5272
J . Phys. Chem. 1994, 98, 5212-5278
Characterization of Transients Produced in Aqueous Medium by Pulse Radiolytic Oxidation of 3,5-Diiodotyrosine T. N. Das' and K. I. Priyadarsini Chemistry Division, Bhabha Atomic Research Centre, Bombay 400 085, India Received: November 2, 1993; In Final Form: March 3, 1994"
Using the technique of pulse radiolysis, oxidation studies of 3,5-diiodotyrosine (DTR) with radicals generated in phosphate-buffered aqueous medium are reported. Secondary oxidizing radicals Sod'-, Br', N3*, Tl(OH)+, (DMS)2+, CCl302*, (CNS)**-, C12'-, and T12+oxidize D T R in neutral, mildly alkaline, and acidic solutions by an electron-transfer process to generate a phenoxyl-type radical transient with peak absorptions observed a t 280 and 350 nm and e350 = 4400 f 300 dm3 mol-' cm-l. In acidic pH, another transient peak a t 310 nm with 6310 = 3500 f 150 dm3 mol-' cm-l and a pKa value of 6.5 is observed due to the simultaneous formation of a radical cation intermediate. The formation rate constant values of these two transients lie between 5 X lo8 and 2 X 1010 dm3 mol-' s-l for these secondary oxidizing radicals as well as for the primary 'OH/'O- radicals. The 'OH radical additionally generates a secondary transient in deoxygenated solutions (kf 1.7 X lo7 dm3 mol-' s-l a t p H = 8) with its peak absorption centered a t 650 nm, which shows two pK, values of 3.9 and 7. It has been assigned the dimer-type structure arising from the D T R reaction with its hydroxyl radical adduct transient. From the equilibrium studies with dehydrocysteine (thiyl) as the oxidizing radical, the reduction potential value for the couple DTR+/DTR has been measured to be +0.78 f 0.04 V vs NHE.
Introduction
Experimental Section
In vertebrates, the thyroid gland concentrates absorbed iodine (as iodide from the plasma) and uses the same for synthesis of growth hormones triiodothyronine (T3) and thyroxine (T4) that are essential for metabolism, expression of specific genes at nuclear level, and a host of other biological activities.1,2 During synthesis, iodination of tyrosine residues of the protein thyroglobulin leads to the formation of the intermediate 3,5-diiodotyrosine (DTR). During normal operation of a nuclear reactor as well as under any accident scenario, radioactive iodine, 1311, which is a major fission product, gets released into the e n v i r ~ n m e n t . In ~ ?case ~ of excessive release of 1311 (natural half-life = 8 days5),the chances of mammalian population in the surrounding areas getting affected become very high. Environmental I3lI, like any other isotope of iodine, after entering the living animal body quickly finds its way into the thyroid. It has an effective biological half-life of 12 days and thyroid residence time of 120 days.5 Thus, a large amount of radioactive iodine absorption may lead to a situation where the conditions in the thyroid gland as well as in the surrounding plasma resemble an aqueous matrix under steady-state radiolysis. In other words, it may lead to a situation whereradiation-induced chemical effects due to primary and secondary radicals derived from water radiolysis gain importance. Thus, synthesis and stability of DTR, T3, and T4 may be affected. It is therefore necessary to understand the subsequent chemical changes of these species in aqueous medium under redox conditions and gain insight for any possible control of the resulting damage and discover repair mechanisms, if any. In the available literature, although some studies are reported on the actions of a- and X-radiation on DTR at lwo pH6a.b and also on steady-state y-radiolysis of DTR in aqueous-alcohol solution^,^ no details are yet available on the transient behavior arising from radiationinduced chemical changes of DTR, T3, or T4 in aqueous medium. In this paper, first in the series of pulse radiolysis studies on the redox behavior of these compounds of interest, detailed studies on oxidation of DTR with primary and secondary radicals derived in buffered aqueous medium and the characteristics of the resulting transients are discussed. The one-electron reduction potential value for the couple DTR+/DTR at neutral pH is also presented.
