Metabolism of 2

8/2/2019 Metabolism of 2

http://slidepdf.com/reader/full/metabolism-of-2 1/15

Vol. 47

175ETABOLISM OF 2-NAPHTHYLAMINE

SUMMARY

1. The administration of 2-naphthylamine torats, either by subcutaneous injection or by stomachtube, is followed by the excretion in the urine of 2-naphthylamine, 2-acetamido-6-hydroxynaphthalene and 2-amino -1 -naphthylsulphuric acid.

2. When rats are dosed with 2-acetamidonaph-thalene by subcutaneous injection or by stomachtube, the urine contains 2-acetamido-6-hydroxy-naphthalene. The urine of rats given 2-acetamido-naphthalene by stomach tube contains smallamounts of the unchanged compound and 2-naphthylamine, but these compounds have not beenfound in the urine of rats dosed with 2-acetamido-naphthalene by subcutaneous injection.

3. The possible steps in the metabolic conversion

of 2-naphthylamine to the derivatives excreted inthe urine are discussed.

The authors wish to thank Dr J. A. McCarter forhis assistance in the development of the methodused for the isolation of 2-amino-1-naphthylsulphuric acid. All the elementary micro-analyses were carried out by Mr Michael Edson. Anaccount of the work described in the present paperformed part of a thesis presented to the Universityof Toronto (Manson, 1947), and a preliminary reportof the investigation was presented to a meeting of the American Chemical Society in St Louis,Missouri, on 7 September 1948. One of us (L. A. M.)wishes to thank the National Research Council of Canada for the award of a Scholarship during thetenure of which the work was carried out.

REFERENCES

Bonser, G. M. (1943). J. Path. Bad. 55, 1.Dobriner, K., Hofmann, K. & Rhoads, C. P. (1941).Science, 93, 600.Elson, L. A., Goulden, F. & Warren, F. L. (1946).Biochem. J. 40, xxix.Engel, H. (1920). Zbl. OewHyg. 8, 81.Engel, H. (1924). Zbl. OewHyg. 12, 35.Grandmougin, E. (1906). Ber. dtsch. chem. Ges. 39,2494.

Greenstein, J. P. (1947). Biochemistry of Cancer. New York: Academic Press Inc.Hueper, W. C., Wiley, F. H. & Wolfe, H. D. (1938).J. indu8tr. Hyg. 20, 46. Kuchenbecker, A. (1920). Zbl . OewHyg. 8, 69.Laidlaw, J. C. (1947). Thesis, University of Toronto. Manson, L. A. (1947). Thesis,University of Toronto. Wiley, F. H. (1938). J.bid. Chem. 124, 627.Windaus, A. (1924). Ber. dtsch . chem. Oes. 57, 1731.

The Fermentation Process in Tea Manufacture11. OXIDATION OF SUBSTRATES BY TEA OXIDASE

By E. A. H. ROBERTS AND D. J. WOODTocklai Experimental Station , Indian Tea Association , Cinnamara , Assam , India

(.Received 1 February 1950)

Considerable differences in opinion are on record asto what substrates are acted upon by the teaoxidase, and also as to their relative rates of oxidation. Thus Deb & Roberts (1940) claimed that,in addition to o-dihydroxypolyphenols, p -

phenylenediamine, ascorbic acid and quinol are alsooxidized, whereas Lamb & Sreerangachar (1940a)stated that o- dihydroxyphenols only are oxidized.Li & Bonner (1947) claim to have shown thatalthough p-phenylenediamine is oxidized, theoxidation is catalysed by a water-solublethermostable system. As originally suggested byLamb & Sreerangachar (1940a, 6), it is now clearthat some of these apparent differences inspecificity are due to adsorption of substrates bycrude enzyme preparations. Elimination of suchimpurities results in the loss of the enzyme’s abilityto catalyse oxidation of quinol and ascorbic acid, but

not of p -phenylenediamine. The use of enzymes con-taining adsorbed polyphenols is also shown to affectresults obtained with catechol as a substrate.

Again, whereas Harrison & Roberts (1939), usingsubstrates at 0-01 M-concentration, showed that tea

catechins (tannins) are oxidized about three timesas rapidly as catechol, Sreerangachar’s (19436)manometric work indicated that catechol isoxidized faster than tea catechins, when bothsubstrates are at 0*05 M-concentration. On theother hand, his results (1943a) with the ascorbicacid oxidation method, indicate that catechins areoxidized faster than catechol at all concentrationsinvestigated.

In order to reconcile the above differences, adetailed investigation of substrate specificity hasbeen carried out, using several different types of enzyme preparation.



176 E. A. H. ROBERTS AND D. J. WOOD 1950 METHODS AND MATERIALS

Rates of oxidation and of carbon dioxide output Rates were determined manometrically, observingthe usual precautions (Dixon, 1934). The bathtemperature was 32°. The total volume of reactantsin each Warburg vessel was 3 ml. For continuousrecords of C02 output the direct method wasemployed, calculating the C02 from the difference in02 uptake, with and without KOH. With crudeenzyme preparations retention of C02 sometimesresulted in appreciable errors, in which case a moreaccurate value of the C02 output was obtained bythe first method of Dickens and Simer (Dixon,1934).

Enzyme preparations

Crude enzyme . Fresh tea-leaf was finely ground in

a domestic mincing machine, using an attachmentfor reducing nuts to a paste. The mince was washedseveral times with ice-cold acetone until thewashings were colourless. The resulting powder wasdried and stored in vacuo. It showed a small residualrate of uptake without added substrates.

Washed enzyme. Crude enzyme was washed withseveral changes of distilled water, until the aqueousextract no longer gave a positive test for amino-acids with ninhydrin. It was then washed withacetone, and dried and stored in vacuo. Residualrespiration was negligible.

Chloroplast suspension and acetone-dried chloroplasts. Leaf was finely ground, mixed with 10% (w/v)glucose and squeezed through cloth. Cellular debriswas removed by centrifuging for 2 min. at thelowest speed on an International Clinical

Centrifuge, after which chloroplasts were spundown at the highest speed. The chloroplasts werewashed once with 10% glucose and then suspended

in an appropriate volume of 10 % glucose.

Microscopic examina-Substrates

Polyphenols were dissolved in pH 5-6 buffer(phosphate or phthalate) in such concentration thatthe desired amount of substrate could be containedin 0-5 ml. Gallic, protocatechuic and ascorbic acids,and ^-phenylenediamine hydrochloride wereneutralized with NaOH before adding buffer.

