Gut, 1964, 5, 295

Studies on the mechanism of destructionof the toxic action of wheat glutenin coeliac disease by crude papain

M. MESSER, CHARLOTTE M. ANDERSON, AND LOIS HUBBARD

From the Gastroenterological Research Unit,Royal Children's Hospital Research Foundation,

Melbourne, Australia

EDITORIAL SYNOPSIS The toxic action of wheat gluten on two patients was eliminated after pre-digestion of gluten by crude papain. This change is thought to be due to an enzyme which liberatesammonia from gluten. It is not thought that this particular mechanism operates in the normalintestinal cell but it is envisaged that one of the peptide bonds of the coeliac active constituent(possibly a N-glutaminyl peptide) is normally split by a specific intestinal peptidase which is defectivein coeliac patients.

Previous authors have shown that the harmfulaction of dietary wheat gluten in coeliac disease ornon-tropical sprue is not eliminated by predigestingthe gluten with either pancreatin or pepsin ortrypsin (Alvey, Anderson, and Freeman, 1957;Frazer, Fletcher, Ross, Shaw, Sammons, andSchneider, 1959; Krainick, Mohn, and Fischer, 1959;van Roon, Haex, Seeder, and de Jong, 1960). Ofvarious enzymes from animal sources, only anextract of hog intestinal mucosa has so far beenshown to abolish the toxic action of gluten incoeliac disease (Frazer, 1956).

It has been reported, however (Krainick et al.,1959), that after wheat gluten has been digested bycrude papain (a commercial product prepared fromthe latex of the unripe papaya) it is no longerharmful when fed to coeliac patients. This pheno-menon has not been investigated further, and it isnot known whether the 'detoxification' is caused bypapain itself or by another enzyme contained in thecrude product, such as chymopapain (Jansen andBalls, 1941) or an enzyme acting on peptides.The present studies were undertaken in order to

confirm and extend these results of Krainick et al.(1959), in the hope that knowledge of the mechanismof action of crude papain on gluten might yieldinformation concerning the chemical structure ofthe coeliac-active constituent of the protein.To this end we have conducted experiments in

which gluten which had been predigested either bycrude papain or by purified constituents of papaya

latex (papain and chymopapain) was fed to threecoeliac patients.We have also carried out certain investigations

into the chemical mechanism of the digestion ofgluten by crude papain, concerned mainly with thequestion of the origin of the free ammonia which isformed during this digestion (Krainick et al., 1959).These investigations led to the discovery of a newenzyme contained in papaya latex which actsspecifically on N-glutaminyl peptides (Messer, 1963);the possible role of this enzyme in the detoxificationof gluten by crude papain was given special attention.

PATIENTS, METHODS, AND MATERIALS

COELIAC PATIENTS The methods used in the diagnosis ofcoeliac patients, the collection of their stools duringfeeding investigations, and the estimation of stool fatwere the same as those described in pIevious papers(Anderson, Frazer, French, Gerrard, Sammons, andSmellie, 1952; Messer and Anderson, 1961). Enzymaticgluten digests were administered in three doses per day,either neat or mixed with milk. When the patients wereon a gluten-containing diet, meals were prepared by thediet kitchen to contain about 9 g. wheat gluten per day.

GLUTEN Gliadin-enriched wheat gluten was supplied byBarret's Food Co. Pty. Ltd., Melbourne, who state thatit was prepared by stirring commercial dried gluten in0-01 M acetic acid, centrifuging, and precipitating thegluten from the supernatant solution with sodiumchloride, followed by freeze-drying. Its amide content was2-76 mmoles per gram dry weight.

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PEPTIDES L-Glutaminyl-L-asparagine was supplied byDr. J. M. Swan, C.S.I.R.O., Melbourne, and L-glutaminylL-leucine by Drs. G. Amiard and R. Heymes, Roussel-Uclaf, Paris. Small amounts of the correspondingpyrrolidone carboxylyl peptides were prepared for use aschromatographic markers by heating the N-glutaminylpeptides for 60 minutes at 100°C. in 0-1 M phosphatebuffer, pH 8 (Waelsch, 1952). Glycyl-L-glutamine wassupplied by Dr. J. M. Swan. All other peptides wereobtained from Mann Research Laboratories, New York.

ENZYMES Crude papain was obtained from E. MerckA.G., Darmstadt, and crude ficin and bromelin fromMann Research Laboratories; crystalline papain, pepsin,trypsin, and chymotrypsin were products of SigmaChemical Co., St. Louis.

