Radical-induced oxidation of glutathione in alkaline aqueous solution

0146-5724/88 $3.00+0.00 Pergamon Press plc

Radiat. Phys. Chem. Vol. 32, No. 2, pp. 163-167, 1988 Int. J. Radiat. Appl. Instrum. Part C

Printed in Great Britain

R A D I C A L - I N D U C E D O X I D A T I O N OF G L U T A T H I O N E IN A L K A L I N E A Q U E O U S SOLUTION TRYGVE E. ERIKSEN and GUNILLA FRANSSON Department of Nuclear Chemistry, The Royal Institute of Technology, S-100 44 Stockholm 70, Sweden (Received 10 June 1987)

Abstract--The reactions of N~, CO~- and "ROH radicals with glutathione have been studied in basic aqueous solution. Whereas only the sulphur centered GS' (GSSG:) radical is produced by the one-electron oxidants N; and CO~-, a strongly reducing radical is formed in pH-dependent yield by "ROH and "OH. The data are explained by pH-dependent conformational changes of glutathione on deprotonation of the amino and thiol groups.

INTRODUCTION In earlier papers tl'2) we described our studies on the OH induced oxidation of the sulphydryl containing tripeptide glutathione, 7-glu-cys-gly (GSH), in basic aqueous solution. One important aspect of our studies was the observation that the nature and yields of the intermediates formed are highly dependent on the protonation state of the - S H and - N H ~ groups, having pKa values 9.2 and 9.5 (3'4) respectively.

neous decarboxylation "~-amino" radical

and

formation

of

an

'CH--CH2-- I

I

NH2 In a 7-radiolysis study (2'6) it was found that whereas the decarboxylation of ophthalmic acid (GCH 3) and S-methylated glutathione (GSCH3) is quite effective this reaction is of minor importance in the case of

0

0

II

II

- O2CCH--CH2--CH2--C - - N H - - C H - - C - - N H - - C H : - C O f . INH~"

(1)

~H2

I

SH The reactions expected to be dominating for OH induced oxidation of glutathione can, in analogy with the general reaction scheme for thiols, be summarized by reactions (2-4). "OH + GSH---~GS" + H20

(2)

"OH + GS----~GS" + O H -

(3)

GS" + G S - ~ G S S G =

(4)

The formation of the GS' radical in pulse radiolysis experiments was followed by monitoring the well characterized optical absorption of GSSG= with 2m~x at 420 rim. In the pH range 8-10.5 the reaction mechanism was, however, found to be more complicated than described by reactions (2-4). The most important feature is a decrease in the GS" yield and the formation of two strongly reducing radicals. The deearboxylation of amino acids has been discussed by MSnig et al. in a recent paper°) and the suggested mechanism involves "OH addition to the lone pair of the amino-nitrogen followed by sponta-

glutathione, G(CO2) being 3.3, 3.3 and 0.5 molecules/100 eV respectively at pH 10.5. The lower CO2 yield for glutathione as compared to G C H 3 and GSCH3, with the same backbone structure, reflects the higher overall reactivity of GSH towards "OH due to the sulphydryl group. In studies of electron transfer from radicals formed on pulse radiolysis of N20 saturated solutions of GSH to p-nitro acetophenone (PNAP) the yield of strongly reducing radicals was found to correspond to G ( P N A P - ) = 2.8 at pH 10.2 i.e. another strongly reducing radical is formed in addition to the ~-amino radical. In experiments with N 2 0 saturated solutions of GSCH 3 and G C H 3 we found CO2 and PNAP= to be formed in equal amounts. The formation of a reducing radical by hydrogen abstraction from the carbon in ~-position to the amino group, as suggested by Sjoberg,") is thus not likely and the formation of a carbon centered radical by a mechanism involving pH-dependent conformational changes of glutathione and hydrogen 163

164

TRYGVE E. ERIKSEN a n d GUNILLA FRANSSON

abstraction from the carbon in ~t-position to the sulphur has been discussed in a recent paper, t2) In the present paper we give a detailed report on the reactions of N 3, C O ; a n d hydroxy-alkyl radicals ('ROH) with glutathione in alkaline aqueous solution and the effect of pH on radical yields.

