Radiation induced decomposition of tryptophan in deaerated aqueous solutions

Radiat. Phy~, Chem. Vol. 22 No $-5 pp. 771-778 1983

~/83/09771-08503.00/0 © 1983 Pergamon Press Ltd

Printed in Great Britain

RADIATION INDUCED DECOMPOSITIONOF TRYPTOPHAN IN DEAERATEDAQUEOUS SOLUTIONS

Lj.Josimovi~, S.Jovanovi~ and l.~udina Boris Kidri~ I n s t i t u t e of Nuclear Sciences, Vin~a, 11001Beograd,Yugoslavia

ABSTRACT The effect of absorbed dose on the decomposition of tryptophan under gamma i r r a d i a t i o n has been investigated. Neutral deaerated and N20 saturated solutions of various tryptophan concentrations were analysed. In both cases the decomposition yield of tryptophan decreases as ~rradiation dose increases. From the observed dependance i n i t i a l yields GO(-Trp) = 3.35 and 4.75 were estimated for deaerated and N20 saturated solutions respectively. Estimated i n i t i al yields are in a good agreement with values calculated according to the reaction scheme proposed. INTRODUCTION The chemical study of reaction products and reaction mechanisms in the radiolysis of amino acids is of great interest for radiation s t e r i l i z a t i o n of protein rich food. I t is an important source of information whether or not toxic substances are formed in irradiated food products. Tryptophan is a constituent of many proteins and is one of the most important amino acids for evaluating quality of a protein. The radiation chemistry of tryptophan has been studied by several authors (Braams, 1966; Adams, 1972; Singh and co-workers, 1981). However, some aspects of the radiolysis remain obscure, although the study by Armstrong and Swallow (1969) has done much to c l a r i f y the mechanism. In the present work, which is a part of a wider study on the effects of irradiation on tryptophan, an attempt is made to complete informations on the decomposition of tryptophan under gamma i r r a d i a t i o n at low dose rate. The effect of increasing absorbed dose on the decomposition of tryptophan in aqueous solutions has been followed. Neutral deaerated and N20 saturated solutions of various tryptophan concentrations were analysed. Results obtained enabled us to get a better insight into the mechanism of tryptophan radiolysis. EXPERIMENTAL L-tryptophan (Trp) (fuer biochemische Zwecke, Herck) was used without additional purification Solutions were prepared using water purified by t r i p l e d i s t i l l a t i o n . The experiments were carried out at neutral pH. Solutions at pH 7 were buffered with phosphate buffer (2 xlO-3 mol dm-3) or tryptophan solutions of natural pH 6.0 were used. Solutions were deaerated by bubbling with argon stated to contain 2 ppm of oxygen (Tehnogas, Pan~evo). Nitrous oxide was of medical grade (Lek, Ljubljana). In the experiments with nitrous oxide the gas was continuosly bubbled through the solution during i r r a d i a t i o n . Irradiations were performed in a 1.85xi014 Bq 60Co gamma source. Low dose rates of the order of 0.5 and 2.5 Gy s-~ were used. Th~ dosimetry was carried out by a Fricke dosimeter: G(Fe3+)= 15.6 and am= 2197 dm~ mol -= cm- . The absorbed doses varied from 0.1 to 50 kGy.

771

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LJ. JOSIMOVIC,

S. JOVANOVlC AND I. CUDINA

Tryptophan remaining after irradiation was separated from products by heating irradiated solutions at 206 oc and by thin layer chromatography (TLC). For experiments with thin layer chromatography glass plates lOx20 cm coated with 0.25 mm layers of Aluminium oxide 60, F254 (Serva, Heilderberg) were used. As a developing system, 2~methyl-l~propanol, g l a c i a l acetic acid, water (5:1:2, v / v / v ) was applied. 0.5 ml of a I0 - j mol dm-~ s o l u t i o n of t r y p t o phan ( u n i r r a d i a t e d and i r r a d i a t e d ) was deposited as a f r o n t on Al203-plates. A f t e r development spot i d e n t i f i e d as tryptophan, by i t s fluorescence and blue colour developed with E h r l i c h reagent, was scraped i n t o c e n t r i f u g e tube to which d i s t i l l e d water was added. A f t e r the tube was centrifuged the absorbancy of the s o l u t i o n was measured at 278 nm. A Varian 634 S spectrophotometer with lO mm optical cells was used for absorption measurements RESULTS AND DISCUSSION Determination of Tryptophan The spectra of tryptophan irradiated with d i f f e r e n t absorbed doses are presented in Fig. I. In irradiated solution the characteristic maximum of tryptophan at 278 nm decreases, while the absorbancy both in the short and long region of the spectrum increases. The overlapping of

T ~"-.

