11109
J. Phys. Chem. 1994, 98, 11109- 11114
Aqueous Medium Pulse and Steady State Radiation Chemical Studies on the Reduction of 3,5-Diiodotyrosine T. N. Das Chemistry Division, Bhabha Atomic Research Centre, Bombay 400 085, India Received: June 16, 1994; In Final Form: August 16, 1994@
Reduction reactions of 3,5-diiodotyrosine (DTR) are reported in phosphate-buffered aqueous medium. Steady state y-radiolysis and electron pulse irradiation have been used to study reduction mechanisms of DTR in the presence of primary radicals, ea¶- and H', and secondary radicals derived from oxygen, H O i and Oz-, in acidic, neutral, and alkaline media. The rate constant for H' addition to the DTR phenolic chromophore is estimated to be k = 2.4 x lo9 at pH 1.8 and 1.6 x lo9 dm3 mol-' s-' at pH 5; a cyclohexadienyl type transient with peak absorption at 385 nm and €385 = 900 f 200 dm3 mol-' cm-' is produced in both cases. Reactions of ea¶- on the other hand show a marked pH dependence. Electron addition to the phenolic chromophore between pH 4 and 12 produces a transient species that absorbs below 300 nm with A,,,= at 280 nm and €280 = 9300 f 700 dm3 mol-' cm-' with concurrent liberation of I-. G(1-) is constant over the pH range and is estimated to be 0.11 f 0.01 pM Gy-'. In addition, in acidic and neutral media, ammonia elimination occurs with G(NH3) = 0.19 at pH 4 and 0.12 pM Gy-' at pH 7. The rate constants for these processes are intermediate to the rate, 8.0 x lo9 dm3 mol-' s-l for eq- addition to the -NH3+ group (subsequently leading to deamination) and 1.55 x 1O'O dm3 mol-' s-' for addition to the phenolic chromophore. DTR does not react with HO2* and 0 2 - radicals. The reducing a-hydroxy radical derived from isopropyl alcohol, (H3C)2C'OH, and carboxyl radical, COZ-, do not react with DTR by either electron transfer, H-abstraction, or addition to the phenolic ring, indicating a reduction potential value of the DTR-/DTR couple more negative than -2.0 V vs NHE.
Introduction
In our previous study,' the pulse radiolytic oxidation reactions of 3,5-diiodotyrosine (DTR) in aqueous medium along with its biological significance have been discussed. Leakage of radioactive iodine (mainly l3'I) into the environment from nuclear reactors and other man-made devices2s3and its subsequent uptake by the mammalian population is expected to affect the thyroid hormone precursor DTR due to the resulting radiation-induced chemical reactions in the thyroid and p l a ~ m a . ~ The radiation-induced primary reducing radicals formed in aqueous matrix, eq- and H', are the major reductants expected under such a situation. In the presence of dissolved oxygen in the plasma, because of its ubiquitous nature, these radicals further give rise to oxygen radicals HO2' and 0 2 - , according to reactions 1-3. These resulting radicals, due to their reducing nature, may also affect DTR stability.
H' + 0,
+
+
eaq- 0,
HO,'
-
(1)
0,-
pK, = 4.7
HO,'
O,-,
+ H+
(3)
To understand the reductive pathways for DTR, either in the presence of ionizing radiation or in the case of thermal chemical reactions, its reactions with each of these individual reducing radicals in an aqueous matrix need to be studied in detail. Some studies are reported on the actions of a- and X-radiation on DTR at low pH5a3band also on steady state y-radiolysis of DTR in aqueous-alcohol solutions,6but no details are available on @
the transient behavior arising from radiation-induced reductive chemical changes of DTR, especially in aqueous medium near neutral pH, the matrix of concern in mammalian thyroid and plasma. Due to the importance of DTR in regulation of hormone concentrationsthat affect different metabolic functions, this study was conducted to characterize the various transients and to map the reduction pathways as a function of pH, both in the presence and in the absence of dissolved oxygen. To study DTR reactions with the radicals of interest, the following reactions were used for their g e n e r a t i ~ n .At ~ ~ pH below 3.5, 0.1 M tert-butyl alcohol in deoxygenated solutions was used as the scavenger for 'OH radicals according to reaction 4 and H' reactions could be studied. The resultant ,&hydroxy carbon-centered radical derived from tert-butyl alcohol was observed to be unreactive toward DTR. The G(H') value at pH 2 was taken as 0.35 pM Gy-' from the literature.7b
Abstract published in Advance ACS Absrrarrs, October 1, 1994.
