at the site of tumor formation. cluded in this category are the polycyclic aromatic hydrocarbons (l), the aminoazo dyes (2), N-(2.fluorenyl)acetamide (3), and the nitrogen and the sulfur mustards (4). With the exception of the mustards, binding of these agents appears to depend on prior metabolism to as yet ill-defined metabolites. In the case of AFAr the magnitude of binding to rat liver proteins in vitro paralleled the relative magnitude of hydroxylation (5). Since liver homogenates fortified with the cofactors required for hydroxylation gave an appreciable value of proteinbound radioactivity when incubated with carbon 14-labeled AFA, whereas only low values were detectable with unfortified homogenates, it has been suggested that hydroxylation is prerequisite to protein binding (6). The protein-bound radioactivity was not removable by drastic means such as solution of the protein in dilute sodium hydroxide followed by reprecipitation with acid, so that the binding referred to here is probably due to strong covalent linkages. A likely reaction path leading ultimately to covalently bound carcinogen-protein derivatives is through the intermediate formation of a quinoneimine or an N-acyl quinoneimine (quinoneimide) (5). This pathway, which requires a further oxidation of the hydroxylated metabolites, is plausible from a consideration of the structures and reactivities of the oxidized intermediThe ates, but heretofore has lacked direct experimental support. validity of this mechanism therefore required experimental proof that such quinonoid intermediates are indeed generated enzymatically from the hydroxylated metabolites, and that these The present report prointermediates in turn bind to proteins. vides evidence that the cytochrome c - cytochrome oxidase system might perform these reactions in the cell, and that these oxidized products bind readily to bovine serum albumin in a model system in vitro. * Supported by grants from the National Cancer Institute, United States Public Health Service (C-2571), and the Minnesota Division of the American Cancer Society. 1 The abbreviations used are: AFA, N-@fluorenvl)acetamide: OFA, 2.amino-1-fluorenol; NQI, 2.amino-1;4-naphthoquinone.Nal (I-hydroxy-2.naphthyI)imine.
EXPERIMENTAL
Materials and Methods-Mitochondria from kidney and liver of Holtzman albino rats were isolated according to Hogeboom (7). Horse heart cytochrome c was purchased from Sigma Chemical Company and standardized according to the conventional procedure (8). Cytochrome oxidase from pig heart which yielded clear solutions was prepared according to Smith and Stotz (9) and assayed spectrophotometrically (10). The specific activity of the enzyme was 4.0 X 1O-3 sec.? pg. protein-l (2.5 X 10-Z sec.-l pg. N-l) or 75 per cent of the Smith and Stotz preparation. Microsomal cytochrome was prepared according to Strittmatter and Ball (11). Catalase, obtained from Worthington Biochemical Corporation, was assayed to have a specific activity of 1.58 X 105 M-I sec.-l (12). N-(1-hydroxy-2-fluorenyl)acetamide, l-fluorenol, OFA (13), 2-amino-7-fluorenol (14), 2-amino-5-fluorenol (15), and 2-aminofluorene (16) were prepared by published procedures. 3-Hydroxy-4-aminobiphenyl, m.p. 185-187”, was prepared by reduction of 3-hydroxy-4-nitrobiphenyl (17) with zinc dust, ethanol and calcium chloride (16). The melting point of the 3-hydroxy-4-aminobiphenyl prepared by catalytic reduction of the nitro derivative was 185-186” (18). 1-Amino-2-naphthol, 4-amino-l-naphthol, o-aminophenol, or their respective hydrochlorides, and cu-naphthol were commercial products which were recrystallized. 2-Amino-1-naphthol was prepared as its hydrochloride by reduction of 2-nitroso-1-naphthol with aqueous ethanolic sodium hydrosulfite, a procedure adapted from Fiescr (19) for the reduction of 2,-I-dinitro-1-rnaphthol, followed by recrystallization of the free aminonaphthol from hydrochloric acid. Aniline was freshly distilled from zinc dust. l-Imino-l , 2-naphthoquinone was prepared by oxidation of 1-amino-2-naphthol in ether with lead dioxide (20). Hydroxy-L-proline, crystalline bovine serum albumin, and glycylglycine were obtained from Mann Research Laboratories and Nutritional Biochemicals Corporation. The albumin had a water content of 9.4 per cent and all calculations are based on a molecular weight of 69,000. N&I was prepared by oxidation of 2-amino-1-naphthol in methanolic tricthylamine with oxygen. The air oxidation of 2-amino-lnaphthol in ammoniacal solution and the isolation of a blue product was first reported by Liebermann (21) who regarded the oxidized material as an o-quinoneimine (“imidoxynaphthalene”). The probable structure was later assigned to this product by
1593
1594
o-Aminophenol
Oxidation
by Cytochrome c-Cytochrome Oxidase.