/../"

,~

r--

~

..Q

°°o

\\'... o°.,

I

I

240

260

I

280

1

300 A[om]

Fig. l Absorption spectra of lO-2 mol dm-3 solution of tryptophan; unirradiated; - - - irradiated, D=50 kGy; . . . irradiated, D=IO0 kGy. the tryptophan maximum and absorption bands of r a d i o l y t i c products makes i t d i f f i c u l t to calculate accurately the tryptophan decay. The same d i f f i c u l t y arises when Ehrlich reagent is used, since some of the products gave positive reaction with the reagent (Jovanovid and Josimovid, 1982). Therefore, to determine the concentration of tryptophan remaining after i r radiation i t was necessary to separate products from undecomposed tryptophan. Thin layer chromatography method could be used, but for our purposes, i . e . to analyse solutions of various tryptophan concentrations and under d i f f e r e n t conditions i t seemed that thin layer chromatography would be a rather time consuming procedure. An attempt was therefore made to separate tryptophan in another way, while thin layer chromatography was applied to check results thus obtained.

Radiation induced decomposition of tryptophan

773

Dry-heating procedure. During irradiation in neutral media the i n i t i a l l y colourless tryptophan solution turned yellow. I f the irradiated solution was evaporated to dryness at about 90oc and then heated at 200°C, cooled and the residue dissolved in water, again a colourless solution was obtained. I t displayed an UV spectrum characteristic for tryptophan. However, a brown residue remained strongly stuck to the bottom of the vessel. Since tryptophan decomposes at 289oc we have assumed that under heating the irradiated solution to a lower temperature, tryptophan would not be damaged while r a d i o l y t i c products "polymerized" and remained as a residue insoluble in water. In order to determine optimum conditions for dry heating the effect of the temperature and of the heating time on tryptophan and products was investigated. Best "polymerization" of products was achieved at 206oc. At lower temperatures the brown residue p a r t i a l l y dissolves in water. The effect of the heating time was therefore f o l l o wed at 206oc, by measuring the absorbance at 278 nm of the solution after heating. Results thus obtained are presented in Fig. 2.

Am

O.5 i

o

0.4

03

Q2 0.1

I

2o

I

4o

I

6o

8b

160

'

Fig. 2 The effect of heating time on 10-2 mol dm-3 solution of tryptophan at 206oc; o - unirradiated; e - irradiated. Unirradiated and irradiated solutions display essentially the same dependance on the heating time. The difference exists for the f i r s t 60 minutes, the time necessary to remove radiolyt i c products. From 60 to 100 minutes both curves have plateaus, which indicates that in i r radiated solution only tryptophan is l e f t . Under prolonged heating tryptophan begins to decompose. According to ~hese findings irradiated solutions were after evaporating to dryness, heated at 206oc for 70 minutes. For each set of irradiated samples unirradiated solution of corresponding tryptophan concentration was treated in an identical manner. The loss of t r y ptophan was determined from the difference between absorbances of unirradiated and irradiated solution after heating, measured at 278 nm, with an experimental error better than ~I0%. Thin layer chromatography. To check the results obtained by dry heating the loss of tryptophan during irradiation has been estimated from the decrease in intensity of the absorption at 278 nm after elution from thin layer chromatograms. The chromatography was carried out by n-butanol, glacial acetic acid, water (4:1:5, v/v/v, upper phase), as a developer, used to determine the decomposition yield of tryptophan in 2xi0 -2 mol dm-3 solution (Armstrong and Swallow, 1969). Since i t did not prove to be enough e f f i c i e n t to separate tryptophan from products, we have also applied 2-methyl-l-propanol, glacial acetic acid, water (5:1:2,v/v/v)