0022-365419412098-11109$04.50/0
In deoxygenated solutions at pH 5 , H' radical was generated by reaction of eq- with 1 M KH2HP04 according to reaction 5 .8
H,PO,-
+ eaq- - H' + HPO,~-
(5)
To study the reactions of eaq- between pH 4 and 12, isopropyl alcohol was used as the scavenger of both 'OH and H' radicals according to reaction 6. This method to scavenge both H' and 'OH radicals was successful, because the resulting a-hydroxy radical, (H3C)zC'OH, was observed to be unreactive toward DTR, as discussed later in detail. 0 1994 American Chemical Society
11110 J. Phys. Chem., Vol. 98, No. 43, I994 (H,C),CHOH
+ 'OH(H')
-
(H,C),C*OH
Das
+ H20(H2) (6)
To study 0 2 - reactions with DTR in oxygen-saturated, neutral, or mildly alkaline solutions, formate anion was used to scavenge both 'OH and H'radicals according to reactions 7 and 8. As discussed later in the text, the intermediate carboxyl radical, COz-, although a strong reductant, was observed to be unreactive toward DTR. It was confirmed from prior saturation of the matrix containing formate anion with N20, when no reactions of DTR was observed. In this case, the eaq- was transformed into 'OH radicals according to reaction 9. Hence, all the primary radicals of water radiolysis were quantitatively converted to the carboxyl radical.
+ HCOO- - H,O(H,) + C0,c0,- + 0, CO, + 0,-
'OH (IT)
N,O
-
+ eq- + H 2 0
N,
+ OH- + *OH
(7)
(9)
HO2' radical was generated in 02-saturated acidic solutions containing 0.1 M tert-butyl alcohol according to reactions 1,2, 3, and 4 above.
Experimental Section 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 by passing deionized water through Barnstead Nanopure cartridge filtration systems to remove ionic and organic impurities. Stock solutions of DTR were prepared by shaking an excess of it in deoxygenated water for a couple of hours and subsequently filtering off the undissolved residue. At room temperature (25 "C), the DTR concentration obtained was 3 x lo-, M, and it remained stable for a long time in the dark. Iolar grade gases N2, NzO, and 0 2 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 M phosphate buffer (equimolar mixture of Na2HP04*H20and KHzPO& and the pH of the solutions were adjusted to the required value either with HC104 or with NaOH. For transient spectral measurements, the concentration of DTR used was in the range (1 -5) x M, while for kinetic studies the same was varied between 5 x and 1 x M. All UV-vis spectra were measured on a Hitachi Model 330 spectrophotometer. The detailed experimental facility for pulse radiolysis study has been described earlier.9 Single electron pulses of 7 MeV energy were used in the experiments, and the selected pulse widths were either of 50 ns or 2 ps duration, depending on the nature of information sought. Maximum doses obtainable with 50 ns pulses were 15 Gy, and for a 2 ps pulse it was 7-8 times higher. The dosimetry was performed with air-saturated 0.01 M KCNS solution, with the GE value for (CNS)2- radical = 2.23 x m2 J-* at 500 nm.lo All subsequent G-values in the text are reported in units of pM Gy-'. Transient spectral measurements were performed using a continuous flow arrangement. The kinetic spectrophotometric detection system covered the wavelength range from 250 to 850 nm. Between 300 and 780 nm, a resolution of ~3 nm in wavelength settings was achieved in these measurements. Cells with an optical path length of 1 cm were used for these measurements. Buffered deoxygenated solutions containing DTR (1 x M) and tertbutyl alcohol (0.25 M) used in the pulsed measurements with a total absorbed dose of ~ 3 5 Gy 0 (three pulses of 2 ps duration)
0.8 ---\I
I
'"0
P
Q
0.0 300
360
A
420
20 40 60 80 If0 120 [I-](IO" mol dm )
I
480
$40
/
600
w a v e l e n g t h (nm) Figure 1. Absorption spectra of AgI colloidal solution in deoxygenated M phosphate buffer, 0.25 M aqueous matrix containing 2.5 x tert-butyl alcohol, and 1 x M DTR; spectra taken 25 min after addition of 5 x mL of 0.1 M AgN03 solution to 5 mL of sample: (1) 100 pM I- (as KI); (2) 10 pM I- (as KI); and (3) irradiated DTR sample (without KI), pH 7.3, absorbed dose 450 Gy. Inset A: Linear variation of absorbance at 425 nm with added KI in the nonirradiated matrix. Inset B: Buildup and decay of absorbance with time at 425 nm after mixing of AgN03 solution with the matrix containing I-.