Adrenaline was first dissolved in the calculatedquantity of HC1.

Tea catechins were prepared as described byHarrison & Roberts (1939), except that the greenleaf infusion was given a preliminary extractionwith CHC13, before treating with ethyl acetate. Thepreparation obtained probably contains all thesubstances referred to by Bradfield (1946). A two-way paper chromatogram indicates that the mixture

may contain as many as thirteen components(unpublished observations).

Merck’s catechin (from Catechu) has beenemployed as a substrate. On a paper chromatogramit is indistinguishable from one of the components of tea catechin, and gives a green spot (R F 0-57 withphenol at 30°) when sprayed with FeCl3.

RESULTS Oxidation of catechol Apparent evolution of carbon dioxide . In the oxidation

of catechol (15 mg.) by crude enzyme the rate of 0 2 uptake falls off with time, and the C02 output,determined by the direct method, is surprisinglyhigh, the R.Q. after 1 hr. amounting to 0*40.Results in Table 1 show this to be due to twofactors, retention of C02 by the enzyme, and

impurities in the enzyme, removed by waterwashing. Employing the

Table 1. Effect of type of enzyme and technique on carbon dioxide output in enzymic oxidation of catechol

(40 mg. enzyme +15 mg. catechol in pH 5*6 buffer.)

tion showed the preparation to consist mainly of intact and broken chloroplasts, together with somelarger fragments.

To obtain an acetone-dried preparation thechloroplast suspension was spun down, washed withwater, and then treated with ice-cold acetone. Theresultant granular powder was dried in vacuo. Inactivation was appreciable, varying from 50 to 90% on occasions.

‘ Water-soluble enzyme .’ Leaf was finely ground,

mixed with a small quantity of 10% glucose, andsqueezed through cloth. The filtered juice was thencentrifuged at a high speed for 30 min. and thesupernatant separated.Dickens and Simer technique the observed R.Q. ismuch lower (0-12), and exhaustive washing of theenzyme reduces the C02 output to a value not appre-ciably greater than zero. An explanation of theorigin of this COa is given later in this paper.

Form of oxygen-uptake curve . Whenever catechol is

Crude enzyme------------------------ A ---------- — --------------- N Washed enzymeDirect method Dickens & Simer method Dickens & Simer method

Time(min.)

{fj\. 02) (f-1. C02) (/U. 02) (#a. C02) (fj. O.) A (jd. co2)

10 44 22 42 — 62 — 20 74 28 91 — 130 — 30 100 42 145 — 198 —

45 128 50 232 — 296 — 60 158 61 326 39 386 3



Vol. 47 TEA OXIDASE SUBSTRATES 177

oxidized at pH 5*4-5* 8 by the washed enzyme, and

in most cases when a chloroplast suspension oracetone- treated chloroplast material is the enzymesource, the 02 uptake curve is initially autocatalytic(Table 2).



178 E. A. H. ROBERTS AND D. J. WOOD 1950

Time(min.)

(3) A (11)

(15) Chloroplasts 2 ml.+ catechol (15 mg.)

chloroplasts (10mg.) + catechol (15

10 18 30 66 66 4320 43 73 144 159 10030 69 126 258 254 16440 100 184 356 349 234

r '1—

1—---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Washed enzyme (40 mg.) + catechol (mg.) Acetone-dried

Table 2. Autocatalytic oxygen uptake with catechol as substrate

02 uptake (/¿I.) ______________ _______________ A______________ k

The autocatalytic nature of catechol oxidation bythe tea oxidase has already been reported byRoberts & Wood (1950), and is also evident in thecase of Ceylon leaf (see Sreerangachar’s diagrams,

19436).When other oxidizable substances are present theeffect may be obscured for reasons which will bedealt with later. This probably accounts for the factthat rates with crude enzyme preparations, whichcontain adsorbed polyphenols, are not always auto-catalytic. The failure to observe the effect in thecase of the Dickens and Simer experiment in Table1 is due to the rate of uptake having reached alimiting value for the type of vessel used.

After the initial autocatalytic phase the rate of uptake slows down, and because of the low rates of oxidation at low substrate concentrations completeoxidation of catechol takes a considerable time. Inone experiment oxidation of catechol (2*2 mg.) bywashed enzyme (40 mg.) resulted in an uptake of 409 ¡A. (1*83 atoms) Oa in 320 min. Given a longerperiod of shaking the uptake would probably haveapproximated closer to the 2 atoms 02/mol. found byLudwig & Nelson (1939) for tyrosinase.

This contrasts with the consumption of 1 atom02/mol. reported by Harrison & Roberts (1939),when catechol (1-1 mg.) was added to a tea mince.

As shown later, there are complications with mixedsubstrates which, combined with the low rates of uptake with 1*1 mg. catechol, particularly in thelater stages, may lead to premature conclusionsthat oxidation is complete.

Our earlier work (Roberts, 1939, 1940a) indicatedthat the first product of oxidation of catechol is o-benzoquinone, and this was confirmed by Lamb &Sreerangachar (1940a), who isolated the corre-sponding anilino-quinone. The uptake of a secondatom of 02 is not due to autoxidation of the o-quinone, as Wagreich & Nelson (1938) have shownthat o-benzoquinone only consumes 02 in thepresence of an oxidase such as tyrosinase. Theseauthors consider it likely that some catechol isregenerated as a consequence of the disappearanceof the o-quinone in aqueous solution according tothe scheme

2 o-benzoquinone + H20 —► p-hydroxy-o-quinone+ catechol,

Biochem. 1950, 47

but this view can neither be reconciled with theautocatalytic nature of the uptake curve with thetea oxidase, nor with the finding that addition of ascorbic acid (3 mg.) initially depresses the rate of

uptake, as reported by Roberts & Wood (1950). Thisdepression of the rate of uptake by ascorbic acid istemporary, for as soon as the ascorbic acid isoxidized (190 fil. 02) the rate of uptake becomesautocatalytic. Our belief is that o-benzoquinonereacts, without the uptake of 02, to form apolyphenol more rapidly oxidized by the tea oxidasethan catechol, although the polyphenol producedcannot, at present, be identified. The failure toobserve similar phenomena with tyrosinase isunderstandable if tyrosinase oxidizes catechol morerapidly than the new polyphenol.