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as a developing system. Both systems were applied to glass plates coated with s i l i c a gel and aluminium oxide. The former system revealed on s i l i c a plates a sharp spot of tryptophan and considerable t a i l i n g of products, caused very l i k e l y by t h e i r degradation during the separation. Even worse separation was obtained on s i l i c a plates when 2-methyl-l-propanol, glacial acetic acid, water was used as a developer. Best resolution and no t a i l i n g of products was obtained on plates coated w~th alumiQium oxide and 2-methyl-l-propanol, glacial acetic acid, water as a developer. A lO"~ mol dm-~ solution of tryptophan, both deaerated and N20 saturated was analysed in this way. Results obtained, together with the decomposition yields estimated by dry heating, are given in Table I. I t can be seen from Table l that the decomposition yields of tryptophan determined by two procedures are in a good agreement, i . e . the results obtained are within an experimental error of ± lO %. TABLE 1 Yields for the decomposition of tryptophan in aqueous solution (10-3 mol dm-3) irradiation dose in kGy O.8 1.2 1.5 2.5 3.0

G(-Trp) molecules lO0 eV-l deaerated N20-saturated heating 2.04 1.65 1.66 I. 30 1.20

TLC

heating

TLC

2. O0 1.60 1.40 l . 42 l .31

3.60 3.46 2.94

3.56 3.20 2.91

-

Mechanism of Tr~ptophan Radiol~sis In the radiolysis of water following reactive species are formed (Dragani~ and Dragani~, 1971): H20--~Am--eaq (2.7),

OH (2.8),

H (0.55)

(1)

and a l l of them react rapidly with tryptophan, mainly by addition at various positions in the indole ring. On the other hand, chemical studies show that the destruction of tryptophan is considerably less than GOH + GH + Ge_ = 6.05; under gamma radiolysis at low dose rates tryptophan is lost only with G~O.7 (ArmStrong and Swallow, 1969). I t was proposed that in the radiolysis of tryptophan, similar to other unsaturated ring systems, a reconstitution reaction is involved (Nakken, 1964; Boguta and Dancewicz, 1981). Besides, to account for this rather low decomposition y i e l d , i t was suggested that under gamma irradiation radical-radical reactions are disfavoured, and tryptophan-radicals often react with stable r a d i o l y t i c products (Armstrong and Swallow, 1969). However, the proposed reaction mechanism was based on the results obtained under irradiation at high absorbed doses, i . e . in the dose range from lO to lO0 kGy. On presumption that reactions of tryptophan-radicals with stable products would be less effective under low absorbed doses we have investigated the gamma radiolysis of tryptophan in a much wider dose range, from O.l to 50 kGy. According to irradiation doses used, the i n i t i a l concentration of tryptophan was varied from 5xlO-4 to lO-2 mol dm-3. Results obtained for deaerated solutions are presented in Figs. 3 and 4. The dependance of G(-Trp) on irradiation dose is given by curve A in Fig. 3. I t can be seen that the decompos i t i o n yield has a rather sharp decrease up to a dose of about l kGy, while for higher doses i t reaches almost constant value (Fig. 4). This is consistent with the fact, previously suggested (Armstrong and Swallow, 1969), that tryptophan radicals react with stable products regenerating tryptophan. However, to evaluate a reaction mechanism, the nature of tryptophan radicals as well as of stable products should be known. The r e a c t i v i t y patterns of the primary radicals generated in the radiolysis of water are rather well known. Hydroxyl radicals are least selective in attacking tryptophan,forming three ring adducts, while H atoms form two, and hydrated electron one, which protonates to give H-adduct (Armstrong and Swallow, 1969i Faraggi, 1977). According to this finding essentially

Radiation induced decomposition of tryptophan

775

two reactive species should be considered, namely tryptophan-OH adducts (Trp-OH) and t r y p t o phan-H adducts (Trp-H).

4I

OA

3.3

d.

,-, m

V

Fig. 3

I

I

I

I

2

4.

6

8

I

10 D [kG3

Deaerated solutions of tryptophan: A- decomposition y i e l d as a function of i r r a d i a t i o n dose; o - 5xlO -4 mol dm-3; x - lO-3 mol dm-3; a - 5xi0-3 mol dm-3; • - lO-2 mol dm-3; B - absorbance at 500 nm of lO"2 mol dm-3 solution as a function of i r r a d i a t i o n dose.