were also collected, and the concentrations of stable end products NH3 and I- were measured at varying solution pH. In an identical matrix containing DTR, steady state y-radiolysis experiments were carried out using a source at a dose rate of 650 Gy h-' (dose by Fricke dosimetry). The total absorbed dose in these experiments was always maintained at a level such that ~ 1 0 concentration % of the reducing radical of interest was obtained as compared to DTR concentration. This was necessary to keep all reactions between the radicals and between radicals and reaction intermediates or products to a minimum. In these experiments to study DTR reactions with eaq-, for quantitative evaluation, the resulting ammonia gas from the irradiated sample was first transferred to a 5 mL, ~ 0 . 0 M 1 H2so4 absorber solution, by steam distillation in the presence of alkali" and later treated with an alkaline solution of mercuric iodide (Hgb2-). The concentration of the amino-mercuric iodide complex was measured at 410-440 nm. Calibration of yields of NH3 was done by standard addition of (NH&S04 to nonirradiated DTR solutions; the errors in these measurements were estimated to be within the range of &lo%. Iodine liberated in the form of I- in these experiments was precipitated as AgI, by addition of Ag+ at pH 2-3, and was measured gravimetrically. Due to low concentrations of I- (few tens of pM), the errors in these measurements were in the range of f 2 0 % , as checked by standard addition of KI in nonirradiated DTR solutions in the matrix of actual studies. For better accuracy, this method was modified as described below. Prior to gravimetric estimation, in this range of I- concentration, colloidal AgI was formed initially on mixing of 50 pL of 0.2 M AgN03 solution to 10 mL of the sample. This colloidal form persisted for almost 15 h, and later complete precipitation occurred. Although the colloidal form showed a continuous and rapid increase of optical absorption below 500 nm mainly due to increased scattering in the UV region, a distinct peak with ,A at 425 nm was obtained in its absorption spectrum. Figure 1 shows the spectra of AgI obtained under different conditions. The calibration curve for A425m vs I- concentration obtained with standard KI solutions in nonirradiated DTR solutions is shown in Figure 1, inset A. From the linear variation of absorbance at 425 nm for I- concentration between 10 and 100 M, this method was found to be best suited for Iestimation in the present set of experiments. The pH of the
J. Phys. Chem., Vol. 98, No. 43, I994 11111
Reduction of 3,5-Diiodotyrosine
r
0.025
-
1
1
0.015
0.020
L 0.01 2
0.01 5 c
- 0.009
0
$0.01 0
ti
0.005
0.000
--
- 0.006
L
300
350
- 0.003 '
400 300 350 400 wavelength (nm)
,;oo.
000
Figure 2. Transient spectra arising due to DTR reaction with H'at pH 1.8 (A) and pH 5 (B) in deoxygenated aqueous matrix containing 1 x lo-* M phosphate buffer, 1 x lo-' M tert-butyl alcohol, and 5 x M for A and 12 x M DTR with 2 ps pulses. [H'] = 23 x M for B. irradiated solution was adjusted between 2 and 3 units prior to addition of AgN03 solution. As shown in Figure 1, inset B, the absorption due to colloidal AgI showed an initial rapid increase after mixing of solutions, and later, it remained stable for almost 1 h, allowing measurements to be completed conveniently. The errors in these measurements were below the level of &4%. All subsequent I- estimations reported in the text were made using this method. DTR shows absorption bands between 270 and 330 nm (Amax = 286 nm at pH 4 and 310 nm at pH 7.5). With the change of solution pH from 1.5 to 12, successive deprotonation from -COOH, $-OH, and -NH3+ groups takes place with respective pKa values 2, 6.4, and 8.7.12 While the two iodine substituents in the phenolic ring in DTR are responsible for lowering its ($-OH) pKa as compared to tyrosine (pK, = 10.07), the pKa of either -COOH or -NH3+ groups are not affected to the same extent.12 Similarly, the absorption characteristics of the phenolic chromophore in DTR are not affected due to the protonation of these two groups. Since below pH 