Details of the pulse radiolysis experimental setup used have been described earlier.8a Single pulses of 7-MeV electrons were used in the experiments, and the selected pulse widths were of either 50-11s or 2-ps duration depending on the nature of information sought. Typical maximum doses obtained with 50ns pulses were 15 Gy, and for 2 - ~ pulse s it was 7-8 times higher. The dosimetry was performed with air-saturated 0.01 mol dm-3 KCNS solution with a G(CNS)2-t value of 2.23 X 1 0 4 m2 J-1 at 500 nme9 (All subsequent G values are reported in units of pM/Gy.) For laser flash photolysis experiments, 248-nm photons of 20-11s duration and 50-mJ energy from a KrF excimer laser were used, and the energy calibration was performed using biphenyl solution in methanoLSb Transient spectral measurements were performed using a continuous flow arrangement. The kinetic spectrophotometric detection systems covered the wavelength range from 250 to 800 nm. Cells with optical path length of 1 cm were used for these measurements. For steady-state y-radiolysis experiments a 6oCo source was employed, and the dosimetry was carried out following the standard Fricke method. 3,5-Diiodotyrosine (DTR) obtained from Eastman Kodak was used without further purification. All other chemicals used were of AR or similar grades available. All solutions were prepared in water purified previously be passing deionized water through Barnstead Nanopure cartridge filtration systems to remove ionic and organic impurities. Stock solutions of DTR were prepared by shaking excess of it in deoxygenated water for couple of hours and subsequently filtering off the undissolved residue. At room temperature (25 "C),the DTR concentration obtained was 3 X 10-3mol dm-3, and it remained stable for a long time in the dark. Iolar grade gases N2, 0 2 , and N20 obtained from M/s Indian Oxygen Ltd. were used for saturating the aqueous solutions employing appropriate pretraps as required. Unless stated otherwise, all solutions were prepared in 0.01 mol dm-3 phosphate buffer (equimolar mixture of Na2HP04.H20and KH2P04),and the pH's of the solutions were adjusted to the required value with either HC104or NaOH. For transient spectral measurements, the concentration of DTR used as approximately 2 X 1 0 4 mol dm-3 while for kinetic studies the same was varied between 5 X
Abstract published in Aduance ACS Abstracts, April 15, 1994.
0022-3654/94/2098-5212$04.50/0
0 1994 American Chemical Society
The Journal of Physical Chemistry, Vol. 98, No. 20, 1994 5273
Pulse Radiolytic Oxidation of 3,5-Diiodotyrosine 10-5 and 1 X 10-3 mol dm-3. All UV-vis ground-state spectra were measured on a Hitachi Model 330 spectrophotometer. DTR shows prominent absorption bands between 270 and 330 nm. In acidic pH the peak is centered at 286 nm with a broad profile. It shifts to 310 nm in neutral and alkaline pH, and its profile changes to a sharp one. Above 340 nm, DTR does not show appreciable absorption. With the change of solution pH value from 1.5 to 12, successive deprotonation from COOH, OH, and NH3+ groups have been reported to occur with respective pKa values 2, 6.4, and 8.7.10 The presence of two electronwithdrawing iodine atoms a t positions ortho to the phenolic OH in DTR are responsible for lowering the pKa value due to it (OH) as compared to tyrosine (pK,(OH) = 10.07) where such substitutions are absent. On the other hand, pK, of either COOH or NH3+ groups is not affected to the same extent by the iodine substitutions as compared to the case of tyrosine.IO Similarly, the absorption characteristics of the iodinated phenolic chromophore in DTR are not affected due to the protonation of these two groups. In the subsequent oxidative studies, the presence of these two iodine atoms at the 3- and 5-positions is expected to affect the transient properties as compared to the case of tyrosine. One-Electron Oxidation. For oxidation studies using either *OH/*O-or other secondary radicals, generated via 'OH (or e,, for SO4- radical) reaction of suitable substrates, at acidic or neutral/alkaline pH, N20 was used as e,, scavenger according to eq 1 (at solution p H below 3, correction for the partial loss of e,, due to its protonation step was made following the reactivity parameters of these individual reactionsll).
eaq
-
-.
+ N,O
H20
N,
+ '0--
OH- + 'OH
(1)
While the G ( 0 H ) value has been taken as 0.57 pM/Gy under N2O saturation, individual G(radica1) has been calculated from the reactivity factor (~(oH+s)[S]) for each substrate (S) employed.12 Under this condition the G(H) value is 0.06, and any effect due to it was neglected. In acidic pH, solutions were sometimes saturated with oxygen, as discussed later, to scavenge both H* and eaq-. Under this condition the G(HO2) value is larger than G ( 0 H ) (G(0H) = 0.29 and G(H02) = 0.35)." For spectral measurements, the effect due to the HO2' radical was subtracted to obtain a qualitative result of DTR reaction with oxidizing radicals at this pH. Various secondary oxidizing radicals used were generated by the reaction of 'OH radicals or eaq-with appropriate solutes according to well-established reactions13aand are briefly described in eqs 2-14.