There is little destruction of enzyme apparent asa result of catechol oxidation. This is in contrast tomushroom tyrosinase, and to the leaf oxidases of tobacco (unpublished observations) and Atropa

12

Substrate (mg./3ml.)Fig. 1. Variation in initial rate of oxidation with

substrate concentration. Curve 1: mixed teacatechins + 3 mg ascorbic acid. Curve 2: Merck’scatechin + 3 mg. ascorbic acid. Curve 3: pyrogallol +3 mg. ascorbic acid. Curve 4: catechol + 3 mg.ascorbic acid. Curve 5: p-phenylenediamine. Curve6: gallic acid + 3 mg. ascorbic acid. pH 5*6 and 40mg. washed enzyme in all cases.




(50 mg. washed enzyme, pH 5-6.)

belladonna (James, Roberts, Beevers & deKock,

1948), where enzyme inactivation in the presence of catechol is appreciable.Effect of substrate concentration. The effect of sub-

strate concentration is shown in Fig. 1. A trueestimate of the rate of oxidation of catechol is onlyobtained when ascorbic acid (3 mg.) is added to thesystem, as the o-benzoquinone is then reduced im-mediately on formation, and there is nocomplication due to the subsequent stages of catechol oxidation. The approximately linear rate of uptake for the first 190 fA. 02 gives the rate of oxidation of catechol at the particular concentrationunder study. After an uptake of 190^1. the ratealways becomes autocatalytic. .

The initial rates increase with substrate concen-

tration, with K m about 0*025m and an optimal con-centration of about 0*1 2 M. Sreerangachar (1943 a)also determined the optimal substrateconcentration for catechol by estimating theresidual ascorbic acid after aeration of a suspensionof acetone-washed leaf powder (20 mg.) in water (10ml.), containing ascorbic acid (5 mg.) and varyingquantities of catechol. His optimal concentration forcatechol was 25mg./10ml. (0*023m). In thistechnique diffusion of Oa may become a limitingfactor when the amount of catechol exceeds 25 mg.

Oxidation of pyrogallol

Table 3 illustrates the oxidation of pyrogallol (10mg.) at pH 5*6. The B.Q. in various experiments

ranged from 0*48 to 0*57. Approximately completeoxidation of pyrogallol within a reasonable time isonly obtained at low substrate concentrations. With2*5 mg. substrate an uptake of 488 /¿I. Oa (2*20atoms 02/mol.) was measured after shaking for 7 hr.with washed enzyme (60 mg.) at pH 5*6. Duringthis period an output of 234 /xl. COa was also

observed.

If the contents of the Warburg vessels, after such

a period of shaking, are extracted with ether, the

extract is a bright yellow and presumably containspurpurogallin. The 02 uptake and C02 outputfigures quoted above, however, indicate thatoxidation does not stop short at the purpurogallinstage.

The uptake of 02 is initially almost linear, but therate decreases as substrate is consumed. There is noautocatalytic phase and addition of ascorbic acidhas no inhibitory effect, but merely prolongs thelinear phase and initially depresses C02 output(Table 3).

The effect of the concentration of pyrogallol, andother substrates, in the presence of ascorbic acid isshown in Fig. 1. K m is about 0*015M with optimalrates at about 0*1M. Oxidation of pyrogallol is

always faster, at equivalent concentrations, thanthat of catechol.

It is difficult to reconcile these findings with thoseof Sreerangachar (19436), who showed that at 0*05

M concentration catechol is oxidized faster thanpyrogallol unless the potassium hydroxide wasomitted from the inner cups of his Warburg vessels,as has admittedly been done in some of his experi-ments. In this case the C02 liberated from pyro-gallol would give fictitiously low values for the rateof oxidation.

Oxidation of gaUic acid As shown by Harrison & Roberts (1939) oxidation

of gallic acid is slow (cf. Fig. 1). With gallic acid (20mg.) and washed enzyme (50 mg.) 02 uptake and C02

output totalled 40 and 33 /xl., respectively, after 1hr.

Other simple phenols Adrenaline is only oxidized at about one-quarter

the rate at which catechol is oxidized, both sub-strates being studied at 0*033m concentration.Phloroglucinol is hardly oxidized at all, in contrastto its rapid oxidation by the belladonna oxidase(James et al . 1948). Tyrosine (0*01 and 0*025M) hasno significant effect, either on initial rate or total 02 uptake, when added to a tea-leaf mince or a systemcontaining washed enzyme (40 mg.) and tea cate-chins (10 mg.). It follows that tyrosine is neitheroxidized directly by the tea oxidase nor by the o-quinones produced in tea fermentation. The above

observations confirm earlier statements made byLamb & Sreerangachar (1940a) and Li & Bonner(1947).

Tea catechins Production of carbon dioxide. Earlier work (Roberts,

1940a; Lamb & Sreerangachar, 1940a) is inagreement that the primary product of oxidation of a tea catechin is an o-quinone. Evidence has alreadybeen presented (Harrison & Roberts, 1939; Barua &Roberts, 1940; Roberts, 1949) that the primaryoxidation products undergo a condensation -polymerization, and this view is accepted by otherworkers in the field.

Tim

(id. 02) A t A (min.) (*a. co2) (td. ( fd. C02)

10 105 50 117 520 220 118 247 4130 320 165 358 9460 568 292 653 25090 760 394 886 375120 925 481 1078 472

Table 3. Effect of addition of ascorbic acid on enzymic oxidation of pyrogallol

Pyrogallol 10 mg. Pyrogallol 10 mg.+ ascorbic acid (3 mg.)




It has been further suggested (Roberts & Sarnia,

1940; Roberts, 19416) that the o-quinones mayfunction as hydrogen acceptors and bring aboutsecondary oxidation of carbohydrates, and possibly




After uptake of 300/J.02

of amino-acids. The 02 uptake and C02 output of

fermenting tea-leaf could be accounted for quanti-tatively on the assumptions: (1) that the catechinsin green leaf are a mixture of epicatechin and gallo-catechin (mean mol.wt. 300) or their low polymers;(2) that no oxidation of catechins takes place beyondthe quinone stage; and (3) that the C02 is formed asa result of glucose oxidation.

Bradfield (1946), Bradfield, Penney & Wright(1947) , and Bradfield & Penney (1948) have estab-lished the main component of the tea catechins to begallo-catechin gallate (mol.wt. 458), and othergalloyl esters also occur. The average molecularweight of the catechins is therefore greater than400, so that the above calculations are invalidated.