Semi-pure, non-crystalline chymopapain was preparedby the following modification of the method of Jansenand Balls (1941). Finely ground granular dried papayalatex (Wheeler and Huisking Pty. Ltd., London), 80 g.,was stirred with 800 ml. water for 30 minutes. Thesuspension was centrifuged and the supernatant solutionadjusted to pH 2-0 and held at 37°C. for 80 minutes. Itwas then centrifuged and the supernatant solutionadjusted to pH 40 and half saturated with sodiumchloride. After centrifugation the clear supernatantsolution was fully saturated with sodium chloride andadjusted to pH 2-0. It was stood in the refrigeratorovernight and then centrifuged. The white precipitatewas chymopapain (about 10 g.). The main differencesbetween this method and that of Jansen and Balls (1941)are the use of dried instead of fresh papaya latex as thestarting product and the 80-minute treatment atpH 2-0 at370C. This treatment was necessary in order to destroy alldeamidase and glutamine cyclotransferase activities (seeDiscussion). The treatment also inactivated papain. Thechief contaminant of our chymopapain is probablylysozyme (Ebata and Yasunobu, 1962).The activities of the crude papain, chymopapain, and

crystalline papain towards benzoyl arginine amide weremeasured at 25°C., pH 7-2 in the presence of 5 mMcysteine, 1 mM ethylene diamine tetra-acetic acid(E.D.T.A.), and 50 mM substrate. They were found to be0 40, 0.33, and 2-7 jgmoles/min./mg. enzyme protein,respectively.

PREPARATION OF ENZYMATIC DIGESTS OF GLUTEN

CRUDE PAPAIN DIGEST Crude papain, 40 g., was stirredfor 30 minutes in 2 1. cysteine (10 mM) and E.D.T.A.(2 mM), pH 7*5. The suspension was filtered and thefiltrate, which contained about 20 g. dissolved crudepapain, was vigorously stirred by a magnetic bar while100 g. gluten was slowly sprinkled onto the surface; thisprocedure prevented the formation of lumps. During theaddition of gluten the pH was maintained above 5.5 bydropwise additions of 5 M NaOH solution. Thirtyminutes after all the gluten had been added the mixturewas adjusted to pH 5-5 and poured into a 5 1. flasktogether with 2 1. water and 40 ml. toluene. The flask wasplaced in a large bath kept at 37°C. and the mixturestirred continuously by a magnetic bar for 42 hours. The

digest was then filtered and the clear, orange filtrateadjusted to pH 10 and evaporated in vacuo at 37°C. to avolume of 1 1. by means of a rotary evaporator. The darkorange ammonia- and toluene-free product was adjustedto pH 6 5, filtered and stored at - 20C. until use.The total amount of insoluble material (mainly lipid)

removed during the procedure was about 5 g.; the finalproduct was therefore about 9.5% with respect to digestedgluten.

CRYSTALLINE PAPAIN DIGEST This was prepared in thesame way, except that 2 g. crystalline papain was usedper 100 g. gluten. The final product was yellow.

CHYMOPAPAIN DIGEST Since native gluten was found tobe digested by chymopapain with great difficulty, it waspredigested by pepsin; 1 g. crystalline pepsin was dissolvedin 2 1. cysteine (5 mM) and E.D.T.A. (1 mM), pH 2-0,and 100 g. gluten was added. After two hours' stirringthe mixture had become homogeneous and was adjustedto pH 5-5. Cysteine (5 mM), and E.D.T.A. (1 mM),pH 5.5, 21., containing 10 g. dissolved chymopapain, wasadded and the mixture readjusted to pH 5.5. The rest ofthe procedure was as described for crude papain. Thefinal product was orange.

Measurements of the formation of ninhydrin-reactivematerial showed that under the above conditions thedigestion was over 90% complete in each case.

OTHER METHODS The amide content of gluten and ofgluten digests was estimated by alkaline hydrolysis atroom temperature (Stegemann, 1958).Enzyme protein was determined by the biuret method

described by Aldridge (1957).Free ammonia and ninhydrin-reactive material in the

gluten digests were estimated as follows. Of the digest,0-10 ml. was treated with 0-1 ml. saturated K2C03solution in the outer well of a Conway microdiffusionunit and the ammonia liberated into 1-00 ml. 0.1 Nsulphuric acid. After 60 minutes the acid was analysedfor ammonia (Brown, Duda, Korkes, and Handler, 1957)and the contents of the outer well diluted to 25-0 ml. andanalysed for ninhydrin-reactive material by the methodof Cocking and Yemm (1954). Standard mixtures ofammonium chloride and leucine were run through thewhole procedure.

Paper chromatography (descending) was carried outon Whatman no. 1 paper with the following solventsystems; n-butanol: acetic acid : water (4:1:1), s-butanol:t-butanol: 2-butanone: water (4:4:8:5) and propanol:water (4:1). For two-dimensional chromatography thefirst two systems were used (Ambe and Tappel, 1961).