8.7, in addition to the phenolic chromophore, the protonated amino group in DTR also offers itself as a possible site for electron attack, the actual one-electron reduction of DTR is expected to be a complex process, strongly dependent on the matrix pH. Detailed studies with individual radicals are presented below. Reactions of DTR with H . The transient absorption spectrum of H' reaction with DTR at pH 1.8 in deoxygenated solution is shown in Figure 2A. Its molar absorptivity, E value at Amax (385 nm), has been estimated to be 900 f 200 dm3mol-l cm-'. It was computed from relation 10, where E P is the molar absorptivity of DTR at 385 nm (close to zero), AR is the experimentally observed transient absorbance at 385 nm, and 6 is its radiation chemical yield, as discussed earlier (0.35 at pH 1.8). The value of (GE)of (SCN)2- radical is 2.23 x m2 J-l at 500 nm.'O E
= ~p
+ (AR(G€)(sCN,,-)/(A~sCN~*-GR)
(10)
The spectral profile of the transient produced from H' reaction with DTR at pH 5 is also shown in Figure 2B for comparison. Both these spectra show multiple peaks between 330 and 410 nm, indicating the formation of similar transients. From the reported H' reactions with phenolic compounds including t y r o ~ i n e , l ~it~is- ~expected that the major reaction pathway followed in this case will be the addition of H' to the phenolic ring, giving rise to the cyclohexadienyl type adduct radical. The observed multiple peak profile of this transient in the microsecond time scale suggests more than one site for the H' attachment in the transient phenolic ring. After the initial H'
TABLE 1: Spectral and Kinetic Parameters of Transients Produced from DTR Reductiona reducing kf/109 E radicals (dm3mol-' s-l) (dm3 mol-' cm-I) decay (s-l) H' 2.4 at pH 1.8 900 f 200 pseudo first order 1.6 at pH 5 at 385 nm ( 5 f 0.5) x 104 esq23.5 at pH 5 16.3 at pH 7 8.3 at pH 11 9300 f 700 at 280 nm COz-, (CH&C'OH, and H02'/02- radicals do not react.
]
approach, its redistribution over the different sites probably takes place within the duration of the pulse. An attempt was made using 50 ns electron pulses, to check the possible buildup of the multiple peak profile observed in the microsecond time scale from an initial expected broad peak profile in the nanosecond time scale, supporting this hypothesis. Time-resolved spectral measurements in the fastest possible time scale of 100 ns/ division however failed to provide a satisfactory explanation for the same, as the multiple peak profile was present even at this lowest possible time scale of measurement allowed by the experimental facility. The measured rate constant, as shown in Table 1, for the H' addition to DTR has a value of 2.4 x lo9 dm3 mol-' s-l at pH 1.8, decreasing marginally to 1.6 x lo9 dm3 mol-' s-l at pH 5 . These were estimated from the change in the formation kinetics of the transient at 385 nm in the presence of different concentrations of DTR in the appropriate matrix. A comparison of the H' reaction rate constant values of DTR and tyrosine (2 x lo9 dm3 mol-' s-l at pH 0.9)13bsuggests that the iodine substituents in DTR do not have any significant influence on the kinetics of this addition process. The decay of this cyclohexadienyl type radical transient always showed a solventassisted pseudo first order kinetics ( k = (5 f 0.5) x lo4 s-l) measured at different peak wavelengths and solution pH, indicating a negligible tendency toward dimerization or addition to a DTR molecule. For measurement of the decay kinetics, the H'adduct yield was varied between 5 x lop6and 5 x M and DTR concentration was changed from 5 x to 2 x M. In steady state y-radiolysis experiments, increase in optical absorption due to any permanent product was not observed at wavelengths higher than 300 nm. Indirectly it suggests that the cyclohexadienyl type radical forms a cyclohexadiene type end product absorbing below 300 nm by a Hor OH-abstraction from water. Due to strong DTR ground state absorption below 300 nm, however, it was not possible to confirm this hypothesis. No significant yields of H2, NH3, or I- were obtained in these