X-
+ 'OH X'
-
X'
-
+ OH-
+ x- x,-
(2) (3)
Here X-represents the halides C1-, B r , and I- and pseudohalides N3- (reaction 2 alone) and SCN- a t concentration of 5 X 10-2 mol dm-3 for C1- and 2 X 10-2 mol dm-3 for others. While for Clz-, Orsaturated acidic medium (pH = 2) is required due to its slow reaction with 'OH at higher pH, for N3*, N2O-saturated solution at pH > 4 is essential as below this pH value HN3 reaction with *OHis not quantitative and it also reacts with Other radicals derived from B r , SCN-, and I- can be studied in acidic, neutral, or mildly alkaline solution saturated with N20. Thallium (Tl+), used at the concentration of 5 X 10-3 mol dm-3, gives rise to two different oxidizing radicals in acidic and near neutral pH as shown in eq 4.14
T1+ + 'OH
-
pK. = 4.6
OH- + T12+ + Tl(OH)+
(4)
Bromineatom was formed by reaction 5 in N2O-saturated solution
a t pH 7 and at 1,2-dibromoethane (DBE)15 concentration 4 mol dm-3. CH2BrCH$r
+
*OH
CH2=CHBr
-I
CH2BrCHBr'
+ H20
X
(5)
+ Br*
Dimethyl sulfide (DMS), a t concentration level of 1 X 10-2mol dm-3, on oxidation with 'OH produced dimerized dimethyl sulfide cation radical in neutral and acidic solutions according to eqs 6 and 7.16
CH3SCH, + *OH
-
CH3S'+CH3+ OH-
-
CH3SCH3+ CH3S'+CH3
(CH3SCH3),'+
(6) (7)
Haloperoxyl radical CC1302'(G = 0.64) was produced from the reactions of H', 'OH, and e,, with C C 4 and isopropyl alcohol in oxygenated solutions at neutral p H as represented in eqs 8-1 1.I7
CCl, (CH,),CHOH (CH,),COH'
-
+ eaq-
+ H'('0H) + CCl,
-
CC1,'
CCl,'
+ C1-
(CH,),COH'
(8)
+ H,(H20) (9)
CCl,'
+ 0,
+ (CH3),C0 + HCl
-
CC130,'
(10) (1 1)
Sulfate radicals were produced from e,, reaction with 5 X mol dm-3 S2082-in deoxygenated solutions at neutral pH as represented in eq 13.13, The 'OH radicals were scavenged by 0.1 mol dm-3 tert-butyl alcohol as in eq 12. The resulting radical was observed to be unreactive toward DTR.
eaq
-so,- + so:-
- + s,o,*-
For equilibrium studies, the thiyl radical was produced from *OH reaction of cysteine under N2O saturation at neutral pH in 5 X l e 2mol dm" phosphate buffer solution, as represented in eq 14. A higher concentration of buffer was necessary to keep the solution p H in check for high cysteine concentrations employed. %H
/coo-
+ HSCYCH
"H3+
-
/coo-
*SCH,CH
+ HO ,
(14)
"H3+
Results and Discussion
In Table 1, the spectral and kinetic parameters of the transients produced from DTR with various oxidizing radicals are listed. The transient spectral data have been corrected for ground-state DTR bleaching between 250 and 350 nm in all cases unless stated otherwise. Individual studies are discussed below in detail. Reactions with Secondary Oxidizing Radicals. DTR oxidation with chloride and azide radicals produced a transient absorption spectrum with peaks at 280, 310, and 350 nm, with a shoulder at 405 nm (Figure 1). With theseoxidizing radicals, the formation rates of transients, with peak absorptions at 280, 310, and 350 nm, were observed to approach the diffusion-controlled limit over the working pH range indicating their primary nature. Their respective rates were estimated from the buildup of the signals at these peak wavelengths. In neutral and alkaline pH, due to substantial DTR ground-state absorption at 3 10 nm, bleaching signals were obtained around 3 10 nm. While qualitative kinetic measurements were performed with the same, for spectral
5214
The Journal of Physical Chemistry, Vol. 98, No. 20, 1994 SCHEME 1
TABLE 1: Spectral and Kinetic Parameters of Transients Produced from DTR Oxidation oxidizing radical
kf(350 nm)/109 ldm3 mol-' s-'1
3500 (310 nm)
2.6. OH 1.8 (3 io nm) 8.0, pH 2 19, pH 8 5.1, pH 4 8.6, pH 9 6, PH 8 1.7, pH 8 0.034, pH 7
4130 4270 4300 4230 4540 3700
4.0, pH 7 1 .O,pH 2
(3660)4 4000 3460 (310 nm)
0.013, pH 8 (650 nm) 0.0064, pH 4 (650 nm) 14, pH 4 23, pH 8 5.5, pH 4 (310 nm) 2.1, pH 12.8
0 V
a a
0.016 0.01 2
3850
1 Jb I
0.008
0.004 0.000
t1
240
r
H~N;
decay [s-l] 2klcld (or k d c k1#(310 nm) = 1 . 1 x 104 6.3 X lo5 7 . 3 x 105 5 x 105
CH
[-e-] (-H+ at pH < 6.4)
H I@
-
6 X lo5 4 x 105 k1,(350 nm) = 9 x 102 6.6 X lo5 7 x 105 kf4(310 nm) = 1.5 x 104 kf4(650 nm) = 2.5 X lo2
,coo-
6.-
OH
DTR
J H ~ N +coo\c'H
I
CH2
H@Y I
0. phenoxyl-type radical
I
4540 4450 3200 (310 nm)b
0.020 -
W
c(350 nm) Idmj mol-' cm-'1
Das and Priyadarsini
7.5 x 105 (av for pH 2-1 1) kf4(310 nm) = 1 x 104 5.7 x 105
a
U
1
,
295
350
,I 405
460
W a v e l e n g t h (nm) Figure 1. Transientspectra of semioxidized DTRobtainedusing secondary radicals at neutral and acidic pH at a normalized G(radica1) = 0.57 and and TI2+ (X). At pH = dose = 10 Gy. At pH = 2, radicals Cl2- (0) 8, radicals Br' (0) and N3' (0).