This led us to reconsider the origin of the C0 2 in

fermentation. Contrary to Sreerangachar’s (1941)findings, appreciable quantities of C02 had been ob-served during oxidation of tea catechin by a crudeenzyme preparation (unpublished observation), butthis had been ascribed to associated impurities inboth enzyme and substrate. The fact that C02 isamongst the oxidation products of both pyrogalloland gallic acid suggested, however, that C02 mightalso be formed in the oxidation of tea catechins.

Using a washed enzyme and the Dickens andSimer technique, R.Q.’S varying from 0*33 to 0*46were obtained with 10 mg. tea catechin assubstrate, while with 15 mg. catechol the R.Q.varied from 0-00 to 0*04. The C02 must come,therefore, from the tea catechins or from some

associated impurity. As identical R.Q.’S are obtainedwith green-leaf infusions and tea-catechinpreparations (to be reported in a latercommunication) the COa presumably comes fromthe catechins.

Table 4 records the effect of variation of substrateconcentration on the R.Q. That the R.Q. decreaseswith increasing substrate concentration is apparentwhen taking total uptakes into consideration, but itis even more noticeable in the early stages. (Thetotal uptake with 60 mg. substrate after shaking for9 hr. does not represent complete oxidation of thecatechins owing to extensive enzymic inactivation.)

R.Q. of the order 0-1. His quoted manometric figures

are somewhat irregular, and, with an R.Q. of 0*1,differences in Oa uptake with and withoutpotassium hydroxide, would fall within the limits of his experimental error.

Form of oxygen-uptake curve . 02 uptake initiallyfollows an approximately linear course with someslight falling off, due to enzymic inactivation (Fig.2).

This inactivation is more evident at high substrateconcentrations as shown in Table 4. The uptakewith 60 mg. substrate almost ceases with about 40% of the substrate unoxidized. With 30 mg.substrate, or less, an approximately linear course ismaintained until the unoxidized substrateconcentration has fallen to about 3 mg./3 ml. (seealso Fig. 1). Earlier work (Roberts, 19406) alsoindicated a concentration of substrate of about thisorder was required to saturate the enzyme. The K m is probably less than

Sreerangachar’s (1941) failure to observe C02 out-put in tea-catechin oxidation now becomes under-standable. He employed 2 ml. of a 2*8% solution (56mg.) and restricted measurements to the first 250-350 /¿l. Oa, for which our results indicate an 0*001

M which is much lower than for catechol or pyrogallol. Sreerangachar (1943 a) concluded that the optimalconcentration of tea catechins is 20 mg./10 ml. Hismixture of catechins and ascorbic acid, however,

12-2

Time (min.)Fig. 2. Effect of added ascorbic acid on oxidation of tea catechins. Curves 1 and 2: 0a uptake and C02 output with 10 mg. tea catechin and 40 mg. washedenzyme, pH 5-6. Curves 3 and 4 : as curves 1 and 2,

together with 3 mg. ascorbic acid.

(mg.) (/xl. 02) (Ml- COa) (R.Q.) (#a- co2) (R.Q.)10 346 152 0-44 116 0-3920 759 270 0-36 64 0-2140 1348 432 0-32 45 01560 1216 316 0-26 33 011

Table 4. Effect of tea catechin concentration on R.Q.

(40 mg. washed enzyme + tea catechins; total vol. 3 ml.,pH 5*6.)

Teacatechins

After 9 hr.shaking




contained 5 mg. ascorbic acid only, and his results,

with 20 mg. tea catechins, show that 5*02 mg.ascorbic acid have been oxidized. The results onlyshow, therefore, that the rate of oxidation increaseswith substrate concentration up to 20 mg./10 ml.,and no conclusions can be drawn as to the oxidationof the substrate at higher concentrations, apartfrom the objections raised to this method in anearlier part of this communication.

Like pyrogallol the rate is unaffected by additionof ascorbic acid (cf. Fig. 2). The effect, as with teafermentation (cf. Roberts, 1939) is to prolong theinitial linear portion of the uptake curve by anamount equivalent to the added ascorbic acid.

Merck's catechin Only negligible amounts of C02 are produced

during the oxidation of this catechin by the washedenzyme. The rate of uptake is initially linear, with asmooth decrease as the concentration of unoxidizedsubstrate decreases (cf. Fig. 6). Fig. 1 shows thatthe enzyme is saturated with its substrate at 10mg./3 ml. concentration, but under optimal conditionsrate of oxidation is equal to that with mixed teacatechins. K m is about 0-002m. The total 02 uptakefor complete oxidation amounts to 450 /zl. for 10mg. catechin, which is rather more than required(381 ¡A.) if each molecule took up 1 atom 02.

Ascorbic acid , quinol and p -phenylenediamine Appreciable oxidation of ascorbic acid and quinol

is only observed with a crude enzyme preparation(cf. Deb & Roberts, 1940). With a washed enzymethere is practically no oxidation of ascorbic acid,and only slight activity towards quinol, but p- phenylenediamine continues to be oxidized to anappreciable extent (Table 5). As the washed enzymehas no self-respiration and promotes little or nooxidation of ascorbic acid, it must be almost freefrom adsorbed polyphenols. The oxidation of p- phenylenediamine, which is considerably morerapid than the control without enzyme, has beenobserved with several different enzymepreparations and must therefore be considered to becatalysed by the tea oxidase.

The rate of enzymic oxidation of p -phenylenedi -amine increases rapidly with substrate concentra-tion, but a limit is put to this increase by its solu-bility in water. At 40 mg./3 ml. the rate of uptake is

nearly half that observed with catechins at optimal

concentration (Fig. 1).Li & Bonner (1947) noted some oxidation of p-

phenylenediamine by chloroplast preparations, butclaimed to have established more rapid oxidation bya water-soluble thermostable catalyst. Our water-soluble enzyme is considered to be equivalent totheir soluble enzyme. It contains more than

sufficient tea catechins to saturate the enzyme sothat self-respiration is high, which incidentally con-tradicts their finding that oxidation of catechol is

slow. Addition of p-phenylenediamine (15 mg.) de-presses the rate of uptake (Fig. 3) so that we fail toconfirm that the water-soluble enzyme oxidizes p- phenylenediamine more rapidly than catechol. Thedepression in uptake is not inconsistent with en-zymic oxidation of p -phenylenediamine as the datain Table 6 show a mixed substrate effect with p- phenylenediamine and catechins. A mixture withcatechin is oxidized at a rate intermediate betweenthat with the two substrates taken singly, so thataddition of p -phenylenediamine to the enzymesaturated with tea catechins would be expected todepress the rate of uptake.