RESULTS OF FEEDING INVESTIGATIONSWITH COELIAC PATIENTS

PATIENT 1 This boy had been diagnosed as sufferingfrom coeliac disease at the age of 13 months. Before thathe had passed pale, bulky stools for several months andhis abdomen had become distended. Investigationsrevealed marked steatorrhoea, microcytic anaemia, and aflat duodenal mucosa. He responded very well to a

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Mechanism of destruction of the toxic action of wheat gluten in coeliac disease by crude papain 297

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FIG. 1. Effects offeedingwheat gluten predigested bycrude and pure papain on thedaily excretion offat (three-day mean) ofpatient 1. Thedaily fat intake averaged45 g. Dotted linecorresponds to upper limitof normal faecal fat(4.5 g. per day).

gluten-free diet. After some months he was readmittedand gluten was added to his diet; after nine days hedeveloped steatorrhoea (Fig. 1). When the steatorrhoeahad subsided again on a gluten-free diet the patient wasfed daily 9 g. of gluten which had been predigested bycrude papain, his diet containing no other gluten.During the three weeks on this diet no steatorrhoea orother symptoms of coeliac disease were observed. Thepatient was then fed 9 g. per day of gluten which had beenpredigested by crystalline (pure) papain; within 11 dayshis stools became pale, bulky, and soft, and showedmarked steatorrhoea (Fig. 1). This feeding was stopped

after three weeks, whereupon the patient's stools graduallyreturned to normality during the following period.

These results show that this patient reactedunfavourably to gluten predigested by pure papainbut not to that predigested by crude papain.

PATIENT 2 This patient was a pale, fair-haired boy of10 months who had failed to thrive in the precedingthree months and had developed during the same timeloose, pale, frequent stools and a distended abdomen.

FIG. 2. Effects offeedingwheat gluten predigested bycrude and pure papain on

the body weight and dailyexcretion offat (three-daymean) ofpatient 2. The dailyfat intake averaged 45 g.Dotted line corresponds toupper limit of normal faecalfat (4S5 g. per day).

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M. Messer, Charlotte M. Anderson, and Lois Hubbard

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FIG. 3. Effects offeedingwheat gluten predigested bychymopapain on the bodyweight and daily excretionoffat (three-day mean) ofpatient 3. The fat intakeaveraged 40 g. per day. Stoolcollections were incompleteso results were not recordedduring the period of diarrhoea.

He had steatorrhoea (average of 13 g. stool fat per day),mild microcytic anaemia, and a flat duodenal mucosa.He was placed on a gluten-free diet and also fed 6 g. per

day of the crude papain-digested gluten (Fig. 2).During the succeeding six weeks he improved considerablyclinically, gaining 2-3 kg. despite an attack of otitis mediaand severe teething troubles. However, although thedegree of steatorrhoea progressively lessened it did notdisappear, an average of about 7 g. fat being excreted perday. The crude papain digest was then stopped andafter a gap of two weeks, during which the fat in hisstools remained the same, the patient was fed 6 g. per dayof crystalline papain digest (Fig. 2). After eight daysthe stools became more bulky, loose and pale, and theirfat content increased to an average of 11 g. per day. Thepatient's weight fluctuated but overall was stationaryduring this feeding. The crystalline papain digest wasstopped after three weeks; the child then gained weightand was allowed to go home. He continued to gainweight and made a good response to a gluten-free diet athome.

It was felt that the crude papain digest of glutenwas not deleterious to this child because he continuedto gain weight and his steatorrhoea decreased even

in the presence of quite severe secondary infections.The crystalline papain digest, however, had a

considerable deleterious effect clinically and on hisdegree of steatorrhoea.

PATIENT 3 This boy was admitted at the age of 2- years,severely ill with symptoms and signs of coeliac disease,including failure to gain weight, very loose, creamy stools,marked temperamental difficulties, and a distendedabdomen. Steatorrhoea and the flat pattern of theduodenal mucosa were demonstrated. The patient was ina state of 'coeliac crisis' for some days, and thereaftermade a sure but slow response to a gluten-free diet,two months elapsing befoie his stools approachednormality; they remained rather soft and pale. As wewere unable to detain this child for much longer inhospital, he was given 7 g. per day of chymopapain-digested gluten at this stage when his stools had not quiteattained a normal fat content (Fig. 3). During the31 days on this digest no gross change in his clinicalcondition was observed, but in comparison with theprevious period the steatorrhoea did not continue toimprove and his weight gain, which had been fairly satis-factory before the chymopapain-digested gluten had beengiven, became slow and irregular (Fig. 3). After threeweeks on this digest the stools became quite fluid andcreamy and the stool collections were incomplete.

It was felt that this patient reacted unfavourablyto the chymopapain-digested gluten, though theresponse was not very marked and therefore notabsolutely conclusive. Hence we prefer to maintainan open mind about the coeliac activity of thechymopapain-digested gluten.