measurements, suitable correction was made assuming negligible radical-radical reactions at the applied dose (experimentally set reactivity parameter of radical + DTR reactions >> reactivity parameter of all radical + radical reactions). When Br2- was used as the oxidizing radical, the transient absorption peak overlapped with the absorption maxima of Brz- at 360 nm; thus, quantitative evaluation of the formation kinetics or absorption parameters was not possible. The decay trace at 350 nm then showed an almost time-independent flat profile indicating merger of decay signal due to Br2-and formation signal due to the transient of DTR. In the previous study,'* oxidation of DTR only with Br2- has been carried out and the bimolecular rate constant measured (1.3 X lo9 and dm3mol-' s-I, pH = 8); however, neither the wavelength of measurement nor the actual details have been reported. With 12- as the oxidant, due to similar reasons as in the case of Br2- above, quantitative results could not be obtained although in acidic pH, the bimolecular formation rate constant of the transient absorbing at 310 nm approached the diffusioncontrolled limit. Results obtained with (CNS)2- radical have not been included in Figure 1 for sake of clarity as similar results were obtained as in the case of N3' as the oxidizing radical.
From the transient molar absorptivity values at 350 nm, measured by keeping the electron pulse dose as low as possible to minimize radical-radical reactions, it is inferred that the oxidation of DTR with these radicals was complete and the G(DTR),, was equal to the G(radica1). Depending on the matrix pH (e.g., acidic for C1- and neutral for N3- and CNS-), the observed transient peaks at 280 and 350 nm indicate the formation of the same transient in all these cases. Comparing these results with the reported oxidation studies of tyrosine and similar phenolic c o m p ~ u n d s , an ~ ~oxygen-centered ~-~ phenoxyl-type transient (I)2o is expected as shown in Scheme 1. While at pH > 6.4, I was formed directly from the phenoxide anion, the same was formed at lower pH by simultaneous deprotonation of the phenolic hydrogen. A phenoxy1 radical-type transient produced from oxidation of tyrosine shows absorption maxima at =41019hJ* and 270 nml9h while from some other phenolic compounds an additional peak is observed at ~ 3 0 nm.19c 0 In the case of DTR, two iodine substituents in semioxidized DTR shift the A, due to these two peaks to 350 and 280 nm. During one-electron oxidation, the possibility of a direct *-electron transfer from DTR phenolic ring to any of these oneelectron oxidants also exists. As presented in Scheme 2, this process leads to the formation of a radical cation intermediate (11) retaining the enolic-type hydrogen on the ring. Unlike tyrosine, where such a transient has not been reported in the literature, the presence of two iodine substituents is expected to stabilize such a cationic intermediate by resonance delocalization of the resulting positive charge. The feasibility of enolic deprotonation is expected to give rise to a transient pKa similar to that of a DTR ground-state molecule. The formation of an additional transient peak (Amaxat310 nm) with propertiesentirely different from the phenoxyl-type transient (I) supports this possibility. Similar results obtained in all the above three cases (with variation of transient absorbance at 310 nm at different pH) and absence of any additional transient peak between 250 and 800 nm indicate the oxidation process to occur by these two modes, giving rise to transients I and 11. The absorption spectrum of transient produced from DTR reaction with Br' radical at neutral pH was very similar to the one discussed above for the N3' radical. Its formation rate constant was measured by following the buildup of signal at 350 nm, and a value of 6 X l o 9 dm3 mol-I s-l was obtained. From its measured extinction coefficient value in NzO-saturated solution it was evident that reaction of DTR with Br' was exclusively by oxidation process, and no addition of Br' to the ring occurred. Transient decay kinetics from Table 1 support this observation. Oxidation study of DTR using thallium was possible both in acidic and neutral pH due to its respective oxidation states TV+/
The Journal of Physical Chemistry, Vol. 98, No. 20, 1994 5215
Pulse Radiolytic Oxidation of 3,5-Diiodotyrosine
0.035 I
SCHEME 2 -1
PI
DTR
t H ~ N \,coo-
7"
H N :,
,coo-
H ~ N \,coo-
FH
FH
H~N,
,coo-
FH
1
0.028
T 0 0
I
A
0.021
W
2
0.014
0.007 0.000 radical cation II (resonancestabilized)
H:N\
,coo-
240
304
368
432
496
W a v e l e n g t h (nm)
560
Figure 2. Time-resolved transient absorption spectra of semioxidized DTR at pH 7 in a matrix of 47% 2-propanol,4% CCl,, and 51% water containing 1 X moldm-3phosphate buffer and saturated with oxygen. DTR concentration used = 5 X 10-4 mol dm-', and absorbed dose = 14 Gy for 50-11s pulse: (0) 1, (A) 2, (a) 3, and (X) 4 ps.