O

"" 2 X c~

C)

1

EXPERIMENTAL zeo

300

34o

3so

42o

46o

500

Glutathione (GSH) (Fluka p.a.), Ophthalmic acid (GCH3) (Serva p.a.), S-methylated giutathione (GSCH3) (Sigma p.a.), N,N,N',N'-tetra methyl-l,4 phenylene diamine (TMPD) and all other chemicals (Merck p.a.) were used as received. The solutions were prepared from water which was doubly distilled in quartz and made oxygen free by purging with Ar (AGA-SR-quality, containing < l ppm 02). The pH was adjusted with NaOH and HC104 and the solutions were saturated with N20 (AGA) immediately before irradiation. The added N 20 converts the reducing hydrated electrons into 'OH radicals via N20 + e~q---~N2 + 'OH + O H - . In solutions containing high concentrations of azide, carbonate or an alcohol (e.g. methanol) the 'OH radicals are converted quantitatively into N], CO;, or "CH2OH radicals via 'OH + N f , CO ]- , CH3OH--~N3, CO~CH2OH + O H - , H20. These reactions take place prior to other possible reactions and thus >90% of all primary reactive species are "OH, N3, C O ; or 'CH2OH respectively. The pulse radiolysis experiments were carried out by applying 0.2-1/~s long electron pulses from a 7 MeV microtron accelerator. The dose per pulse (5-20 Gy) was measured with a secondary emission chamber, which was previously calibrated with a N20 saturated aqueous 10 -2 M KSCN solution with GE(CNS)f=4,64 x l04 at 500nm. Details of the pulse radiolysis set up are given elsewhere. (7) Data storage and analysis were based on a Biomation 8100 transient recorder and a PDP 11/40 computer. The signal/noise ratio of small signals was improved by superposition of several signals. Steady state irradiations were performed in an AECL 6°Co source with dose rate 43 Gy m i n - ' . The CO2 yield was determined gas chromatographically using an AGAArgograph equipped with a 6 ft x 1/8 in., 150-200 mesh Porapak Q column, t6) All experiments were carried out at ambient temperature (20°C).

where OD0 is the TMPD "+ absorption when (GSH) = O. The rate constants obtained are summarized in Table 1. The optical absorption at 420 and 280 nm at varying pH following the reactions between glutathione and pulse radiolytically produced N3 and CO~- are depicted in Fig. 2.

RESULTS AND DISCUSSION

Table 1. Reaction parameters

The transient absorption spectra obtained on pulse radiolysis of N20 saturated basic solutions of glutathione are dominated by an absorption band peaking at 420nm ( E = 8 x I 0 3 M -~ em -~) unambiguously assigned to the GSSG:- radical ion and an absorption band with a maximum at about 270 nm. The initial absorption in the 270 nm region is followed by an exponential build up of an absorp-

X(nm)

Fig. I. Transient spectrum on pulse irradiation of N20 saturated 5 x 10-3mol dm -3 GSH solution at pH 10.5. Dose 10 Gy. ©. End of pulse: A, 300#s after pulse.

tion band with continuously increasing intensity from 350nm towards u.v. (Fig. 1). The change in the 270 nm absorption is not concurrent with the decay of the G S S G : absorption and has been assigned to a monomolecular transformation of a primary formed reducing radical. (~) To determine the rates for the reactions between radicals and glutathione the rate of the complex formation GS" + G S - ~ G S S G ~ (k4 = 6 x l0 s M -j s -Z, k_ 4 = 1.4 x 105s - j ) must be taken into account. In the case of 'OH,N~ and CO~- the complex formation will be rate determining at all pH, whereas for the alcohol radicals the rate constants can be determined directly from the temporal change in the 420 nm absorption. The rates for the reactions of N 3 and C O ; with glutathione were measured in competition kinetic experiments using N,N,N',N'-tetra methyl-l,4 phenylene diamine. (TMPD) is easily oxidized to TMPD '+ which has a strong absorption band peaking at 645 nm, (s) E = 13000 M-~cm ~. By titrating TMPD into N20 saturated GSH solutions containing high concentrations of N f or COlrespectively and monitoring the TMPD "+ yield the rates were calculated using the equation 1/OD = 1/OD 0 +

1/ODo[kGsH.(GSH)/kTMPD.(TMPD)]

Reactions "CH2OH+ GSH-*GS" CH3CH2~OH+ GSH--*GS" (CH3)2~H2t~OH+ GSH--,GS' N~+ TMPD--TMPD"+ CO~+ TMPD-*TMPD"+ N~+ GS--*GS" CO~-+GS -*GS"

Rate constants (M ms ~) 1.0 x l0s 1.4 x l0s 5.0 x l0T (5.4 + 0 . 2 ) × 10 9 (4.5 _+0.2) x l09 (2.7 +0.3 × l09 (7.1 +0.6) x l0s