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Mechanism of destruction of the toxic action of wheat gluten in coeliac disease by crude papain 299

Unfortunately no further patients were availableat this stage for long-term fat balance and feedingstudies.

Table I summarizes our results concerning thetoxicity or otherwise of the gluten digests with theforegoing three patients.

TABLE ISUMMARY OF RESULTS OF EXPERIMENTS WITH WHEAT GLUTEN

PREDIGESTED BY PAPAYA LATEX ENZYMES FEDTO COELIAC PATIENTS

Patient No. Crude Papain Pure Papain ChymopapainDigest Digest Digest

23

Non-toxic ToxicNon-toxic Toxic

Toxic (?)

CHEMICAL STUDIES

The general purpose of these studies was to determinewhether crude papain contains an enzyme, other thanpapain and chymopapain, which could be respons-ible for destroying the coeliac activity of wheatgluten. As it turned out the investigation becameconcerned mainly with whether crude papaincontains a deamidase. Krainick et al. (1959) hadobserved the liberation of a relatively large amountof free ammonia from gluten by crude papain, andDamodaran and Ananta-Narayanan (1938), whohad made a similar observation with other proteins,had in fact postulated that papain contains adeamidase. In view of the evidence of van de Kamerand Weijers (1955) that glutamide amide might playa role in the coeliac activity of gluten, these observ-ations seemed to us to be highly relevant. In additionto this question of deamidase activity of crudepapain, we gave some attention to the possible roleof specific peptidases in the gluten- 'detoxifying'action of crude papain; this aspect had been stressedby Krainick et al. (1959).

RELATIVE DEGREE OF DIGESTION OF GLUTEN BY VARIOUSPROTEASES It was of interest to compare the degreeof digestion of gluten by a number of proteolyticenzymes. To this end we measured the amount ofninhydrin-reactive material, i.e., amino-acids andpeptides with free alpha-amino groups, formed ineach case during the digestion of the protein.

It was found that the plant enzymes used (papain,chymopapain, ficin, and bromelin) liberated muchmore ninhydrin-reactive material than did any ofthe animal proteases (Table II). It is evident that theformer produce a considerably greater degree ofdigestion of the protein.

It is also noteworthy that appreciably lessninhydrin-reactive material was produced by crudepapain than by either pure papain or chymopapain,

TABLE IIFORMATION OF NINHYDRIN-REACTIVE MATERIAL AND OF

AMMONIA DURING DIGESTION OF WHEAT GLUTENBY PROTEOLYTIC ENZYMES

Enzyme Amount pH Ninhydrin- Ammonia(mg./g. reactive Material (mmoleslg.gluten) (leucine gluten)

equivalents,excludingammonia)(mmolesfg. gluten)

Crude papain 200 5-5 1-42 1-28Crystalline papain 20 5-5 1-78 0-12Chymopapain1 100 5-5 1-60 0-11Crude ficin 200 6 1-91 0.34Bromelin 200 6 1-55 0-14Crystalline pepsin 20 2 0 44 0-16Crystalline trypsin1 20 8 0-27 0-08Crystalline chymo- 20 8 0.77 0.07

trypsin

1Chymopapain and trypsin were added after two hours' predigestionby pepsin.

Digestion by trypsin and chy,notrypsin was done in the presence of10 mM CaCI2, by the plant enzymes in the presence of 5 mM cysteineand 1 mM E.D.T.A. Crude papain, ficin, and bromelin were dialysedagainst 1 mM E.D.T.A. before use. All digestions were done over42 hours at 37°C. in a volume of 40 ml. per g. gluten; toluene wasadded as preservative. Figures are corrected for moisture content ofgluten and for ninhydrin-reactive material and ammonia formed inthe absence of gluten.

despite the fact that crude papain contains a mixtureof these two enzymes.

AMMONIA FORMATION DURING THE ACTION OF

PROTEASES ON GLUTEN Table II shows that whereasa large amount of free ammonia was producedduring the action of crude papain on wheat gluten,relatively little was produced by other proteolyticenzymes, including two constituents of crudepapain (papain and chymopapain) and two otherplant proteases (ficin and bromelin).

Determination of the total amide content of thedigest showed that the crude papain had produced adecrease in amide-nitrogen equal to the increase inammonia-nitrogen (1-28 mmoles/g.). Therefore theammonia liberated by crude papain was derivedfrom the amide groups of gluten; it represented46% of the total amide content.During early experiments with chymopapain

it was found that unless the enzyme had been treatedat pH 2 at 37°C. for at least 80 minutes, up to0.8 mmoles of ammonia per gram gluten wasproduced during its action on gluten.These results suggest that crude papain contains a

deamidase which is distinct from both papain andchymopapain and is unstable at pH 2. None of theother proteases tried, with the possible exception officin, had deamidase activity.