experiments. Reactions of DTR with HO$ and 02-Radicals. The reactions of DTR with HOz' and 0 2 - radicals at pH 2 and 7.5, respectively, were studied in oxygen-saturated solutions. In pulse radiolysis experiments, the decay rate of the signal at 260 nm due to either of these oxygen radicals did not show any change either in the absence or in the presence of DTR, at a concentration level of 5 x M in the matrix. Similarly, no transient spectrum was observed over the entire wavelength region, even for a high absorbed dose (100 Gy) at a DTR concentration level of 2 x M in the appropriate matrix. These observations indicate that both these oxygen radicals are unreactive toward DTR and neither electron exchange nor their addition to the phenolic ring takes place. In steady state experiments, the irradiated sample spectra, as expected, did not show any appreciable change between N20-saturated and 0 2 saturated solutions containing 2 x lo-' M HC02-, indicating even abstraction of hydrogen atom from the aliphatic side chain
Das
11112 J. Phys. Chem., Vol. 98, No. 43, 1994 SCHEME 1 H
N+ COO-
pKar6.4
zwltter-ion
11. N+ COO-
113N+ COO-
1
" I
YH
N
1.1
pKar8.7
,
H2N
COO-
YH
COO-
YH I
dl -ani on
mono-anion
SCHEME 2 H N+ COO-
H N+ COO-
\CH
\CH
I
I
:$: +
ei
H N+ COO-
Yn
I
__$
.Q I
0-
:$:
I
0
J
r l
0
(I)
- ~ f r i mH ~ o
stable products
by 0 2 - / H 0 2 * was minimum. Since these oxygen radicals are also produced inside living systems by enzymatic reactions in the absence of r a d i a t i ~ n ,these ~ ~ ~ results .~ indicate no possible loss of DTR due to their presence in the matrix. Detailed pulse and steady state radiolysis studies with stronger reducing radicals like C02- and (H3C)zC'OH under appropriate conditions also suggest that one-electron reduction of DTR or DTR- does not take place in an aqueous matrix at room temperature at any pH. No change in the decay rates of either of these radicals, measured at 260 nm, was observed in the absence or presence of DTR in the matrix, and no new transient peaks were observed between 250 and 800 nm. No bleaching signals were observed due to DTR depletion in the matrix at appropriate wavelengths, supporting the above observations. Steady state y-radiolysis studies also support these observations. Solvated Electron, eaq-, Reactions. The pKa values of 6.4 and 8.7, respectively, for deprotonation of phenolic -OH and the -NH3+ groups for DTR suggest that between the working pH range of 4 and 12 for studying eaq- reactions, DTR exists in three different forms, as shown in Scheme 1. The deprotonation of phenolic -OH leads to the formation of phenoxide (mono) anion where the negative charge is delocalized over the entire ring, thus increasing the electron density over it. The loss of another proton near pH 8.7 from the amino group to form the dianion however does not affect the electron density in the ring. Therefore, following the reaction pattern for other aromatic amino a ~ i d ~ the , ~possible ~ ~ , sites ~ ~ for ~ eq* ~ attack on DTR are (i) the amino group and (ii) the ring. The relative proportions and the rates of eaq- attack on either of these two sites will be a function of the matrix pH which results in the structural changes. It is expected that, with the formation of mono- and dianions with increasing solution pH, the overall reaction rate will decrease. As the pH changes, the reaction pathways and the end products are also expected to change. While the eaq- attack on the ring is expected to lead to a change in the spectral characteristics of the transient, the neutralization of positive charge on -NH3+ may not be readily visible in the wavelength range of measurement, as the resulting aliphatic radical after possible deamination is not expected to absorb in the near-UV. The individual results are discussed below.
+I-
a 0.008 0.004 0.000
I&,
240
265
,'"iB-1
290
315
wavelength (nm)
340
Figure 3. Transient spectra arising due to DTR reaction with eaq-at pH 11 (0),pH 7.6 (O), and pH 4.3 (A) in deoxygenated aqueous matrix containing 0.01 M phosphate buffer, 0.1 M tea-butyl alcohol, and 2 x M DTR with 50 ns pulses. [e,-] = 5.5 x M.