Radt (22). There is, however, some doubt as to the purity of Liebermann’s product since the elemental analysis was unsatisfactory. Moreover, the presence of ammonia during the air oxidation may introduce side reactions involving addition of ammonia to the aromatic system (23). Even under the present conditions where the use of ammonia was avoided, the oxidation product was a mixture which required fractionation by column chromatography. To a solution of 1.98 gm. (5.0 mmoles) of 2-amino-1-naphthol hydrochloride in 10 ml. of absolute methanol were added 10 ml. of a 1 M solution of anhydrous triethylamine in ether. The mixture was thoroughly shaken and the triethylamine hydrochloride was precipitated by addition of 100 ml. of anhydrous ether. The precipitate was removed by filtration, washed with ether, and the combined filtrates concentrated to incipient dryness under reduced pressure. The residue was taken up in 125 ml. of absolute methanol, 0.70 ml. (5.0 mmoles) of anhydrous triethylamine was added, and oxygen saturated with methanol was passed into the solution for 3 hours. The dark violet solution which contained some dark violet precipitate was then concentrated to dryness under reduced pressure. Fractional crystallization of the residue from chloroform-petroleum ether (30-60’) gave amorphous violet solids with variable melting points ranging from 80-150” for different fractions. The combined solids were dissolved in 25 ml. of chloroform and subjected to chromatography on a 1.7 X 27 cm. column of acid-washed, activated alumina. Development and elution with chloroform gave four distinct blue-violet bands, the boundaries of which were readily seen under an infrared lamp. The two lower bands were eluted separately and the eluates concentrated to approximately 15 ml. under reduced pressure. Addition of petroleum ether (30-60”) precipitated 0.34 and 0.13 gm. of violet solids from Fractions 1 and 2. Bands 3 and 4 were tightly adsorbed and were not eluted. Fraction 1 when again subjected to chromatography as described above moved as a single band, the trailing end of which was discarded. Evaporation of the eluent followed by recrystallization of the residue from chloroform-petroleum ether gave 0.23 gm. of a dark violet solid, m.p. 180-184’; x~~~‘~ 547 rnp (e, 10,200), x ES:” 244, 306, 540 rnp (not sufficiently soluble in ethanol or methanol for calculation of c).
I
Vol. 234, No. G
cytochrome c in 100 ~1. of water, and 25 ~1. of the cytochrome oxidasepreparation(0.93pg. protein N per ~1.)and the mixtures were incubated in air at 37”. After incubation, the mixtures were extracted successivelywith 3.0 ml. followed by 2.0 ml. of chloroform. The chloroform extract wasdried over a few crystals of anhydrous sodiumsulfate and the volume readjustedto 5.0 ml. with chloroform. The visible spectra (325 to 650 rnp) of the chloroform extracts were recordedon a Beckman model DR spectrophotometer. When the ultraviolet spectrum was taken, the aminophenolwasaddedin one-halfof the above quantity and the spectrumwasdetermineddirectly with useof appropriate spectrophotometricblanks. Occasionally, the mixtures were also extracted with ether. When (a) the chloroform extract did not absorbthroughout the visible region of the spectrum, (5) the ultraviolet spectrum measureddirectly gavea curve superimposable with the spectrum of the aminophenolcontainedin a blank lacking only in cytochromec, or when (c) the ether extract exhibited an ultraviolet spectrumwhich wasidentical with the spectrumobservedin the ether extract of a control mixture lacking only in cytochrome c, it was concludedthat the starting material had remainedunchanged. The congruity of the spectral curvescomparedunder (6) and (c) wasusually within 12 per cent. RESULTS
Oxidation of o-Aminophenols-In preliminary experimentsthe reduction of oxidized cytochrome c in buffered solutions(E” = 0.25 volt at pH 7 (25, 26)) by a number of amino- or amidophenolswas tested (Table I). o-Aminophenolitself as well as the o-aminophenolanaloguesof the naphthaleneand fluorene seriesweregoodreducingagents,while the other isomericaminophenols,with the exception of 4-amino-l-naphthol, did not reduce cytochrome c under these conditions. The N-acetyl derivative of OFA likewisedid not reduce cytochrome c. Under the experimentalconditionsdescribedin Table I, microsomalcytochromewasnot reducedby OFA. Theseobservationsstrongly suggestedthat certain o-aminophenolsshouldbe oxidizable by the cytochrome c-cytochrome oxidasesystem, and the experimentswhich follow were carried out to obtain experimentalsupport for this view.