We find that boiling the soluble enzyme com-pletely inactivates it, and the rate of uptake with

added p -phenylenediamine is then only what is tobe

Fig. 3. Oxidation of ^-phenylenediamine by water-soluble enzyme. Curve 1: 1 ml. water-solubleenzyme diluted to 3 ml. Curve 2: as curve 1 with 15mg. p-phenylenediamine hydrochloride neutralizedto pH 5*4. Curve 3:1 ml. boiled water-solubleenzyme^ Curve 4: as curve 3 with 15 mg. ^-phenylenediamine hydrochloride neutralized to pH5-4.

Table 5. Oxidation of ascorbic acid , quinol and p- phenylenediamine (PPD) (40 mg.

washed enzyme; total vol. 3 ml. at pH 5-6.)Uptakes (/xl. 02)

Time Residual PPD PPD (15 mg.) Quinol Ascorbic acid(min.) respiration (15 mg.) no enzyme (15 mg.) (3 mg.)20 Nil 38 4 10 340 Nil 79 7 26 660 Nil 110 9 33 10




++e«oo+ — H — _ O I 00 CO © <N 00 <30

CO io co co co >-< oo »oos CO 1 (N CO CO <N CO <N

«i +

I T* I , CnTÍh" r-I ÎO r-H CO 00 1-H »2 05

1lO

1I> CO 05 CO 05

I

rOS3 OQ n

zi

£

SI2d''

V-HH

2 .a i

-I4 I§12 ¡Ho ------

%

i ► I* H tiit2 o OD S

I'isH —

> O <N<N < )<Neoo>5 H <N

• 3 -q' hH

oq 48 £

.3 rO

0 GQ

*3 eS c8

oo +-10O

fi13

I I 1 co © 1

30

a •e

H0 — O

M Z $ S

t .- J ‘^1 O eg ®■£HP

lOCOO- 05 COCO CO

I a co

a

lO <NGSIC

II SI 58

0 GQ

© o

Fig. 4. Effect of addition of gallic acid to the enzymicoxidation of catechol. Uptakes are recorded forsuccessive 10 min. intervals. Curve 1: 02 uptake with40 mg. washed enzyme+ 11 mg. catechol at pH 5-6.Curve 2: as curve 1 together with 1*7 mg. gallic acid.Curve 3: as curve 2, C02 output.

the observed rate of uptake will be diminished.Support for this view is afforded by the finding that inthe oxidation of catechol, mixed with a small amountonly of gallic acid, the rate of uptake accelerates aftercomplete oxidation of the gallic acid (Fig. 4). This effect

is analogous to that observed when ascorbic acid isadded to catechol.

As the initial rate of oxidation of a mixture of polyphenols is the same as the rate with the morerapidly oxidized polyphenol, it seems logical to con-clude that in these initial stages the more rapidlyoxidized substrate is the only one to be oxidized. Thisimplies successive rather than simultaneous oxidationof mixed substrates, although it must be emphasizedthat we believe there is a considerable overlap in suchcases. Evidence for the preferential oxidation of onesubstrate in the presence of another

expected from autoxidation (Fig. 3). Our findings inthis direction, therefore, are completely at variancewith those of Li & Bonner (1947).

Mixed substrate tests Initial rates of reaction. Whenever one or both sub-

strates are at optimal concentration (except p~ phenylenediamine) addition of the more slowlyoxidized substrate has no depressant effect upon therate of uptake, as can be seen by comparing results incolumns 3, 8 and 11 of Table 6. The effect is clearestwhen gallic acid or rutin is the more slowly oxidizedsubstrate, for here the difference in rate of oxidation of the two substrates is greatest. There is an apparentdepression in the rate of uptake, when gallic acid isadded to catechol, but this is probably due to an

oxidation of gallic acid by o-benzoquinone. If o-benzoquinone is reduced in this way, the second stageof catechol oxidation will not take place, and

60 90 Time(min.)

.E o

% 40

120

20

a_ CO !>■

i ¿b a.

1 E3 m

OiOOiOOOOOiOOQiO HHHHHHCOHTH

•S * * -g

2 2 ^ eö Cöj® eéo 'o 8 .2 o o ^ .o ^ o t í

ÜÜ

.23ja^-s<N<N , Tf (M (N (M00 O io ~~~-CO «O |> T*C0C0C0C0<N05©

g I« —

WS+- _

>-H CO05 O5 CO COlO

CO i — i 1 1 ^ ^ i 00 i 00 t*» CO i co <

<N l> 00 <M iO 05 -1 t* 1

CO »O CO 1 CO '

VO lO jÑOÑ^ÑJOO|

(NTJIOCOOÖ5 05 CO CO CO I US <N ©

J II

A eS

£ ü 2 «3% ¿I

H —

00 , lOOCOTfO i ©^ CO rH TP CO

I HÍIHCO g I 05 <N 00CO

_ l COíO» — t<N-Ô5l>00C<I'^'^CO




is provided by the colours produced in the oxidationof mixtures of catechol and tea catechins. Theoxidation products of tea catechins are a brightorange- brown in colour, quite distinct from thoseof catechol. With the mixed substrates, the finalcolour is a dull brown, but in the initial stages,where the rate of uptake is the same as with teacatechin alone, the colour of the mixture isindistinguishable from the bright orange-browncharacteristic of tea catechins alone, which wouldappear to show that tea catechins only are oxidizedduring this period.

Secondary oxidations . With mixed substrates the o-quinone produced by enzymic oxidation of onesubstrate may oxidize the second substrate. This isoften indicated by a marked increase in the outputof C02 (cf. columns 5, 10 and 13 in Table 6). Suchsecondary interactions are non-enzymic and areprobably largely determined by the oxidation-reduction potentials of the substances involved.Thus, although pyrogallol is oxidized more rapidlythan catechol, the latter has the higher E '0, andfurther oxidation of pyrogallol oxidation productsby o- benzoquinone is considered more likely thansecondary oxidation of catechol oxidation productsby the o-quinone of pyrogallol.