(a) Pulse Radiolysis Study. In matrices containing 0.1 M tert-butyl alcohol saturated with N2, with pH varied between the working range of 4 and 12 units, eaq- reaction with DTR produced bleaching signals between 280 and 320 nm, indicating a chemical reaction with DTR. The transient formation kinetics was conveniently measured at 700 nm following the change in decay kinetics of eaq- with increasing amounts of DTR in the matrix. In Figure 3, the absorption spectra of the transients formed from DTR (after suitable correction for its own depletion) at three different solution pH are presented. The experimental conditions such as absorbed dose and DTR concentration were maintained at a level to keep radical-radical reactions below the 5% level as compared to radical-DTR reaction. The eaqaddition to the phenoxide ring of a DTR molecule at pH 7.5 can be represented as in Scheme 2. The radical anion intermediate formed initially is shown within square brackets. In aqueous medium, in strongly acidic pH, this intermediate is expected to get protonated, giving rise to a cyclohexadienyl type radical. In other words, it amounts to the addition of a H'to the ring. As discussed earlier, the DTR+H' transient adduct shows absorption above 300 nm with its Am= at 385 nm. Since between pH 4 and 12, time-resolved transient spectra in this case did not show any peak above 320 nm, the protonation of this radical anion even at acidic pH seems to be insignificant. As an alternative, the radical anion can readily decompose to give rise to iodide anion and the radical
Reduction of 3,5-Diiodotyrosine
J. Phys. Chem., Vol. 98, No. 43, 1994 11113
SCHEME 3 H 'N
H COO-
0-
%.
\CH
0
DTR
f i
a t pH 7 . 6
e
aq
-DTR adduct
2.0
2.5
E
VI
c gl.5
I-
jE
hl W
N
0 1
--
.o
1.5
T
Eo
7
X
Q
40.5
2.0
1.0
7
W
3
5
7
9
11
13
Y
0.5
4
6
8
10
12
PH Figure 4. Variation of transient absorbance at 280 nm with matrix pH after correction due to ground state DTR depletion in deoxygenated aqueous matrix containing 0.01 M phosphate buffer, 0.1 M rert-butyl alcohol, and 2 x loW4 M DTR with 50 ns pulses. [e,-] = 5.5 x M. Transient pK, from plot = 8.2.
PH Figure 5. Variation of the bimolecular rate constant of e,- + DTR reaction with pH measured at 700 nm in deoxygenated matrix containing 0.01 M phosphate buffer, 0.1 M tert-butyl alcohol, and DTR and 2.5 x M with with concentration varied between 5 x 50 ns pulses. [e,-] = 2.2 x M.
I shown above. In similar studies with iodouracil,16avbloss of
s-l, is expected to be entirely due to the addition of ea¶- to the phenoxide ring, this higher value in near neutral and acidic pH is due to the additional reaction channels made available. For ea¶- attack on the phenolic chromophore, the prevailing electron density in it is expected to influence it to a large extent. Below the matrix pH value 6.4, when the fraction of the phenolic form of DTR is more than the phenoxide form, the addition of ea¶to the ring is faster due to comparatively lower electron density in it. A higher value k for the e,,--DTR reaction at pH 5 and below as compared to the value at pH 7.5, as shown in Figure 5, confirms this argument. Since the addition of ea¶- to the protonated amino groups in amino acids generally takes place in acidic and neutral pH with the rate approaching the diffusion controlled limit,13bx15b the same may be expected in the case of DTR. From the estimated value of the rate of ea¶- addition to the phenoxide ring at pH 11 (and with the assumption that this value remains the same even at neutral pH for this reaction channel) and the overall measured reaction rate at mildly acidic and neutral pH, the ea¶- addition to the protonated amino group is estimated to have a rate constant value of 8 x lo9 dm3 mol-' s-'. Following similar arguments, the value of the rate constant for the eq- addition to the phenolic ring in an acidic matrix (pH < 6) was 1.55 x lotodm3 mol-' s-l. The decay of the transients formed after ea¶- addition are expected to follow one or more of the following pathways: (a) for ea¶- addition to the protonated amino group, liberation of ammonia with simultaneous formation of a carbon-centered radical (A') is favored (Scheme 3). Study of formation and decay kinetics of the transient A' was not possible by our kinetic spectrophotometric detection setup. Since the electron addition takes place in the aliphatic side chain, the absorption characteristics of the phenolic chromophore are not disturbed. Thus, estimation of the liberated ammonia as a stable end product was found to be best suited to verify this postulate. Detailed results are discussed later. (b) For eaq- addition to the phenolidphenoxide ring, concurrent iodide liberation from DTR is the facile mode for transient structural rearrangement, as discussed above and as reported in