TABLE I Reduction of jerricytochrome c by amino-and amidophenols The test system contained 1.0 ml. of 0.1 M phosphate buffer of c, 0.057 The diacetyl derivative, preparedby heatingthe product in acetic pH 7.4, 0.057 pmole (or 0.114 pmole) of ferricytochrome anhydride for 1 hour, waspurified by chromatography and re- pmole of amino- or amidophenol,and water to give a volume of by crystallized from chloroform-petroleumether (30-60”) to give 3.0 ml. The reduction was measured spectrophotometrically observing the appearance of the reduced band of cytochrome c at violet needles,m.p. 236-238”, ~Et2.l~507rnp (E, 4,980), x$,:: 232, 303, 487rnp (6, 63,000;20,000;4,300). The infrared spec- 551 rnF; f indicates that cytochrome c was reduced,but spon-
Calculated: C 76.4, H 4.49, N 8.91 Found: C 76.3, H 4.47, N 8.74
trum (Nujol) showedbandsat 5.65p and at 5.85p, corresponding taneously reoxidized after 15minutes. to a phenolicacetate band and the amideI band (24). Compound 2-Amino-l-fluorenol . 2-Amino-5-fluorenol . 2-Amino-‘l-fluorenol . .. The various incubation systemsare describedunder the ap- N-(I-hydroxy-2-fluorenyl)acetamide.. propriate figures or tables. In experimentswhere the soluble 1-Amino-2-naphthol. preparation of cytochrome oxidase was employed, the experi- 2-Amino-1-naphthol. 4-Amino-1-naphthol. . mentalprocedurewasas follows: 0.29 pmoleof the aminophenol o-Aminophenol. .. or the amidophenolin 250~1.of ethanol wasaddedto a mixture 3-Hydroxy-4-aminobiphenyl . .
Calculated: C 72.4,H 4.55,N 7.03 Found: C 72.2, H 4.70, N 6.95
of 2.0 ml. of 0.1 M phosphatebuffer of pH 7.4, 0.029 pmole of
..
. ... . . ... . . ... . .. . .. ... . . . . .... . . . . . . . .. . . .
Reduction
H. T. Nagasawa
June 1959 TABLE
Oxidation
of
OFA by cytochrome from rat kidney
and H. R. Gutmann
II
1.0, c and cortex
time
AAm
min.