On adding catechol (2-2 mg.) to pyrogallol (2-5mg.) after oxidation of the latter substrate hasproceeded almost to completion both Oa uptake andC02 output are stimulated (Fig. 5). The total C02

output is approximately doubled, and as catecholgives no C02 on oxidation, the extra C02 must haveoriginated in secondary oxidation of pyrogallol oxi-dation products. If catechol is added at the sametime as the pyrogallol the C02 output is againapproximately doubled (Table 6 and Fig. 5). Thismight be interpreted by assuming, with Willstatter& Heiss (1923), that catechol can be substituted for

OH( , ) 2

O ™ + i t o '

OH OHOH , "'''«OH

0H+O +pyrogallol in purpurogallin

formation, so that all the pyrogallol moleculeseliminate C02, instead of only half as whenpyrogallol alone is oxidized. The structure forpurpurogallin is that suggested by Barltrop &Nicholson (1948). The position of the hydroxylgroups in the analogue to purpurogallin (reaction2) is assumed.

This interpretation cannot apply when catechol is

added subsequently to the complete oxidation of pyrogallol. It is therefore clear that o-benzoquinone

can oxidize pyrogallol oxidation products a stagefurther, with the total evolution of approximately1 mol. C02/mol. pyrogallol. In further confirmation

HO I I

— H 0 c c r

of this, secondary oxidation of purpurogallin by o-benzoquinone is shown in Table 7 where additionof catechol to purpurogallin definitely stimulatesC02 output. The nature of such oxidationproducts of pyrogallol and purpurogallin isunknown.The above results were all obtained by the directmethod of Warburg, using washed enzymeprepara

O

Time (hr.)Fig. 5. Oxidation of a mixture of catechol andpyrogallol. Curves 1 and 2: 02 uptakes and C02 outputs for a mixture of 2-2 mg. catechol, 2-5 mg.pyrogallol, and 40 mg. washed enzyme at pH 5-6.Curves 3 and 4: as curves 1 and 2 but catecholadded after 4 hr. shaking.

O ----- H




SubstratePyrogallol Teacatechins Gallicacid Gallic acidProtocatechuicacidPurpurogallint

tions which give negligible COa outputs withcatechol as substrate. Table 7 also summarizesthe results of some experiments by the method of Dickens and Simer, where addition of excesscatechol to pyrogallol more than doubles C02 output.

gallol and tea catechins are approximately additivewhen these two substrates are mixed (Table 6), sothat there is no evidence of any interaction betweenthem. On the other hand, tea catechins do inducesecondary oxidation of gallic acid.

Results tabulated in Tables 6 and 7 show that C0 2 output from tea catechins is also very much in-creased by addition of catechol. The increase of COa

evolution in teafermentation whencatechol is added(Roberts, 19416) isnot, therefore,necessarily due to

increasedbreakdown of respiratory substrates, and is probablylargely due to further oxidation of tea catechins,resulting in production of COa in the same way asestablished in the experiments now underdiscussion. The production of C02 when a crudeenzyme acts upon catechol is probably due tooxidation of adsorbed tea catechins, the output of COa being enhanced by the further oxidation of thecatechins by the o-benzoquinone.

Oxidation of gallic acid by o-benzoquinone isapparent from the data in Tables 6 and 7 and Fig. 4.E' Q for gallic acid is higher than that for catechol(Ball & Chen, 1933), but as the primary oxidationproduct of gallic acid is unstable, oxidation of gallicacid may proceed to completion. It is of interest tonote that protocatechuic acid, with a much higherE' 0 , and no pyrogallol grouping, is not appreciablyoxidized in the presence of catechol (Table 7).

Merck’s catechin behaves similarly to catechol inpromoting the further oxidation of pyrogallol, teacatechins and gallic acid, as demonstrated by in-creases in total C02 output (Table 6). The resultswith pyrogallol indicate E' Q for this catechin to beprobably a little lower than that of catechol. Using1*7 mg. of gallic acid +10 mg. catechin the results inFig. 6 show that about 2 atoms of 02 (223 /J.) areconsumed and 1 mol. C02 (203 /¿I.) produced in thecomplete oxidation of gallic acid. This confirms the

statement made earlier that the high E' 0 value for

gallic acid is no obstacle to its complete oxidation bya substance of lower potential.

Mixed tea catechins, as indicated by their second-ary oxidation by Merck’s catechin and tea oxidase,probably have lower mean E' 0 values than Merck’scatechin. Total Oa uptakes and COa outputs of pyro-

Pyrogallol, with probably the lowest E' 0 value of the substances under consideration, has not beenshown to bring about any secondary oxidations.Even gallic acid is completely unaffected.

(mg.) (Ml- 0* C02) (/xl. 02) (#a. co2 (min.1-26 152 72 518 167 1105 110 42 362 150 9020 40 33 330 207 600-85 * * 376 106 80

0-77 * * 498 20 8010 18 5 486 57 90i t Direct of 40 mg. enzyme.

Table 7. Effect of added catechol on carbon dioxide production from polyphenols

(Dickens & Simer technique; 25 mg. washed enzyme; total vol. 3 ml.; pH 5*6.)Without catechol With catechol (15 mg.)

Durationof

ah ».king




Mixed substrates with p -phenylenediamine . No true

mixed substrate effect has yet been demonstratedwhen both substrates are polyphenols oxidizable bythe tea oxidase. On the other hand, as the data inTable 6 show, p -phenylenediamine and catechinsbehave quite normally in this respect. The

Fig. 6. Mixed substrates, Merck’s catechin and gallicacid. Curve 1: 02 uptake with 10 mg. Merck’scatechin +40 mg. washed enzyme at pH 5*6. Curve 2:as curve 1 together with 1*7 mg. gallic acid. Curve 3:as curve 2, C02 output.




typical mixed substrate effect is strong evidence forthe belief that p -phenylenediamine is oxidizedenzymically by the tea oxidase.

DISCUSSION Nature of the tea oxidase With an enzyme washed free from adsorbed sub-strates neither ascorbic acid nor quinol are appreci-ably oxidized, from which it follows that the enzymeis unlikely to be a cytochrome oxidase. Furtherevidence against the identification of the tea oxidasewith cytochrome oxidase is afforded by oxiâtion-reduction potentials. Stotz, Sidwell & Hogness(1938) obtained E' 0 = 0*262V. for cytochrome cbetween pH 5*0 and 8*0. At pH 5-6, E' 0 for catechol=0*466 V. (calculated from Ball & Chen, 1933) so

that at the optimal pH, catechol oxidation by acytochrome system is unlikely to be very rapid,especially as o-benzoquinone is more stable at pH5*6 than at pH 7*3 at which level only a slowoxidation of catechol by the cytochrome system isknown to take place (Slater, 1949). Earlier claims(Roberts, 1940a, 1941a, 1942) are therefore with-drawn.