ACTION OF CRUDE PAPAIN ON GLUTAMINE AND ONGLUTAMINE PEPTIDES In order to elucidate the

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mechanism of the above-mentioned deamidaseactivity of crude papain, further investigations withthis end in view were carried out. Since gluten has avery high glutamine content (about 40%) it seemedthat a possible mechanism was the deamidation offree glutamine formed as an intermediate during thecourse of the digestion. It was found, however, thatboth L- and D-glutamine remained largely un-changed when incubated with crude papain underthe conditions which had been used for the digestionof gluten (42 hours at 37°C. at pH 5.5 in the presenceof 5 mM cysteine and 1 mM E.D.T.A.). Further-more, paper chromatography showed that onlysmall amounts of glutamic acid and pyrrolidonecarboxylic acid (two possible products of thedeamidation of glutamine) were present in the crudepapain digest of gluten. No free glutamine could bedetected.

It was considered that an alternative source ofamide ammonia might be glutamine-containingpeptides. When glycyl-L-glutamine was incubatedwith dialysed crude papain under the foregoingconditions, it remained largely unchanged. However,paper chromatography showed that two peptides inwhich L-glutamine occupies the N-terminal position,viz., L-glutaminyl-L-asparagine and L-glutaminyl-L-leucine, completely disappeared in the presence ofpapain and were replaced by ninhydrin-unreactiveproducts which had the same RFS in three differentchromatographic solvent systems as pyrrolidonecarboxyl-asparagine, and pyrrolidone carboxylyl-leucine, respectively (Table III). Estimation of theamide contents of the solutions showed that withboth peptides an amount of ammonia equal (on amolar basis) to the amount of peptide initially presenthad been liberated. Neither of these changes wereobserved with pure papain or chymopapain, or aftertreatment of crude papain at pH 2 at 37°C. for 80minutes.

TABL]RF VALUES OF NINHYDRIN-1

ACTION OF CRUDE PAPAPEPTII

L-glutaminyl-L-asparagineL-glutaminyl-L-leucine

Glutaminyl peptide, 25 ,moles, wacrude papain for 42 hr. at 37°C. in aof cysteine (5 mM) and E.D.T.A. (1the glutaminyl peptide had disappeidentical with those of pyrrolidoipyrrolidone carboxylyl-L-leucine, re

Solvent 1: n-butanol :acetic acid :wjSolvent 2: s-butanol:t-butanol :2-bhSolvent 3: n-propanol:water, 4: 1.

These results show that crude papain contains anenzyme, other than papain or chymopapain, whichcatalyses the cyclisation of N-glutaminyl peptides tothe corresponding carboxyl peptides plus freeammonia and is unstable at pH 2. Under theconditions used the enzyme had no effect onglutamine or on C-glutaminyl peptides.

FORMATION OF PYRROLIDONE CARBOXYLYL PEPTIDES

DURING DIGESTION OF GLUTEN BY CRUDE PAPAIN The

previous observations on the effect of crude papainon N-glutaminyl peptides strongly suggested thatthe ammonia liberation from gluten by crude papainmight be due to the action of the foregoing enzyme.In that case it should be possible to demonstrate thepresence of pyrrolidone carboxylyl peptides in thedigest.

This part of the investigation is incomplete but thefollowing preliminary observation is noteworthy.A crude papain digest of gluten was dialysed and thedialysate subjected to paper electrophoresis inpyridine acetate buffer at pH 5.7. The negativelycharged material was eluted and subjected to one-dimensional paper chromatography in n-butanol:acetic acid:water (4:1:1). It was found that threemajor and several minor spots stained with starch-iodide following chlorination (Pan and Dutcher,1956) but did not stain with ninhydrin. The RFvalues of the three major spots were 0-13, 0-29, and0-38 respectively.The observation suggests that the crude papain

digest contained peptides which, being acidic andapparently lacking free alpha-amino groups, behavedlike pyrrolidone carboxylyl peptides.ACTION OF CRUDE PAPAIN ON OTHER PEPTIDES It was

of interest to determine whether crude papain actson peptides other than N-glutaminyl peptides.

Samples of dialysed crude papain solution wereincubated at pH 5-5 and 37°C. for 42 hours in thepresence of 5 mM cysteine and 1 mM E.D.T.A. with

E III the following peptides (each at 25 mM): glycyl-glycine, glycyl-L-proline, L-prolylglycine, glycyl-L-

UNREACTIVE PRODUCTS OF leucine, L-leucylglycine, L-leucinamide, L-alanyl-AIN ON GLUTAMINYL glycylglycine and carbobenzoxyglycyl-L-phenyl-DES alanine. These peptides are the main ones commonlyMolvent 1 Solvent 2 Solvent 3 used as substrates for the specific peptidases (Smith,

0.22 0.18 0.14 1951).081 049 055 Paper chromatography showed that except for

glycylglycine and the two proline peptides there was~sincubated with 5 mg. dialysed slight (less than 10 %) hydrolysis of all the peptides

a volume of 1 ml. in the presencemM). At the end of this time all tried. Similar results were obtained, however, whenared. The above RF values are pure papain was used instead of crude papain.,ne carboxylyl-L-asparagine andspectively. There was no hydrolysis of any peptide by crude

papain in an E.D.T.A.-free medium containingater, 4:41:1. 10 mM iodoacetate (an inhibitor of papain andstanone:water, 4:4:8:5.

chymopapain).