CH
0.032
I
D
'70.024 0 0
w W
4
0.01 6
a Tl(OH)+ as discussed earlier. In acidic solution (pH = 2) oxidation of DTR produced the identical transient spectrum as observed with Clz-. The rate constant of its formation, measured at 350 nm, gave a value of 1 X lo9 dm3 mol-' s-I. At neutral pH quantitative evaluation of the rate constant was not possible due to partial spectral overlap due to Tl(OH)+ and the transient absorption spectra in the wavelength range of interest producing complex decay/formation traces. However, after necessary corrections, the qualitative absorption spectra due to the transient were found to be of similar nature as observed at neutral pH. Studies with SO4-required special care as DTR was observed to get oxidized thermally by S20s- employed for generation of SO4-. Working solutions for kinetic measurements of DTR oxidation with S208-were prepared and used immediately while for spectral study partial decomposition of DTR was observed during the course of its measurements, preventing quantitative measurements of spectral parameters. The haloperoxyl radical, CC1302*,reaction with DTR was observed to be relatively slow (kr(350 nm) = 4 X lo8 dm3 mol-' s-I), and a possible reason may be due to its own very high self-reaction rate.17 This is also reflected by its comparatively lower yield of the transient (as represented by low value of e350, Table 1). The spectral profile of the transient produced by CC1302*oxidation, however, was somewhat different from that observed with other oxidants. As shown in Figure 2, the 280-nm peak in this case was absent possibly due to the different nature of the matrix used.17 The presence of 310- and 350-nm peaks, however, confirmed the formation of semioxidized transients by simple electron transfer from DTR. Dimethyl radical cation (DMS)2+ oxidation of DTR confirmed the formation of transient I at 350 nm, although its yield was almost half of the expected value. The kinetic parameters, however, indicate that under oxidizing condition a rapid electron transfer from DTR to a variety of semioxidized RSH substrates may be favored in the thyroid and plasma. Reactions with 'OH/*O-. Oxidation of DTR with *OH/*Oradicals produced transients with similar characteristics as discussed above, indicating transfer of an electron from DTR. In addition to its oxidative properties, hydroxyl radical is expected to add to a phenolic moiety, forming a cyclohexadienyl-type radical intermediate with characteristic absorption in the UV region.lgb,h Such a carbon-centered radical may subsequently follow one or more of the various possible reaction pathways: it may (a)
0.008 0.000 24 40 0 0.030
?
370
500
630
760
Wavelength ( n m j
I
0.025
L
0 0.020
0 v
0.015
a
0.01 0
0.005 0'0002:0
320
400
400 5 6 0
40
720
8 0
W a v e l e n g t h {nm) Figure 3. (a, top) Time-resolved spectra of transient obtained from 'OH reaction with DTR at pH 5 and [DTR] used = 1 X 10-4 mol dm-3 in 1 X 10-2 mol dm-3 phosphate buffer aqueous solution saturated with NzO, 50-11s pulse, ['OH] = 8 X 1od mol dm4. Peaks at 280, 310, 350, and 650 nm. (b, bottom) Time-resolved spectra of transient obtained from '0- reaction with DTR at pH 12.7 and [DTR] used = 3 X 10-4 mol dm-3 in 1 X 1 t 2 mol dm-3 phosphate buffer aqueous solution saturated with N20,50-ns pulse, ['O-] = 7.8 X 10-6 mol dm4. Peaks at 280,350, and 415 nm.