I- from its electron adduct radical anion was found to be dominating over its protonation, and this step was reported to be complete in the nanosecond time scale. Similarly, in DTR, electron addition followed by deiodination (within the pulse duration) gives rise to optical absorption due to the transient I absorbing between 260 and 320 nm. Therefore, the radical anion transient shown within the square brackets was not observed experimentally in the time scale of our measurements. The absorbance values, & 8 m , at different pH are shown in Figure 4. It indicates a sharp increase in alkaline pH with a corresponding pKa value of 8.2 of this transient. In acidic pH, the transient spectral profile was different as compared to that above acidic pH. Its broad profile below 300 nm with a nonzero A value at 250 nm indicates that it (transient 11)corresponds to the protonated form of transient I, eq 11. At
OH (Ill
pH 11, with the assumption that G(1) = G(e,-), the molar absorptivity, €280, was estimated to be 3400 f 260 dm3 mol-' cm-'. More details with respect to the €280 value will be explained in the section below describing steady state results. In Figure 5, the variation of the e,- reaction rate constant with matrix pH is presented. The progressive decrease of k values from 2.35 x 1O1O in acidic pH to 8.3 x lo9 dm3 mol-' s-l in alkaline pH closely follows the increasing negative charge on the DTR molecule as a result of deprotonations of its phenolic and amino groups. The pKa values, 5.9 and 8.5, obtained from this plot closely match the respective pKa values for DTR. While the value of the rate constant at pH 11, i.e. 8.3 x lo9 dm3 mol-'
Das
11114 J. Phys. Chem., Vol. 98, No. 43, 1994 0.35
r
3.0
I
5.3
7.5
9.8
12.0
PH Figure 6. Variation of yields of NH3 and I- with p H in irradiated deoxygenated aqueous matrix containing 2.5 x M phosphate M DTR; total absorbed buffer, 0.25 M teri-butyl alcohol, and 1.5 x dose 350 Gy: (A) e-beam for NH3; (0)y-radiolysis for N H 3 ; (0) e-beam for I-; and (*) y-radiolysis for I-. Yield of hydrated
ele~tron'~ shown for comparison. a similar case in the 1iterat~re.I~ While the liberated I- can be conveniently monitored as a stable end product, the decay of the transients radicals I or I1 at appropriate pH is expected to follow a solvent-assisted pseudo first order kinetics as addition of an -H or -OH would result in the formation of stable end products monoiodotyrosine or 3-hydroxy-5-iodotyrosine,respectively. In irradiated DTR using X-ray,5bformation of iodide ion and monoiodotyrosine has been reported earlier; however, the reaction conditions do not confirm that these arise exclusively due to eaq- reactions. In this study, in the working pH range 4-8, as a result of bleaching signals obtained due to depletion of DTR, the oscilloscope traces thus obtained prevented any direct estimation or confirmation of the order of transient decay kinetics. End product analysis in the form of I-, therefore, offered a convenient alternative to confirm the reaction pathway resulting from eaq- addition to the phenolic chromophore. Analysis of NH3 and I-. The variation of NH3 and I- yields against matrix pH is plotted in Figure 6, for a total absorbed dose of 350 Gy in experiments performed with deoxygenated solutions containing 1.5 x M DTR in 0.025 M phosphate buffer and 0.25 M tert-butyl alcohol. These data are presented from two sets of experiments, namely, pulse radiolysis and y-radioly sis. In pulse radiolysis experiments using 2 ps pulses (three pulses, each resulting in an absorbed dose of ~ 1 1 Gy), 7 the G(NH3) value, as expected from the arguments above, was close to 0 above pH 9, indicating negligible ea¶- addition to the -NH2 group. The observed values of the yield of I- under the same conditions indicate that the ratio G(I-)/G(eaq-) remains almost invariant with changing pH and has an average value of 0.11 f 0.01 pM Gy-'. The 6280 value of transient I reported earlier in pulse radiolysis experiments was estimated with the assumption that G(e,-)/G(transient I) = 1. The actual value of this ratio from final analysis of I- concentrations (same as transient I concentrations) indicates the necessity of steady state product estimations and provides a correction factor of 2.7. Therefore, the corrected value of €280 is 9300 k 700 dm3 mol-' cm-'. The total yields of NH3 and I- show a value close to the hydrated electron yield below pH 6 and decrease continuously with increasing pH. Qualitatively it indicates that the attack of eaq- on DTR is strongly dependent on the nature of the charge on it. With increasing negative charge, the fraction of eaqreacting with DTR decreases. While the I- yields from y-radiolysis experiments match the pulse radiolysis results over the working pH range, the NH3 yields in these two cases show an entirely opposite trend above pH 8. In y-radiolysis, above pH 8, the total yields of NH3 and