Q~

o.4,

o.6,

o.o,

lo, o [,c4

,

E 0 u

Fig. 4

i

I

I

I

lo

~o

~o

~o

I

so D[,~i

Deaerated solutions o f tryptophan: decomposition yield^as a function of i r r a d i a t i o n dose; o - 5xlO-4mol dm-3; x - lO-Jmol dm-3; Q- 5xlO-3mol dm-3; • - lO-Zmol dm-3.

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S. JOVANOVIC,

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Regarding stable r a d i o l y t i c products, we have found (Jovanovi~ and Josimovid, 1982) that recombination of tryptophan-H adducts leads to formation of dimers, while tryptophan-OH adducts give products of both dimeric and monomeric s t r u c t u r e . In the reaction of tryptophan-H r a d i cals and tryptophan-OH radicals tryptophan is regenerated. On the basis of these r e s u l t s , f o l l o w i n g reaction mechanism may be proposed: Trp

+

OH

Trp

+

H(eaq)

Trp-OH

Trp-H Trp-OH

+

+ +

÷

Trp-OH ÷

Trp-H

Trp-OH

Trp-H Trp-H

(2)

÷

OH-Trp

÷

dimer

÷

(3) +

Trp

+

2 Trp

(4) (5)

dimer

÷

H20

(6) +

H20

(7)

Assuming that radical recombinations (reactions (4), (5), (6) and (7)) proceed at approximat e l y equal rates, one t h i r d of tryptophan-OH adducts w i l l react with tryptophan-H adducts to regenerate tryptophan. Since G(Trp-OH) = GOH=2.8 and G(Trp-H)=G e_ + GH = 3.25, to a f i r s t approximation one t h i r d of tryptophan H-adducts w i l l be involveda~n the reaction (7) as w e l l . The decomposition y i e l d of tryptophan would then equal~ G(-Trp) = I / 2 GOH + 2/3 (Ge~q + GH) = 3.56

(8)

From the l i n e a r part of curve A (Fig. 4) the i n i t i a l decomposition y i e l d G°(~Trp) = 3.35 was obtained, what is in a good agreement with calculated value. The decrease of the decomposit i o n y i e l d with absorbed dose could be ascribed to the reaction of tryptophan radicals with some stable products. Such reactions should regenerate tryptophan as G(-Trp) s t e a d i l y decreases. Based on the possible structure of stable r a d i o l y t i c products, and t h e i r chemical properties (Jovanovid and Josimovid, 1982), we assumed that dimeric rather than monomeric products are involved in regeneration reactions. I f the neutral s o l u t i o n , which turned yellow under i r r a d i a t i o n , is a c i d i f i e d , i t changes colour to red, and a well defined maximum at 500 nm appeared. On the basis of the r e s u l t s obtained f o r a c i d i c tryptophan solutions (Jovanovid and Josimovid, 1982), i t is very l i k e l y that the same product, which was ascribed to a dimer, is formed in both acid and neutral media under i r r a d i a t i o n . Since neutral s o l u t i o n does not display a d i s t i n c t absorption due to t h i s product, we have measured the absorbancy at 500 nm a c i d i f y i n g the i r r a d i a t e d s o l u t i o n . The absorbancy was measured in a dose range from 0.25 to I0 kGy, and r e s u l t s obtained are presented by curve B in Fig. 3. Curve B has j u s t the opposite trend from curve A which represents G(-Trp) vs. i r r a d i a t i o n dose. For higher doses, same as the decomposition y i e l d , the measured absorbancy reaches an almost constant value showing t h a t the formation and destruction of t h i s product is in e q u i l i b r i u m . These f i n d i n g s support our assumption that a product of d i meric structure is involved in reactions with tryptophan radicals. The dependance of tryptophan decomposition on absorbed dose is f o r N20 saturated solutions presented in Fig. 5. As can be seen the decomposition y i e l d decreases with increasing i r r a d i a t i o n dose. However, from the l i n e a r part of the curve given in Fig. 5 the i n i t i a l y i e l d G°(-Trp)=4.75 was obtained. In the presence of N20 p r a c t i c a l l y the only reactive species present during i r r a d i a t i o n are OH r a d i c a l s : N20 +

eaq

÷

and therefore G(OH)=GOH + Ge_

N2

+

OH +

OH

(9)