-
1 60
0.5
0”
0.4
5 9
0.3
2 Q
0.2
0. I
Disappearance of aminofluorenol %
0.040 0.355
0.8 0.7 0.6
mitochondria
Conditions: OFA (0.14 rmole), cytochrome c (0.029 pmole), and rat kidney mitochondria (210 pg. of nitrogen) in borate buffer of pH 7.3, to give a volume of 6.0 ml. (final borate concentration = 0.0035 M). The blank contained no cytochrome c. After incubation at 37”, 5.0 ml. of 95 per cent ethanol were added to precipitate the proteins, and the absorbancy due to OFA in the supernatant liquid was measured at 280 mp. AA280 is the difference in the absorbancies of the deproteinized reaction mixtures with and without cytochrome c. Incubation
1595
J 350
370
400
1 95
When mitochondria from rat kidney cortex were added to catalytic amounts of cytochrome c, 95 per cent of OFA was oxidized in 1 hour, as measured spectrophotometrically by the disappearance of the aminofluorenol band at 280 rnp (Table II). The oxidation was dependent on the addition of catalytic quantities of cytochrome c, since no oxidation was detectable in its absence. On increasing the concentration of the aminofluorenol Z- to 3fold, a violet oxidation product was obtained as the incubation progressed. This product which was not formed when cytochrome c was omitted from the system, was readily extractable into chloroform or ethyl acetate, and the visible spectrum exhibited a sharp absorption maximum at 585 rnp and a broad maximum at 355 rnp (Curve A, Fig. 1). Substitution of rat liver mitochondria for kidney mitochondria gave qualitatively similar results. Incubation of liver or kidney mitochondria with o-aminophenol and 3-hydroxy-4-aminobiphenyl gave oxidation products which absorbed maximally at 425 rnp and 413 rnp, respectively (Curves B and C, Fig. 1). No oxidation was detectable in the absence of cytochrome c. Cytochrome c and the soluble cytochrome oxidase (3) oxidized OFA, o-aminophenol, and 3-hydroxy-4-aminobiphenyl to products with spectral characteristics identical with those shown in Fig. 1. These results indicated that the oxidations observed in the presence of mitochondria were probably due to the mitochondrial cytochrome oxidase acting in conjunction with cytochrome c. Since the purified cytochrome oxidase has the advantage of yielding optically clear solutions on which spectrophotometric measurements can be made directly, all subsequent experiments were performed with this preparation. Under these conditions, 2-amino-5-fluorenol and N-(1-hydroxy-2-fluorenyl)acetamide gave no colored oxidation products, and their ultraviolet spectra remained unaltered. 2-Amino-7-fluorenol slowly underwent a spectral change on prolonged incubation with the complete enzyme system (Fig. 2). The slow rate of this reaction when compared with the rapid oxidation of OFA suggested that the former might have been a hydrogen peroxide-linked oxidation rather than a direct oxidation by cytochrome c and cytochrome oxidase. Hydrogen peroxide-initiated oxidations in systems containing cytochrome c, cytochrome oxidase, and ascorbic acid have been observed (27). However, addition of 25 ~1. of a commercial preparation of catalase to the system did not inhibit
450
Wavslenqth
500
550
600
700
, m,u
FIG. 1. The products of the oxidation of o-aminophenols by cytochrome c and kidney mitochondria. Conditions as described in Table II with the following exceptions: 0.29 pmole of the o-aminophenol and 0.029 pmole of cytochrome c were incubated with kidney mitochondria equivalent to 210 pg. of nitrogen in a final volume of 3.9 ml.; incubation time, 1 hour. The visible spectra of the chloroform extracts (3.0 ml.) are shown. Curve A, oxidation product of OFA; Curve B, of o-aminophenol; Curve C, of 3-hydroxy-4-aminobiphenyl.
0.8
0.7 06 0.4 u’ 5 z, zi * 2
0.3 0.2
0.1
I 250
I 260
I 270
Wavelcnqth
/ III 280 290 300310320 , m,u