Oxidation of glutathione

165

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A

A

A

•o4 ~<

x

3

8 c 0

o .Q <~

oz .D

<1

2

/-. /o'" 8

A

9

10

11

12

I

I

7

8

[

I

I

I

I

9

10

11

12

pH

pH

Fig. 2. Optical absorption on pulse irradiation of N20 saturated 5 x 10-3 mol dm -3 GSH in 0.5 mol dm -3 azide and carbonate solution. Dose 10 Gy. Azide: (©) 420 nm; (0) 280 rim; carbonate: (/X) 420 nm;(&) 280nm. The 420 nm absorption, recorded directly after the pulse, has been corrected for the equilibrium (4) using /(4=4.4 x I & M -~. The absorption at 280nm was recorded 300/is after the pulse, i.e. after the build up. The optical absorption at 420 and 280 nm obtained in experiments with alcohol radicals produced on pulse radiolysis of aqueous solutions of glutathione containing high concentrations of methanol, npropanol and t-butanol are depeicted in Figs 3-5. In the pH range 8.5-9.5 the decrease in the 420 nm absorption is paralleled by increasing absorption at 280 nm. At pH > 10 both absorption bands decrease with increasing pH. The absence of the long lived 280 nm absorption on "OH induced oxidation of ophthalmic acid

Fig. 4. Optical absorption on pulse irradiation of N20 saturated 5 x 10-3 GSH solution containing 2 mol dm -3 n-propanol. Dose l0 Gy. A, 420 nm; &, 280 nm. (GCH3) and S-methylated glutathione (GSCH3) in basic solution(2) clearly indicates that the sulphydryl group is directly involved in the formation of the reducing radical. The "OH induced decarboxylation of GSH, GSCH a and GCH 3 has been shown to take place with G values of 0.5, 3.3 and 3.3 respectively. The low CO2 yields for the reactions of N~, and alcohol radicals with GSH, GSCH 3 and GCH 3 (Table 2) show that the formation of the amino radical

"CH---CH2-1 I NH 2

5x

/x-->.xx\ x

o

x

×

%

4 o

x

\×

3

B O .O

o

< 1

/ I

I

I

I

L

I

7

8

9

10

11

12

pH

Fig. 3. Optical absorption of pulse irradiation of N20 saturated 5 x 10-3mol dm -3 GSH solution containing 2 mol dm -3 methanol. Dose 10 Gy. ©, 420 nm; 0 , 280 nm.

I

I

I

1

I

I

7

8

9

10

11

12

pH

Fig. 5. Optical absorption on pulse irradiation of N20 saturated 5 x 10- mol dm -3 GSH solution containing 2 mol dm -3 t-butanol. Dose 10Gy. x, 420nm; ®, 280nm.

166

TRYOVE E. ERIKSEN a n d GUNILLA FRANSSON Table 2. Yields of CO 2 and radicals

Reaction

pH

OH + GSH

G(GSSG:-)

G(Red)i"

G(CO:)

6.2 4.0

0 2.2

-----

-----

6.8 6.2

0 1.2

0 0.5 0 3.3 0 3.3 0 0.3 0.7 0

7.8 10.6 <8.0 10.6 <8 10.6 7.8 10.6 <8 7.8 9.8 10.6 10.7

GSCH~ GCH 3 N~ + GSH GSCH 3 'ROH + GSH

GSCH 3

--

--

5.8 1.2

0 1.7 1.0

--

--

0.3 0.4

The deprotonation reactions of the - S H and the - N H ~ groups are claimed to be relatively independent of each other °,4) but Peters Cm'") demonstrated, using relaxation kinetic methods, that considerable intramolecular proton exchange takes place in the half dissociated form and proposed a reaction scheme for the deprotonation assuming five microscopic dissociation constants. Experimental results from detailed studies by several groups °vl6) on complex formation of metals with sulphur containing ligands have been interpreted using similar deprotonation schemes. ,

t Assuming ~ = 8100 M - era- ' at 280 nm. "ROH denotes radicals from methanol, n-propanol and t-butanol.

G

\

is a minor process and gives support to the mechanism given by Asmus and coworkers. The stability of GSH to oxidation relative to other sulphydryl containing molecules have been studied extensively and Calvin (9) first proposed that this stability results from the interaction of the - S H group with the carbonyl of the y-peptide linkage. The hydroxythiozolidine derivative formed could be stabilized by interaction of the deprotonated amino group and water elimination leading to a pH-dependent dynamic equilibrium and a mechanism for ring opening and closure CH~CH~ r~ HS

_ ICH O2C \ N I - ~

CH~

C. ~ N

o

.lt

/ H

(5)

H S / .CH~-CH~ -..<.y/ CH -O~C~ \

CH~

-..<

C. ~

NH~

,-,

~,

CH-- ~.-NH--CH~'-COT, z z

/

~N

r~

CH--~-NH--CH~-COZ z -2

/

(6)

H

1L-HzH20 /

CHa"CH.,

~"~

O C CH

-2 ~ ~N /

/

S

CH~

~

C

~N

H

v,

CH--C-NH-CH2--CO2

/ (7)

H

In a recent paper t2) on the OH induced oxidation of GSH we proposed that a reducing carbon centered radical could be formed by OH addition to the sulphur or hydrogen abstraction from the carbon in position to the sulphur i.e.