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Mechanism of destruction of the toxic action of wheat gluten in coeliac disease by crude papain 301

These results show that crude papain does notcontain any of the common specific peptidases;in so far as there was any hydrolysis of peptides bycrude papain, this could be ascribed to the actions ofpapain or chymopapain.

AMINO-ACID FORMATION DURING DIGESTION OF GLUTEN

BY CRUDE PAPAIN According to Krainick et al.(1959), crude papain digestion of gluten results inthe formation of appreciable amounts of freeglutamine and proline.The absence of free glutamine from a crude

papain digest of gluten in the present studies hasalready been noted. Nor could free proline bedetected, either by one- or two-dimensional paper

chromatography.No attempt was made to identify other amino-

acids, but the probable presence of appreciableamounts of leucine (or isoleucine) was noted.

DISCUSSION

The results of the feeding investigations show thatwheat gluten retains its harmful action on coeliacpatients after digestion by pure (crystalline) papainbut not after digestion by crude papain. The observ-ation that crude papain destroys the coeliac activityof gluten had previously been made by Krainicket al. (1959). It is evident that crude papain containsa 'gluten-detoxifying' factor, presumably an enzyme,

other than papain itself.Papaya latex contains a variety of enzymes,

including lipase, amylase, lysozyme, and others(Hwang and Ivy, 1951), but it can be assumed thatthe gluten-detoxifying enzyme is one acting on

proteins or peptides.Besides papain the only other protease known to

be contained in papaya latex is chymopapain(Jansen and Balls, 1941; Ebata and Yasunobu, 1962).We tried to determine whether chymopapain-digested gluten retains its coeliac activity (patient 3),but unfortunately the result, though suggestingthat the digest was still toxic, was not entirelyconclusive. The possibility that the gluten-detoxi-fying enzyme is chymopapain therefore remainsopen.

Krainick et al. (1959) considered that the gluten-detoxifying enzyme of crude papain might be a

peptidase, but our results show that crude papaindoes not contain any of the known specific peptidasesand has no action on peptides which could not beascribed to papain itself.

It is conceivable that papaya latex contains otherunknown proteases or peptidases, one of whichmight be the gluten-detoxifying enzyme, whichsplit particular peptides formed as intermediatesduring the digestion of gluten. However, the only

marked action which crude papain was found tohave on peptides was the conversion of N-glutaminylpeptides (L-glutaminyl-L-asparagine and L-glutaminyl-L-leucine) to the corresponding pyrro-lidone carboxylyl peptides plus ammonia. Thisreaction was not observed with either pure papain orchymopapain and is therefore catalysed by anadditional enzyme contained in crude papain. Atthe pH used for the crude papain digestion of gluten(5.5) this enzyme had no action on glutamine, butother investigations have shown that the analogouscyclisation of L-glutamine to ammonium pyrrolidonecarboxylate occurs above pH 7 (Messer, 1963).This new enzyme has not yet been purified; wepropose that for the time being it be called glutaminecyclotransferase.Our results confirm the observation of Krainick

et al. (1959) that crude papain liberates a largeamount (46 %) of amide ammonia from gluten.Other proteases, including papain and chymopapain,liberated relatively minor amounts and it is evidentthat crude papain contains a deamidase distinctfrom these two enzymes. Damodaran and Ananta-Narayanan (1938), on the basis of observations onammonia liberation during the action of crudepapain on casein and edestin, had previouslypostulated the presence of a deamidase in crudepapain.Both this deamidase and glutamine cyclotrans-

ferase were found to be unstable at pH 2, and itseems highly probable that they are one and thesame enzyme. This implies that the ammonialiberated from gluten by crude papain arises throughthe cyclisation of N-glutaminyl peptides formed asintermediates in the digestion shown on page 302.