FIG. 2. Spectral changes during the incubation of 2-amino-7fluorenol with cytochrome c-cytochrome oxidase. Curve A, spectrum of 2-amino-7-fluorenol; Curve B, spectrum after incubation with the enzyme system for 30 minutes; Curve C, spectrum after 2 hours incubation. the oxidation. It is of interest that in the mitochondrial system described in Table II, 2-amino-7-fluorenol likewise disappeared from the supernatant solution. The rate of oxidation of OFA as measured by the color developed during the oxidation was dependent on the enzyme concentration (Fig. 3), and at the two lower concentrations, was, as a first approximation, directly proportional to the quantity of the enzyme. The larger deviations from proportionality at the higher protein concentration may be attributed to binding of an oxidized intermediate to the protein (see below) to give in effect a reduced color yield. These kinetic measurements could not be pursued further since increase in the concentration of cytochrome c interfered with the direct spectrophotometry at 570 rnp, the
o-Aminophenol Oxidation by Cytochrome c-Cytochrome Oxidase. I
1596 0.70 0.60 0.50 2 0.40 2 Q
0.30 0.20 0.10
2
0
4
6
8 t
( Timo
IO
12
14
16
18
i
in Minutes.1
FIG. 3. The rate of oxidation of OFA with increasing cytochrome oxidase concentration. The reaction mixtures contained 2.1 ml. of 0.1 M phosphate buffer of pH 7.4, 0.029 pmole of cytochrome c, variable amounts of cytochrome oxidase expressed as pg. of nitrogen, and 0.29 rmole of OFA in 250 ~1. of ethanol; total volume, 2.45 ml. A&70 = (At - Ao) at 570 mp, where At is the total absorption at time t. Since increasing the amount of cytochrome oxidase gave increasing absorbancies at 570 rnp, this value was determined before addition of OFA and corrected for the volume change on addition of the aminofluorenol (A,,). 1.0 t': 0:G 0.5 3 u 5
ical oxidation of I-amino-2-naphthol and 4-amino-1-naphthol (1-imino-1 ,2-naphthoquinone and 1,4-naphthoquinone imine (20)) as well as their hydrolysis products (1,2-naphthoquinone and 1,4-naphthoquinone) have well defined spectral characteristics (28) and the spectral properties of the oxidation product of 2-amino-1-naphthol have been described in the present work. However, none of these products were detected when the aminonaphthols were incubated with cytochrome c-cytochrome oxidase. The possibility of inhibition of the enzyme system by reaction products or by impurities in the aminonaphthols was ruled out since addition of OFA to the cytochrome c-cytochrome oxidase system after preincubation with 4-amino-1-naphthol for 30 minutes gave rise to the characteristic spectrum of oxidized OFA (Fig. 1). This apparently anomalous behavior of the aminonaphthols suggests the possibility that cytochrome oxidase may be reversing the oxidative process by transferring electrons from cytochrome oxidase to the quinoneimine. This possibility is supported by the repeated observation that oxidation of 2-amino-1-naphthol by air was inhibited as much as 55 per cent in the presence of cytochrome c and cytochrome oxidase. Further experiments are required to settle this point. Although 2-amino-1-naphthol was not directly oxidized by the enzyme system oxidation of this compound did occur when equimolar quantities of 2-amino-I-naphthol and OFA were incubated simultaneously with cytochrome c and cytochrome oxidase. The spectrum of the oxidation product was identical with that of N&I (Fig. 4). It would therefore appear that the oxidation of this aminonaphthol was coupled to the oxidation of the aminofluorenol as follows:
0.4
r
OH
0.3
0
1
enzymatic
-2 2 Q
Vol. 234, No. 6
0.2
0.1
I
I
I
350
370
400
I
450
Wavclsnqth
I
I
I
I
500
550
600
700
nonenzymatic
r
0
1
0
, m,u
FIG. 4. The spectrum of synthetic N&I in chloroform (Curve A) compared with the spectrum of a chloroform extract after incubation of equimolar amounts of 2-amino-1-naphthol and OFA with cytochrome c-cytochrome oxidase (Curve B).
wave length at which the oxidation product of OFA exhibited maximal absorption.2 Speti$tity-The three isomeric aminonaphthols, 1-amino-2naphthol, 4-amino-1-naphthol, and 2-amino-l-naphthol, readily reduced cytochrome c as shown in Table I. It was therefore anticipated that addition of cytochrome oxidase would result in the formation of oxidation products in quantities sufficient for spectral characterization as was the case for o-aminophenol, 3hydroxy-4-aminobiphenyl, and OFA. The products of the chem2 In one experiment where the cytochrome c concentration was increased lo-fold, some violet color developed independently of the addition of cytochrome oxidase. This is to be expected with very large quantities of cytochrome c since 82 per cent of the added cytochrome c was in the form of ferricytochrome c.