eliminate a n iodine atom to form a stable compound hydroxyiodo tyrosine, (b) add to a DTR molecule to form a dimer-type intermediate, or (c) extract a hydrogen atom from the matrix to form a cyclohexadiene-type stable product. In this study, when transient formation from DTR oxidation with secondary radicals was compared with 'OH radical reactions, indeed some differences were observed in the yield as well as spectral characteristics of the transients formed. As shown in Figure 3a, for 'OH radical as the oxidant, on a comparatively slower time scale ( 4 0 times slower as compared to the formation of peaks a t 310 or 350 nm), a new transient peak formation with a broad profile and absorption maxima centered at 650 nm was observed at all pH. In case of oxygen saturation (to scavenge H atoms a t acidic pH or even at higher pH for comparison) this peak was not observed, indicating the involvement of a carbon-
Das and Priyadarsini
The Journal of Physical Chemistry, Vol. 98, No. 20, 1994
5216
20
I
1
0.03
2.7 n v)
n v)
, I
I 2.4
T 0
-
v
15
I
0 2.1
7
X
X
W
5
0
W
10
0
1.8
2
Y
0
I
t
-
-
350 n m
'
0.02
a a 0.0 1
5E In CI
Y
5
2
4
6
8
10
1.5
12
PH Figure4. Variation of formationrateconstantsof transientswith solution pH. [DTR] = 2 X lO-"oldm-3in 1 X 10-2moldm-3phosphate buffered aqueous solution saturated with N20, 50-11spulse, ['OH] = 7.6 X 1V mol dm-3. (a) Transient with peak absorptionat 650 nm. (b) Transient with peak absorption at 350 nm. Inset: variation of DTR absorption at 310 nm with pH (pK, = 6.4 due to phenolic OH/phenoxide anion) for comparison.
SCHEME 3 H3N' COOYH
OH proxy radical intermediate
H3N;
,COO-
FH
OH
DTR
H3N:
,COOCH
OH DTR-OH adduct (A)
I
DTR
centered radical intermediate during its formation. At neutral and alkaline pH, the rate of formation of this transient increased as compared to acidic pH as shown in Figure 4. The variation of the formation rate constant with solution pH strongly indicates involvement of DTR in the reaction scheme with its phenoxide form reacting faster than the phenolic form.18 Qualitatively, this can be explained as follows: in the phenoxide form of DTR the electron density in the ring is expected to be more than in the case of the phenolic form. Since *OH behaves as an electrophile in its reactions, its addition to the electron-rich ring in the first case becomes faster in the initial step of its formation. Although the resulting adduct (A), Scheme 3, is expected to show absorption in the UV region, its actual presence as a primary transient is
0.00
PH Figure 5. Variation of transient absorbance measured at 3 10,350, and 650 nm with solution pH. [DTR] = 2 X l ( r mol dm-3 in 1 X mol dm-3 phosphate buffer aqueous solution saturated with N20,50-ns pulse, ['OH] = 7.6 X 1V mol d r 3 . pK. (310 nm) = 6.5 and pK, (650 nm) = 3.9 and 7. probably masked by the other two transients I and I1 absorbing in the same region. Only after it reacts further with DTR to form the secondary transient will its presence become noticeable. The observed details of this peak are presented in Table 1. This peak was not observed when '0-was used as the oxidant (at pH > 12.5) as such additions of nucleophilic '0- to phenolic chromophores are not known to take place. Hence, formation of this transient is not expected and indeed not observed as seen from Figure 3b. From its formation kinetics, the observed increased rate with increase in DTR concentration confirmed the proposed reaction Scheme 3, while from the observed decay parameters it was concluded that the peak at 650 nm is due to an intermediate and not a stable product (as also confirmed by steady-state y-radiolysis experiments). In short, the electrophilic 'OH radical in addition to oxidizing DTR by electron transfer, as expected, first forms an adduct with cyclohexadienyl-type radical structure A that reacts further with another DTR molecule to form the adduct radical B. In the presence of dissolved oxygen the adduct radical A was preferentially scavenged, reducing the secondary intermediate formation. In the absence of dissolved oxygen, the same radical attached itself to a molecule of DTR (preferably with its phenoxideform) with simultaneous liberation of HzO and forming transient B. The slower time scale of its formation can thus be qualitatively explained. This structure having two enolic OH groups is expected to give rise to two separate pKa values for the corresponding deprotonation processes. In Figure 5 the variation of absorbance of transients with change in solution pH is plotted. It is seen that the transient with peak absorption at 650 nm indeed shows two pK, values, 3.9 and 7. These values indicate that deprotonations of either COOH or NH3+groups do not give rise to these observations as these are expected to occur respectively around solution pH 2 and 9-10 as in case of DTR. These observations further substantiate the assumptions and the reactions indicated in Scheme 3. From Figure 5 it is observed that the absorbance at 350 nm is almost invariant with the change of solution pH. From nanosecond laser flash photolysis study, the spectrum of the phenoxyl-type intermediate I as presented in Figure 6 has been confirmed in oxygen-saturated solutions. In this case, 'DTR produced initially is expected to give rise to the radical I by transferring an electron to molecular 0 2 and simultaneousself-deprotonation as presented in eq 15 and reported in the case of tyrosine.22 The pKa values of phenoxyl-type radicals arising from phenols are generally very low (=O pH unit),20and the same is expected of the radical I produced from DTR oxidation and supported by the absence of any pKa in the working pH range. Thus, from both primary *OHradical and all other secondary radicals used in this study, I was formed as a primary transient with its formation rate constant approaching the diffusion-controlled value. As
Pulse Radiolytic Oxidation of 3,5-Diiodotyrosine
0'0%40 290 340 390 440 490 540 590 6 4 0 O.O1 ~
Wavelength (nm) Figure 6. Phenoxy1 radical (of DTR) transient spectrum observed after 1 ws from 248-nm nanosecond laser flash photolysis study of 6 X lo" mol mol dm-3 phosphate buffered aqueous solution at d m 3 DTR in 1 X le2 pH 7 and saturated with 02.