This scheme could explain the observation thatless ninhydrin-reactive material was producedduring the digestion of gluten by crude papain thanby pure papain or chymopapain (Table II), sincepyrrolidone carboxylyl peptides, which have no freeamino groups, would not be expected to react withninhydrin.The existence of glutamine cyclotransferase

raises the possibility that this enzyme is the gluten-detoxifying factor of crude papain, and that thecoeliac-active constituent of gluten is therefore aN-glutaminyl peptide. This idea is consistent withthe fact that deamidation of gluten by means of hotdilute HCl has been shown to destroy its coeliacactivity (van de Kamer and Weijers, 1955), indicatingthat its coeliac-active constituent must contain atleast one glutamine residue. Before this could beconsidered to be more than a working hypothesis itwould be necessary, however, to purify glutaminecyclotransferase and show that it has no action,e.g., proteolytic, other than on N-glutaminyl

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M. Messer, Charlotte M. Anderson, and Lois Hubbard

Papain,Chymopapain

Gluten ->

CO.NH2

(CT)2NH2-CH-CO.R

Glutaminecyclotranferase

CO

0. / (CH2)2

NH-CH-CO.R

N-Glutaminyl peptides Pyrrolidone carboxyl peptides

plus

Other peptides, amino-acids

R = remainder of peptide

plus

Ammonia

peptides. It would also be desirable to carry outfurther feeding investigations on coeliac patientswith gluten which has been predigested by chymo-papain or a combination of pure papain andchymopapain.

Several authors have postulated that the basicdefect in coeliac disease involves an intestinalmucosal enzyme normally required to complete theintestinal digestion of gluten (see Frazer, 1962).Attempts to identify this enzyme with one of theknown intestinal peptidases by comparing theactions of coeliac and non-coeliac duodenal mucosaon a number of simple peptides have yieldednegative results (Messer, Anderson, and Townley,1961). Therefore the postulated defective enzyme isprobably one which acts specifically on an un-common amino-acid sequence peculiar to gluten.The difficulty of testing this hypothesis has beenthat gluten consists of an infinite variety of amino-acid sequences any of which could conceivably bethe coeliac-active constituent. The present results,which suggest that this constituent might bedistinguished by having glutamine as its N-terminalamino-acid, constitute a rationale for isolating orpreparing N-glutaminyl peptides contained in purepapain and chymopapain digests of gluten, andcomparing their behaviour in the presence ofcoeliac and non-coeliac duodenal mucosa.To avoid possible misunderstanding we wish to

emphasize that we expect the 'gluten-detoxifying'mechanism of the normal intestine to be quitedifferent from that of crude papain. There is noevidence, either for the occurrence of an enzymelike glutamine cyclotransferase in the intestinalmucosa, or for the deamidation of gluten or ofpeptides derived from gluten during normaldigestion. We rather envisage that one of the peptidebonds of the coeliac-active constituent (possibly aN-glutaminyl peptide) is normally split by a specificintestinal peptidase which is defective in coeliacpatients.

Krainick et al. (1959) found that their crude papain

digest of gluten contained appreciable amounts offree glutamine and proline, and therefore proposedthat the coeliac-active constituent of gluten nmight bea glutamine-proline polypeptide which is normally'detoxified' in the intestine by a peptidase such asprolidase. We were unable, however, to detect eitherglutamine or proline in the digest; the observationthat the crude papain had no prolidase or prolinaseactivity was consistent with this. Furthermore,previous studies have shown that there is nodifference between coeliac and non-coeliac duodenalmucosa in their prolidase or prolinase activities(Messer et al., 1961). It is nevertheless possible, assuggested by Krainick et al. (1959), that the postu-lated coeliac-active peptide consists mainly ofglutamine and proline, if only for the reason thatthese two amino-acids together constitute over 50%of the protein (Woychik, Boundy, and Dimler, 1961).The apparent resistance of the coeliac-activeconstituent of gluten to a variety of proteases(pepsin, trypsin, pancreatin, pure papain, andprobably also chymopapain) suggests that it has anunusual amino-acid composition; this could well bedue to a high proline content.

SUMMARY

The toxic action of wheat gluten on two coeliacpatients disappeared after predigestion of gluten bycrude papain but not after digestion by crystalline(pure) papain. A similar experiment with chymo-papain-digested gluten was not entirely conclusive,but suggested that it, too, had retained its coeliacactivity.Crude papain was found to contain an enzyme

(deamidase) which liberates free ammonia fromgluten. Evidence is presented suggesting that thisenzyme is identical with one which catalyses theconversion of N-L-glutaminyl peptides to thecorresponding pyrrolidone carboxylyl peptides plusammonia. The enzyme has been named glutaminecyclotransferase.

302

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Mechanism of destruction of the toxic action of wheat gluten in coeliac disease by crude papain 303

It is proposed that glutamine cyclotransferasemight be the factor in crude papain which eliminatesthe coeliac activity of gluten, and that this activitymight therefore be due to an amino-acid sequence(peptide) contained in gluten which has L-glutamineas its N-terminal amino-acid.