)c_/

s

%

x.#.

H

o

s

,, , CH-C~' .OH,~ C/ ~

aH O \_ ,~. ./~--~--/ H

(8)

5-

/NH~

°,

/NH

2

71 / NH G \

2

SH

~ /

(9)

It has been concluded °5) that the sulphydryl is approximately 1.6-1.9 times as acidic as the glutamyl amino group. The pH-dependent distribution of glutathione among the various protonated forms based on the microscopic dissociation constants give a maximal fraction of NHEGSH (0.2) at approximately pH 9-9.5 "5) and the hydroxythiazolidine form, given in formula (8), should have the same pH dependence. The N~ and CO~- radicals are efficient one electron oxidants and, in contrast to OH, have little tendency to form intermediate adducts. These radicals are therefore expected to react predominantly with the NH~-GS- form, thus producing GS'. The experimental results are in accordance with this assumption. The slight decrease in the GSSG ~ yield in the pH range 8.5-10 corresponds to G(H') and clearly indicates that the reducing radical is not formed in a secondary reaction following electron transfer from the dissociated thiol group. The decrease in the 280 nm absorption at high pH in the experiments with alcohol radicals can not be explained by deprotonation of the hydroxygroup of the radicals as the pK, values vary by at least one unit. "7) The "ROH radicals might be expected to react preferentially with the NH2GSH form and produce the reducing radical (8) with maximum yield at pH 9-9.5. The experimental data in this work thus give support to the recently proposed reaction mechanism involving addition to the sulphur or hydrogen abstraction from the carbon in position to the sulphur leading to the formation of a carbon centered radical.

Oxidation of glutathione The reaction mechanism is thus tentatively written, assuming N H 2 G S H to have the structure given in reaction (7). R"

NH + GSH-.GS" NH~ GS-

•oH, N~,col

(10) , GS"

"OH , ~-aminoradical

(11) (12)

NH2GSH-~ OH', "ROH

radical (8)

(13)

R" = "OH, N 3, CO~-, "ROH. Acknowledgements--The authors would like to thank Drs J.

Lind and G. Merenyi for valuable discussions. The financial support of the Swedish Natural Science Council is gratefully acknowledged. REFERENCES

1. L. Sjoberg, T. E. Eriksen and L. Revesz, Radiat. Res. 1982,89, 255. 2. T. E. Efiksen and G. Fransson, Z Chem. Soc. Perkm TranslI, (accepted).

167

3. G. Jung, E. Breitmaier and W. Vcelter, Eur. J. Biochim. 1972, 24, 438. 4. T. N. Huckerby and A. J. Tudor, J. Chem. Soc. Perkin. Trans H 1985, 759. 5. J. Monig, R. Chapman and K.-D. Asmus, J. Phys. Chem. 1985, 89, 3139. 6. G. Fransson and T. E. Eriksen, (to be published). 7. T. E. Eriksen, J. Lind and T. Reitberger, Chem. Script. 1976, 10, 5. 8. J. Steigman and W. Crankright, J. Am. Chem. Soc. 1970, 92, 673. 9. M. Calvin, in Glutathione (Edited by S. Colowick, D. R. Schwarz, A. Lazarow, E. Stadman, E. Racker and H. Waelsch). 1954. 10. F. Peters, Dissertation, Braunschweig, 1971. 11. G. Maass and F. Peters, Angew. Chem. Int. Ed. 1972, 11, 429. 12. R. B. Martin and J. T. Edsall, Bull. Soc. Chim. Biol. 1958, 40, 115. 13. D. L. Rabenstein, J. Am. Chem. Soc. 1973, 95, 2397. 14. J. E. Letter Jr and R. B. Jordan, J. Am. Chem. Soc. 1975, 97, 2381. 15. D. L. Rabenstein and R. Guevremont, Metal Ions in Biological Systems (Edited by H. Sigel) pp. 103-141. Marcel Decker, New York, 1979. 16. R. S. Reid and D. L. Rabenstein, J. Am. Chem. Soc. 1982, 104, 6733. 17. A. J. Swallow Prog. React. Kinet. 1978, 9, 210.