The tea oxidase differs in many respects fromtyrosinase; tyrosine is not oxidized, nor is theenzyme appreciably inactivated as a result of cate-chol oxidation. A further characteristic is its powerof oxidizing p -phenylenediamine and of oxidizingsubstrates with a pyrogallol grouping more rapidlythan those with a catechol grouping. Although

simple polyphenols, such as pyrogallol and catechol,are oxidized by the enzyme, it seems clear that itstrue substrates are the catechins, for which muchlower Michaelis constants are observed than for thesimpler polyphenols.

Oxidation of substrates

Although there is no reasonable doubt that theprimary product of oxidation with all polyphenols isan o-quinone, subsequent changes are often dis-similar. With catechol, the o-quinone appears toundergo a transformation into another polyphenol,more rapidly oxidized by the tea oxidase than theoriginal catechol. No C02, however, is evolved fromthis, nor from any other polyphenols containingcatechol as opposed to pyrogallol groupings (Merck’s

catechin, adrenaline, protocatechuic acid). WithMerck’s catechin the amount of 02 required for com-plete oxidation is little more than 1 atom per mol.,which suggests that substitution in the 4-positionprevents the formation from the primary o-quinoneof a polyphenol capable of undergoing furtherenzymic oxidation.For pyrogallol the mechanism of Willstatter & Heiss

(1923), as modified by Barltrop & Nicholson(1948) , would appear to explain oxidation as far aspurpurogallin, but it is evident that oxidation doesnot stop at this stage. The formation of C02 in theoxidation of pyrogallol and gallic acid, for which a

similar mechanism is suggested by Willstatter &Heiss (1923), is also readily understandable and therecorded output of C02 in the former case is only alittle greater than that required for 1 mol. C02 forevery 2 mol. pyrogallol.

No simple mechanism for the formation of C02 from tea catechins can be put forward at present. Asshown by Bradfield et al. (1947) the tea catechinsconsist of various isomers of catechin (I), gallo-catechin (II), and their galloyl esters (III) and (IV).The simple catechins, judging from our results withMerck’s catechin, give no COa on oxidation, so thatthe C02 must be formed from one or more of theother three types, all of which contain one or twopyrogallol groupings. In this connexion, however, itmay be pointed out that the total C02 outputs re-corded in Tables 6 and 7 for mixtures of teacatechins and catechol are approximately thoserequired were the C02 to originate from oxidation of the gallic acid residues only. It is not, therefore,necessary to assume any elimination of C02 from,the pyrogallol group in the gallocatechins and theirgallates, although the possibility cannot at presentbe excluded.

The fact that no C02 is evolved in the presence of ascorbic acid proves that C02 formation takes placeat a stage subsequent to the formation of the o-quinone. Until pure substrates can be obtained insufficient quantity for detailed study, there is littleprospect of working out the mechanism of C02 elimination. Judging by the variation in R.Q. withsubstrate concentration (Table 4) the scheme of reaction is likely to be complex.

The relatively low R.Q.’S (0* 1-0*4) suggest eitherthat only some of the catechins lose C02 in thecourse of oxidation, or that elimination of C02 goesonly partly to completion. In the latter case twoseries of condensation products are likely to beformed for each type of catechin, one series derivedfrom catechins which have undergone a relativelysimple oxidation, and the other derived from oxida-tion products which in the course of their oxidationhave expelled 1 mol. COa. The simple catechins (I)account for only a small proportion of the total tea

OH

(I) R=H. (II) R = H.

(Ill) R= galloyl. (IV) R= galloyl.




catechins, so that on the first hypothesis it would benecessary to assume that not all of the catechinscontaining a pyrogallol group yield COa on enzymicoxidation. The great variations in R.Q. with substrateconcentration, temperature (Harrison & Roberts,1939) and dilution (Roberts, 19406) suggest thatelimination of C02 is only partial, a view which isconfirmed by the considerable increases in C02 evo-lution when catechol is added to the system. Thebalance of evidence, therefore, is in favour of thelatter alternative.

The high K m values for catechol and pyrogallolrender them unsuitable substrates for a study of thekinetics of the tea oxidase. The high concentrationsof substrate that have to be used result in completeoxidation taking a disproportionately long time.Catechol also has the objection that rates are auto-catalytic, so that initial rates are difficult tocalculate.

The use of a tea-catechin preparation is also notfree from objection, as we are not dealing with apure substrate nor even one of known or constantcomposition. A simple catechin, such as Merck’scatechin, seems the most suitable substanceavailable at present. The fact that C02 is not formedon its oxidation is an added advantage in someexperiments.

Mixed svhstrates When p -phenylenediamine is mixed with

catechins the rate of uptake observed is

intermediate between the rates for the singlesubstrates. This normal behaviour is not observedwhen two different polyphenols are mixed, for insuch cases the rate is that of the more rapidlyoxidized substrate. An apparent exception to this isprovided when catechol is the faster oxidizedsubstrate, as reduction of the initially formed o-benzoquinone by the second substrate eliminatesthe faster second stage of the reaction (with teaoxidase). It appears that, with mixed polyphenols,the more rapidly oxidized substrate is oxidizedalmost exclusively in the initial stages, providedthat there is no secondary interaction between theo-quinone formed and the more slowly oxidizedsubstrate.

An interesting parallel to this is provided by theobservations of Wieland & Mitchell (1932), whofound that the purine bases were oxidized to uricacid by xanthine oxidase in the presence of acet-aldehyde, without any simultaneous oxidation of thealdehyde. As the enzyme oxidizes both purine basesand aldehydes, this is another example of successiveoxidation of substrates.

The principle of successive oxidation of poly-phenols, and the recognition of interactions betweenoxidation products with mixed substrates, givessome insight into the complicated processes takingplace in tea fermentation, where a mixture of

several catechins and gallic acid is undergoingenzymic oxidation.