We wish to thank Barret's Food Co. Pty. Ltd., Melbourne,for their generous gift of gliadin-enriched wheat gluten;Dr. J. M. Swan, C.S.I.R.O., Melbourne, for gifts ofglycyl-L-glutamine and L-glutaminyl-L-asparagine; andDrs. G. Amiard and R. Heymes, Roussel-Uclaf, Paris,for a sample of L-glutaminyl-L-leucine.We would also like to thank Professor E. C. Webb,

Secretary, Standing Committee on Enzymes, for adviceon the naming of glutamine cyclotransferase, and Dr. F.J. R. Hird, Department of Biochemistry, University ofMelbourne, for his vajuable advice and encouragement.

REFERENCES

Aldridge, W. N. (1957). Liver and brain mitochondria. Biochem. J.,67, 423-431.

Alvey, C., Anderson, C. M., and Freeman, M. (1957). Wheat glutenand coeliac disease. Arch. Dis. Childh., 32, 434-437.

Ambe, K. S., and Tappel, A. L. (1961). Improved separation ofamino-acids with a new solvent system for two-dimensionalpaper chromatography. J. Chromatog., 5, 546-548.

Anderson, C. M., Frazer, A. C., French, J. M., Gerrard, J. W.,Sammons, H. G., and Smellie, J. M. (1952). Coeliac disease:gastro-intestinal studies and the effect of dietary wheat flour.Lancet, 1, 836-842.

Brown, R. H., Duda, G. D., Korkes, S., and Handler, P. (1957).A colorimetric micromethod for determination of ammonia;the ammonia content of rat tissues and human plasma. Arch.Biochem., 66, 301-309.

Cocking, E. C., and Yemm, E. W. (1954). Estimation of amino-acidsby ninhydrin. Biochem. J., 58, 12p.

Damodaran, M., and Ananta-Narayanan, P. (1938). Enzymaticproteolysis. I. Liberation of ammonia from proteins. Ibid., 32,1877-1889.

Ebata, M., and Yasunobu, K. T. (1962). Chymopapain. I. Isolation,crystallization, and preliminary characterization. J. biol. Chem.,237, 1086-1094.

Frazer, A. C. (1956). Discussion on some problems of steatorrhoeaand reduced stature: on the growth defect in coeliac disease.Proc. roy. Soc. Med., 49, 1009-1013.

-(1962). The malabsorption syndrome, with special reference tothe effects of wheat gluten. Advanc. clin. Chem., 5, 69-106.

Fletcher, R. F., Ross, C. A. C., Shaw, B., Sammons, H. G., andSchneider, R. (1959). Gluten-induced enteropathy. The effectof partially digested gluten. Lancet, 2, 252-255.

Hwang, K., and Ivy, A. C. (1951). A review of the literature on thepotential therapeutic significance of papain. Ann. N. Y. Acad.Sci., 54, 161-207.

Jansen, E. F., and Balls, A. K. (1941). Chymopapain: a new crystallineproteinase from papaya latex. J. biol. Chem., 137, 459-460.

Krainick, H. G., and Mohn, G. (1959). Weitere Untersuchungenuber den schadlichen Weizenmehleffekt bei der Coliakie. 2. DieWirkung der enzymatischen Abbauprodukte des Gliadin. Helv.paediat. Acta, 14, 124-140.

Messer, M. (1963). Enzymatic cyclization of L-glutamine andL-glutaminyl peptides. Natuire (Lond.), 197, 1299.

, and Anderson, C. M. (1961). Pancreatic carboxypeptidases Aand B in coeliac disease. Clin. chim. Acta, 6, 276-280.

-, ,. and Townley, R. R. W. (1961). Peptidase activity ofbiopsies of the duodenal mucosa of children with and withoutcoeliac disease. Ibid., 6, 768-775.

Pan, S. C., and Dutcher, J. D. (1956). Separation of acetylatedneomycins B and C by paper chromatography. Analyt. Chem.,28, 836-838

Smith, E. L. (1951). The specificity of certain peptidases. Advanc.Enzymol., 12, 191-257.

Stegemann, H. (1958). Eine Mikrobestimmung von Amid-Stickstoff,speziell in Proteinen. Hoppe-Seylers Z. physiol. Chem., 312,255-263.

van de Kamer, J. H., and Weijers, H. A. (1955). Coeliac disease. V.Some experiments on the cause of the harmful effect of wheatgliadin. Acta paediat. (Uppsala), 44, 465-469.

van Roon, J. H., Haex, A. J. C., Seeder, W. A., and Jong, J. de (1960).Clinical and biochemical analysis of gluten toxicity. I. Experi-entia (Basel), 16, 209.

Waelsch, H. (1952). Certain aspects of intermediary metabolism ofglutamine, asparagine, and glutathione. Advanc. Enzymol., 13,237-319.

Woychik, J. H., Boundy, J. A., and Dimler, R. J. (1961). Wheat glutenproteins. Amino-acid composition of proteins in wheat gluten.J. Agric. Food Chem., 9, 307-310.

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