N&I A similar coupling reaction has been observed by Butenandt et al. (29) in the oxidation of 3-hydroxykynurenine to an ommochrome dyestuff in the presence of tyrosinase. This reaction proceeds only when 3,4-dihydroxyphenylalanine is added to the system, the oxidation of 3,4-dihydroxyphenylalanine to “dopaquinone” being catalyzed by tyrosinase. “Dopaquinone” in turn oxidizes the hydroxykynurenine to the ommochrome. Protein Binding of Enzymatic Oxidation Product of OFA-If during the tyrosinase-catalyzed oxidation of catechol or homocatechol certain amines, amino acids (30), peptides (31), or proteins (32) are added, red-violet adducts instead of yellow o-qui-
June 1959
H. T. Nagasawa
nones are obtained. In order to test whether similar adducts could be formed with oxidized OFA equimolar amounts of hydroxyproline, glycylglycine, aniline, and 2amino-fluorene were added to mixtures containing OFA and cytochrome c and cytochrome oxidase. However, the spectra of the oxidation products under these conditions were in each instance indistinguishable from the blue-violet product obtained without such additions, and the time course of the oxidation was likewise unchanged. Blue-violet oxidation products were also observed on addition of a-naphthol, the coupling agent in the ‘(Nadi” reaction3 or on addition of 1-fluorenol. Mason and Peterson (32) have shown that the o-quinone which is generated in the oxidation of catechol by phenolase combines with the N-terminal proline of salmine to give a red quinoneprotein adduct. In the case of o-quinoneimides model experiments indicate that 1,2-naphthoquinone-1-benzimide, 1,2-naphthoquinone-1-acetimide, and 1,2-fluorenoquinone-2-acetimide undergo a rapid reaction with crystalline bovine serum albumin to give colored quinoneimide-protein complexes (33). The experiments which follow provide evidence that the product of the enzymatic oxidation of OFA likewise combines with crystalline bovine serum albumin. When a constant amount of OFA was oxidized enzymatically in the presence of varying quantities of bovine serum albumin, the amount of blue-violet oxidation product extractable from the incubation mixtures into chloroform diminished with increasing albumin concentration, suggesting binding of the oxidized product to albumin. Curve A, Fig. 5, depicts the relation between the amount of compound bound and the albumin concentration. Binding was defined in terms of the assumption that the amount of the dye which was extractable from mixtures containing no albumin @lo), corresponded to zero per cent protein binding. The per cent binding at albumin concentrations greater than zero was calculated
from the expression
1 - $. 100 where A ( ) is the value of the absorbancy of dye extractable at any given albumin concentration. The observed decrease in extractable dye with increasing protein concentrations might have been due to the following factors: (a) inhibition of the enzymatic oxidation in the presence of albumin, (b) binding of the violet dye by albumin, and (c) binding of a dye precursor by albumin. The first possibility was excluded by the fact that at an albumin concentration at which binding was 80 per cent complete (mole ratio of albumin/aminofluorenol = 0.25), no OFA was detectable in the supernatant liquid of the incubation mixtures by ultraviolet spectrophotometry, whereas 88 to 90 per cent of the oxidizable substrate was recoverable when cytochrome c was omitted. The oxidation was therefore not inhibited and the disappearance of OFA was caused by interaction of the oxidized substrate with albumin. When increasing quantities of albumin were added to mixtures after substrate oxidation had gone to completion (30 minutes), the resulting binding of the dye is shown in Curve B, Fig. 5. The extent of dye binding was independent of the time of incubation (1, 10, or 30 minutes) and this, together with the fact that the proteins retained a blue color after extraction of excess dye, suggests that the dye itself was bound without requiring further
3 p-Aminodimethylaniline + or-naphthol -----+(0) indophenol blue. In the present system, OFA may be considered as the entity which corresponds to p-aminodimethylaniline.
and H. R. Gutmann
0
.25
.50 (Albumin (OFAl
.75
1.0
)
FIG. 5. Binding of oxidized OFA to crystalline bovine serum albumin. Curve A, albumin present during enzymatic oxidation; Curve B, albumin added a&r oxidation was complete. The experimental details are described in the text.
transformation. Thus, the second possibility mentioned above may contribute to the protein binding of oxidized OFA. Nevertheless, the binding of the violet dye accounted for only one-half of the total binding represented by Curve A. Consequently, the difference between Curves A and B must have been due to binding of a precursor of the dye. This difference probably represents a minimal value, since, at least in the initial phase of the oxidation represented by Curve A, the amount of violet dye available for binding is undoubtedly less than under the conditions of the oxidation represented by Curve B. The fact that the proteins were virtually colorless when albumin was present throughout the incubation period, further indicated that the major fraction of the bound compound consisted of a product with spectral characteristics different from that of the violet dye. The results therefore show that the dye precursor as well as the dye may interact with protein. Although the proportion in which the two compounds combine with protein cannot readily be determined, the evidence suggests that the major share of the bound material was contributed by the dye precursor. As will be shown in the companion paper (34), this dye precursor is the o-quinoneimine, 1,2-fluorenoquinone-2-imine.