= 5.5 while GH=O.55. According to t h i s the reaction (7) can be

neglected, and the decomposition y i e l d (reactions (4), (5) and ( 6 ) ) w i l l equal: G(-Trp) = 3/4 GOH +

GH = 4.67

(I0)

Radiation induced decomposition of tryptophan

0.2

0.4

0.6

0.8

I

I

I

I

1.0

777

D [kGy]

I

5

ct E 02

I

I

I

I

I

2

4

6

8

10

Fig, 5 N20-saturated solutions of tryptophan: decomposition yield as a function of irradiation dose; o - 5xlO-2 mol dm- ; x - 10-3 mol dm'~; • - lO-Z mol dm-3. The calculated yield is in a good agreement with the i n i t i a l yield experimentally obtained. The decrease of the decomposition yield of tryptophan is in the presence of N20 same as in deaerated solutions,very l i k e } y due to the reactions of tryptophan radicals with stabile radiol y t i c products. Results obtained by chromatographic analysis of N20 saturated solutions of various tryptophan concentrations (Jovanovid and Josimovid, 1982) support this assumption. The less pronounced decrease observed under these conditions (Fig. 5) indicates, however, that in the presence of N20 less tryptophan is regenerated. Results obtained in the present work show that although tryptophan reacts rapidly with primary radicals produced in the radiolysis of water, i t s decomposition is e f f i c i e n t l y supressed. This is due to the reactions of tryptophan radicals with stable radiolytic products and is especia l l y pronounced in deaerated :olut~ons. Under these conditions even at r e l a t i v e l y low absorbed doses the decomposition yield significantly decreases; at D=lO kGy the decrease of about 70 % was observed. According to this i t can be concluded that among stable products dimers formed by recombination of tryptophan-H adducts are most effective in regenerating tryptophan. Despite this complex reaction mechanism, the fact that tryptophan under gamma irradiation decomposes with a rather low y i e l d , might be very useful with regard to the radiation s t e r i l i zation of protein-rich food. REFERENCES

Adams, G.E. (]972), Radiation chemical mechanisms in radiation biology. In M.Burton, and J.L. Magee (Eds.), Advances in Radiation Chemistry, Vol. 3, Wiley-lnterscience, New York. pp. 125-208. Armstrong, R.C., and A.J.Swallow (1969), Pulse- and gamma radiolysis of aqueous solutions of tryptophan, Radiat.Res., 40, 563-579. Boguta G., and A.M. Dancewicz(~Sl), Radiation-induced dimerization of tyrosine and glycyltyrosine in aqueous solutions, Int.J.Radiat.Biol., 39, 163-174.

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LJ. JOSIMOVIC,

S. JOVANOVlC AND I. CUDINA

Braams, R. (1966), Rate constant of hydrated electron reactions with amino acids. Radiat.Res., 27, 319-329. Draga~d, I.G., and Z.D.Draganid (1971), The Radiation Chemistry of Water. Academic Press, New York. Faraggi, H., and A. Bettelheim (1977), The reaction of the hydrated electron with amino acids, peptides, and proteins in aqueous solutions: tryptophyl peptides..Radiat.Res., 72, 81-88. Jovanovid, S., and Lj.Josimovi~ (1982), Gammaradiolysis of aqueous solutions of try~ophan. In P.Hedvig (Ed.), Proc. 5th Tihany Simposium on Radiation Chemistry, Akademiai Kiado, Budapest, in press. Nakken, K.F. (1964), The action of x-rays on dilute solutions of p-aminobenzoic acid. Radiat. Res., 21, 446-464. Signh,-~., ~ J . B e l l , G.W.Koroll, W.Kremers, and H.Singh(1981), Reaction of tryptophan with singlet oxygen, hydroxyl radical, and superoxide anion. In M.A.J.Rogers, and E.L.Powers (Eds.), Oxygen and Oxy-Radicals in Chemistry and Biology, Academic Press, New York, pp. 461-473.