DTR -3
(triplet DTR)
in Scheme 1, the electron transfer from DTR in phenolic form involves two steps, Le., electron transfer and subsequent deprotonation, while from the phenoxide anion it is a single-step process. Consequently, the rate constant for this process is expected to be proportional to the ratio of phenoxide concentration to the phenol concentration. As seen in Figure 4, the increase in the bimolecular rate constant value of this process closely matched the fraction of DTR in its phenoxide form. Thus, the peaks at 280 and 350 nm represented the formation of the transient I. The decay of the transient peaks at 280/350 nm were always observed to follow second-order kinetics independent of solution pH and DTR concentration. As indicated in Table 1, the measured Zk/dvalues for its decay agreed well with each other for different oxidizing radicals employed in this study. The plausible mechanisms for this behavior are (i) dimerization of thephenoxyl radical transient I and/or (ii) disproportionation of two I radicals. Since dimerization of such radicals is well-known for tyr0sine,~3secondorder decay kinetics observed in the present case strongly suggests this structure. In other words, in the aqueous matrix inside the animal body, the possibility of formation of phenoxyl radicals from DTR by one-electron oxidation process is high. From Figure 5, it is also observed that the peak at 310 nm shows a pKa value of 6.5, almost matching with the pK, of DTR (phenolic OH). Under the experimental conditions, the transient absorption at 3 10 nm always decayed following pseudo-firstorder kinetics irrespective of the oxidizing radical used. The measured pseudo-first-order rate ( k ~ , value ) of 1 X lo4 s-1 at pH 4 suggests a matrix-assisted decay mechanism. The decay characteristics at 3 10 nm are very different from the one observed at 350 nm due to the phenoxyl radical discussed above. Since the measured yields at 3 10 nm due to this transient matched well with each other irrespective of the oxidizing radical employed, its pKa at 6.5 strongly suggests structure 11. Such a cationic radical is expected to interact with the solvent matrix and consequently decay following pseudo-first-order kinetics. The observed decay pattern that remains unchanged in the presence of dissolved oxygen supports its cationic nature.
The Journal of Physical Chemistry, Vol. 98, No. 20, 1994 5277 In a comparative study of DTR oxidation with *OH against other secondary oxidants like N3* and Br' or '0-,a net decrease of 10-15% in the yield of transient at 310 nm was observed for 'OH radical as the oxidant as DTR concentration was varied between 5 X 10-5 to 2 X mol dm-3. Thus, only a small fraction of the DTR formed the 'OH radical adduct (A) while the larger fraction transferred an electron to the OH radical to form I and 11. This observation was found quite different from the results observed earlier in the case of tyrosine where the OH adduct yield was as high as 50%.19h Surprisingly, for DTR oxidation, no change in the yield of the phenoxyl-type transient was observed under these conditions. As expected, for the other secondary oxidizing radicals no adduct formation was observed. In this context, it may be noted that for the one-electron oxidation process it has been assumed that individual yields (G)of either of the phenoxyl radical I or the cationic transient I1 were the same as Gradical. From the observed experimental results it was not possible to measure their individual yield fractions for any of the secondary oxidizing radicals used. Thus, the transient molar extinction coefficient values (e) at 350 and 310 nm were calculated accordingly. In comparison to other oxidizing radicals, as the number of reaction channels for 'OH radical was more, no corrections of the e values for the transient with peak at 650 nm was done, and the value is reported with the assumption that its yield (G value) was same as GOHradical. Reduction Potential of DTR+/DTR Couple. Form the measured rates of oxidation of DTR withvarious radicals as employed above, it was not possible to have a system with possible reversible electron transfer forming an equilibrium. Qualitatively, it indicated a low value (less than 1.O V) for the reduction potential for the DTR+/DTR couple. However, when the thiyl radical derived from cysteine (CySH), reaction 16a, was used as a secondary oxidizing radical at pH 7, an equilibrium system as shown in reaction 16b could be established for reverse electron transfer. CySH
+ 'OH/N,'
-
+ H,O/HN,
(1 6a)
+ D T R + DTR' + CySH
(16b)
CyS' K
CyS' (+H+)
Under the equilibrium conditions, the equilibrium constant K is related to the concentration of various species by the relation.