The oxidation of certain o-aminophenols of the benzene, biphenyl, and fluorene series by cytochrome c and cytochrome oxidase might perhaps have been expected since quinol as well as p-phenylenediamine and its various N-alkyl derivatives are also oxidized by this enzyme system (35). It has generally been assumed that any substance which reduces ferricytochrome G can be more or less completely oxidized by the combined action of cytochrome c and cytochrome oxidase (36). The failure of cytochrome oxidase to promote the oxidation of the isomeric aminonaphthols, even though these compounds reduce ferricytochrome c readily, throws some doubt on the validity of this generalization. Whether the reason for the apparent specificity can be attributed to the transfer of electrons from cytochrome oxidase to an acceptor different from oxygen, i.e. the quinone imine, requires further study. 2-Amino-l-naphthol is oxidized by oxygen to N&I (I), an indophenol with the hydroxyl auxochrome at a position ortho to the azo nitrogen. The absorption maximum of this compound
1598
o-Aminophenol
Oxidation
by
Cytochrome
is at 547 rnp (chloroform).4 By analogy, the enzymatic oxidation product of OFA, A,,,. ‘=‘I3 585 rnp, is probably also an indophenol. Evidence in support of such a structure is presented in the companion paper (34). The stability and spectral properties of the yellow oxidation products of o-aminophenol and 3-hydroxy-4-aminobiphenyl precluded the possibility that they were monomeric o-quinoneimines, and they will be shown to be isophenoxazones (34). The question may be raised whether the dimerization proceeds by an ionic mechanism involving the o-quinoneimine or by a free radical mechanism involving semiquinones. The experimental evidence permits no clear distinction between the two mechanisms; however, the ionic mechanism accounts satisfactorily for the protein interaction in terms of established addition reactions (40). The failure of aniline, 2-aminofluorene, hydroxyproline, and glycylglycine to combine with the o-quinoneimine generated by the enzymatic oxidation of OFA is in marked contrast to the known effectiveness of such compounds in ‘%rapping” o-quinones. The absence of such an interaction is probably due to the very rapid rate of dimerization which may be looked upon as a ‘[trapby a molecule of OFA. ping” of the o-quinoneimine It has recently been reported that the implantation of certain o-aminophenols such as 3-hydroxy-4-aminobiphenyl and a-aminoI-naphthol into the bladder of the mouse gives rise locally to the formation of neoplasms (41). It has also been suggested that the o-aminophenols arising in the metabolism of aromatic amines rather than the aromatic amines themselves are the proximate carcinogenic agents (42). If protein interaction is a requirement P. M., HADLER,
I
Vol. 234, Ko. 6
for carcinogenic action as is becoming increasingly likely (43), the oxidation to o-quinoneimines, either directly by an oxidase or indirectly by a coupled oxidation, and the subsequent binding of these reactive intermediates to proteins might provide the chemical basis for the observed carcinogenic activity of certain o-aminophenols. SUMMARY
1. The direct enzymatic oxidation of certain o-aminophenols by cytochrome c and mitochondria or by cytochrome c and soluble cytochrome oxidase has been studied. o-Aminophenol, 3-hydroxy-4-aminobiphenyl, and 2-amino-l-fluorenol were rapidly oxidized by cytochrome c-cytochrome oxidase and yielded end products with characteristic spectral properties. The isomerit aminonaphthols were not oxidized by cytochrome c-cytochrome oxidase. In the presence of 2-amino-1-fluorenol, 2amino-1-naphthol yielded, through a coupled reaction, an oxidation product which was identified as the indophenol, 2amino-l ,4-naphthoquinone-N4-(1-hy&oxy-2-naphthyl)imine. 2. Data are presented which show that the indophenol derived from the enzymatic oxidation of 2-amino-1-fluorenol, as well as the precursor of this indophenol are bound to bovine serum al.. bumin. The evidence suggests that the major portion of the bound material is contributed by the indophenol precursor. 3. The suggestion is made that the oxidation of certain o-aminophenols and the binding of the resulting o-quinoneimines to cellular proteins may have a bearing on the recognized carcinogenic activity of these compounds.
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