As Merck’s catechin has a higher K m than mixedtea catechins it appears likely that the simple cate-chins and epicatechins (formula I) will be amongthe last to be oxidized during fermentation. Asoxidation in fermentation is normally about 80-90%complete, and these catechins probably constituteless than 10 % of the total catechin content, it is atleast possible that these simple catechins will notbe oxidized to any great extent. Preliminary workwith paper chromatograms, to be published later,also indicates that during fermentation somecatechins are completely oxidized before others.

The possibility of interaction between catechinoxidation products is also indicated by the consider-able increase in C02 output obtained when Merck’scatechin is mixed with tea catechins. Untilsufficient quantities of individual catechins areavailable for their enzymic oxidations to be studied,both singly and in mixtures, the extent to whichsuch interactions go on is unpredictable, but itappears definite that such interactions will followthe primary oxidation to o-quinones, and that theresultant changes in chemical composition will bereflected in differences in the quality of themarketed tea.

# The resvlts of Sreerangachar

Sreerangachar (19436) has studied the relativerates of oxidation of various substrates of the tea

oxidase manometrically, and has also investigatedthe influence of oxidation products of tea poly-phenols on these rates. He states that ‘any compli -cation due to respiration changes was avoided bynot introducing potassium hydroxide into thecentral tube ’. As appreciable quantities of C02 areevolved in the enzymic oxidation of tea catechins,and as these amounts are increased in the presenceof catechol, it is evident that Sreerangachar’sfigures for substrate oxidation by minced leaf underthese conditions are valueless and his conclusionsinvalidated.

SUMMARY

1. The tea oxidase, when washed free from ad-sorbed polyphenols, oxidizes catechol without any

formation of carbon dioxide. The rate is auto-catalytic owing to formation of a more rapidlyoxidized polyphenol from o-benzoquinone. The teaoxidase is not appreciably inactivated as a result of catechol oxidation. K m is high (about 0*025m).

2. Pyrogallol is oxidized, probably to purpuro-gallin, with elimination of one molecule of carbondioxide for every two molecules of pyrogallol, withsome subsequent oxidation beyond the purpuro-gallin stage. Km is 0*01 5M and pyrogallol is oxidizedmore rapidly than catechol at all concentrations.

3. Tea catechins are oxidized more rapidly thansimple polyphenols, with a lower K m (0*001-0-002 M) .




Merck’s catechin produces no carbon dioxide, butmixed tea catechins are oxidized with elimination of carbon dioxide, the amount depending upon sub-strate concentration, time of reaction, etc. Thecarbon dioxide produced probably accounts for muchof the carbon dioxide evolved in tea fermentation.

4. Adrenaline, gallic acid and rutin are oxidizedmore slowly than catechol. Phloroglucinol, tyrosine,ascorbic acid and quinol are either not oxidized orare oxidized very slowly, p -Phenylenediamine isoxidized at an appreciable rate.

5. With a mixture of two polyphenols, the rate of uptake is equal to that of the more rapidly oxidizedsubstrate, and the more slowly oxidized substrateappears initially to undergo no oxidation. Secondaryinteractions between polyphenols and their o-

quinones take place, and the extent of oxidation of substrates such as pyrogallol, gallic acid and teacatechins, can be much increased by adding catecholor Merck’s catechin to the system, judging by theincrease in carbon dioxide output, p -Phenylene-diamine with polyphenols gives normal mixed sub-strate reactions.

6. The effect of these mixed substrate effects onthe mechanism of tea fermentation is discussed.

The authors wish to express their thanks to theChief Scientific Officer, Mr C. J. Harrison, for hisinterest in this work, and to the Indian Tea

Association for permission to publish these results.Thanks are also due to Dr J. F. Couch of the U.S.Department of Agriculture for a generous gift of rutin.

REFERENCES

Ball, E. G. & Chen, T. T. (1933). J. biol. Chem. 102,691.Barltrop, J. A. & Nicholson, J. S. (1948). J. chem.Soc. p. 116.Barua, D. N. & Roberts, E. A. H. (1940). Biochem. J. 34, 1524.Bradfield, A. E. (1946). Chem. Ind. 28, 242.Bradfield, A. E. & Penney, M. (1948). J. chem. Soc. p.2249.Bradfield, A. E., Penney, M. & Wright, W. B.(1947). J. chem. Soc. p. 32.Deb, S. B. & Roberts, E. A. H. (1940). Biochem. J. 34,1507.

Dixon, M. (1934). Manometric Methods. Cambridge:University Press.Harrison, C. J. & Roberts, E. A. H. (1939). Biochem.J. 33, 1408.James, W. 0., Roberts, E. A. H., Beevers, H. &deKock, P. C. (1948). Biochem . J. 43, 626.Lamb, J. & Sreerangachar, H. B. (1940a). Biochem.J. 34, 1472.Lamb, J. & Sreerangachar, H. B. (19406). Biochem.J . 34, 1485.Li, L. P. & Bonner, J. (1947). Biochem. J. 41, 105.Ludwig, B. J. & Nelson, J. M. (1939). J. Amer. chem.Soc. 61, 2601.Roberts, E. A. H. (1939). Biochem. J . 33, 842.Roberts, E. A. H. (1940a). Biochem. J. 34, 500.Roberts, E. A. H. (19406). Biochem. J. 34, 507.Roberts, E. A. H. (1941a). Biochem. J . 35, 1209.

Roberts, E. A. H. (19416). Biochem. J. 35, 1219.Roberts, E. A. H. (1942). Advanc. Enzymöl. 2, 113.Roberts, E. A. H. (1949). Biochem. J. 45, 538.Roberts, E. A. H. & Sarma, S. N. (1940). Biochem. J. 34, 1517.Roberts, E. A. H. & Wood, D. J. (1950). Nature ,Lond., 165, 32.Slater, E. C. (1949). Biochem. J. 44, 305.Sreerangachar, H. B. (1941). Biochem. J. 35,1106. Sreerangachar, H. B. (1943a). Biochem.J. 37, 653. Sreerangachar, H. B. (19436).Biochem. J. 37, 656.Stotz, E., Sidwell, A. E. & Hogness, T. R. (1938). J.biol. Chem. 124, 11.

Wagreich, H. & Nelson, J. M. (1938). J. Amer. chem.Soc. 60, 1545.Wieland, H. & Mitchell, W. (1932). Liebigs Ann. 492,156.Willstätter, R. & Heiss, H. (1923). Liebigs Ann. 433,17.

Documents

Metabolism of 2