K=
[DTR'] [CySH] [CYS'I DTRI
The validity of this equilibrium is based on the following: assumptions: (1) as [CySH] or [DTR] was varied, the total radiolytic yield of radicals taking part in it, i.e., [Re] (=[CyS*] [DTR+]), remained constant under these experimental conditions a t a dose of 8 Gy used; (ii) at thewavelength of measurement for DTR+, i.e., 280 nm, interference due to the absorption of thiyl and its dimer radical (CySS'-Cy) was minimum. Equation 17 can be rearranged with the help of the relation conc = A/tl as
+
-
where AA is the corrected absorbance, CDTR and ~ D T R +are the molar extinction coefficients of DTR and its semioxidized transient, respectively, and the numerical value of I is unity. From relation 18 the value of K was determined by the following graphical method as presented in Figure 7a where the inverse of transient absorbance (l/AA) has been plotted against the ratio ofconcentrations ofcysteineandDTR (=[CySH]/[DTR]). From the measured values of the slope (= 1.05) and the intercept (=263),
5278 The Journal of Physical Chemistry, Vol. 98, No. 20, 199'4 375
,
I
350
325
-
300
275
250
I
I 0
10.0
20
,
CG H]/ [STR]
[
100
80
1
Das and Priyadarsini
In the case of an accident inside a nuclear reactor, all personnel are given a mandatory dose of potassium iodide to block the iodine receptors in the thyroid. Such an intake is also expected to produce and maintain a high level of iodide ions in the plasma that may ultimately produce the secondary oxidants I* and 12via its oxidation with the radiolytically generated 'OH radicals formed due to 1311 decay. From our study is is quite clear that even in the case of 'OH as the oxidant, the yield of the dimer adduct B is low as compared to the yields of semioxidized transients I and I1 that are always produced with secondary oxidants. Thus, it may be possible to minimize DTR loss by oxidation process if the semioxidized DTR in turn acts as an electron acceptor from another substrate present in the surrounding matrix. Nontoxic antioxidant ascorbic acid (at near neutral physiological pH) with a low value of its reduction potential offers itself as a potential chemical for such a study. A detailed study is needed for in-vivo DTR damage control by this method. Acknowledgment. The authors sincerely thank Dr. J. P. Mittal for his encouragement and support during the course of this study. References and Notes
'
0.0 0.00
I 0.01
0.02
[ DT R ]"i"["cY $ ;
0.05
0.06
Figure 7. (a, top) Linear plot of l/AA against [CySH]/[DTR] under equilibrium conditions for [DTR] = 2 X le5mol dm-3 and [CySH] varied from 4 X lo-' to 1.6 X mol dm-.' in 5 X le2mol dm-3 phosphate buffered aqueous solution at p H 7 and a t a dose of 8 Gy. (b, bottom) Linear plot of k,b/[CySH] against [DTR]/[CySH] under equilibrium conditions as in (a).
the value of the equilibrium constant K (=intercept/slope) obtained was 250. The equilibrium constant K was also determined from the observed pseudo-first-order rate constant, k,b, from relation 19 at low dose rate24
where kf and kb are the rate constants for the forward and backward reactions, respectively, for the equilibrium (16b). Assumptions have been made that under the equilibrium condition other loss mechanisms for the various radicals were negligible at the time scale of events for the measurement of equilibrium values of [DTR+]. Figure 7b represents the linear plot of kohl [CySH] against [DTR]/[CySH]. From the measured values of slope (kf = 143 X 109) andintercept (kb= 6 X lo8), thevalueofK(kf/kb) has been measured to be 240. The average value of K was taken as 245, and the reduction potential of the couple DTR+/DTR was determined by the following relation:
The value of E'DTR+,DTRhas been measured to be +0.78 f 0.04 V vs NHE. The value of the reduction for the CyS' (+H+)/ CySH couple has been taken from literature to be +0.92 f 0.04 V.25
Conclusion From the measured one-electron reduction potential value for DTR+/DTR couple in aqueous medium, its immediate oxidation is expected to take place inside a mammalian body by electrontransfer mechanism in the presence of oxidizing radicals like *OH, Clz*-, I2*-, RS', etc., in the surrounding plasma or matrix.
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