I- show a marginally higher value than the hydrated electron yield,7b and from the comparative study, it is implied that the rapid increase in the yield of NH3 above pH 8 may be due to the deamination reactions of secondary intermediates formed during continuous radiolysis. Since DTR was observed to be thermally stable even at pH 12 at the experimental temperature, the resulting NH3 cannot arise from the unreacted DTR in the matrix. While the measured yields of NH3 and I- below pH 8 follow the expected pattern with the distribution of ea¶- attack on two different sites of DTR, the fate of either the deaminated or the deiodinated DTR was not known. The resulting respective aliphatic or aromatic radicals probably formed stable end products by extracting a -H or -OH from the water molecules in the matrix. Product analysis with the help of HPLC may throw light in this direction. Conclusions Unlike one-electron oxidation, one-electron reduction of DTR is observed to be a quite difficult process in an aqueous matrix. From the measured parameters, the reduction potential of the couple DTR/DTR- is expected to have a value more negative than -2.0 V vs NHE. Reducing oxygen radicals have been shown to be unreactive toward DTR at all pH values; thus, in the presence of dissolved oxygen in the plasma and inside the thyroid, actual reduction of DTR either in the presence of ionizing radiation or under thermal conditions is expected to be very low. Biologically, it signifies a low chance of DTR damage inside the mammalian body during radioiodine leakage or under any other reducing conditions. Acknowledgment. The author thanks Drs. J. P. Mittal and K. V. S. Rama Rao for their keen interest and advice during the course of this study and K. I. Priyadarsini for her cooperation in some PR experiments. References and Notes (1) Das, T. N.; Priyadarsini, K. I. J . Phys. Chem. 1994, 98, 5272. (2) Katcoff, S. Nucleonics 1960, 18, 201. (3) National Council of Radiation Protection and Management, NCRP Report No. 92; Bethesda, MD, 1987. (4) Eisenbud, M. Environmental Radioactivity from Natural, Industrial and Military Sources, 3rd ed; Academic Press: London, 1987; p 414. ( 5 ) (a) Yalow, R. S. Radiat. Res. 1959,2,30. (b) Yalow, R. S.;Berson, S. A. Radiat. Res. 1961, 4, 590. (6) Obaid, A. Y.; Basahel, S. N.; Diefallah, E. M. J . Radioanal. Nucl. Chem. 1990, 139, 355. (7) (a) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J . Phys. Chem. Re$ Data 1988, 17, 513. (b) Spinks, J. W. T.; Wood, R. J. Introduction to Radiation Chemistry; John-Wiley & Sons, Inc.: New York, 1990; p 262. (8) Ye, M.; Schuler, R. H. Radiat. Phys. Chem. 1986, 28, 223. (9) Guha, S. N.; Moorthy, P. N.; Kishore, K.; Naik, D. B.; Rao, K. N. Proc. Ind. Acad. Sci. (Chem. Sci.) 1987, 99, 261. (10) Fielden, E. M. The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis; Baxendale, J. H., Busi, F., Eds.; D. Reidel: Dordrecht, Holland, 1984; p 59. (11) Jeffery, G. H.; Bassett, J.; Mendham, J.; Denney, R. C. Vogel's Textbook of Quantitative Chemical Analysis, 5th ed.; ELBS-Longmans: Essex, 1978; p 679. (12) Lange's Handbook of Chemistry, 12th ed.; Dean, J. A., Ed.; McGraw Hill: New York, 1987; Section 5. (13) (a) Navon, G.; Stein, G. Isr. J . Chem. 1964,2, 151. (b) Feitelson, J.; Hayon, E. J. Phys. Chem. 1973, 77, 10. (c) Neta, P.; Huie, R. E.; Ross, A. B. J . Phys. Chem. Re$ Data 1988, 17, 1027. (14) (a) Bielski, B. H. J.; Cabelli, D. E.; Amdi, R. L.; Ross, A. B. J . Phys. Chem. Ref. Data 1985, 14, 1041. (b) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine; Clarendon Press: Oxford, 1985. (15) (a) von Sonntag, C. Chemical Basis of Radiation Biology; Taylor and Francis: London, 1987; p 396. (b) Mittal, J. P.; Hayon, E. J. Phys. Chem. 1974, 78, 1790. (16) (a) Patterson, L. K.; Bansal, K. M. J . Phys. Chem. 1972, 76,2392. (b) Rivera, E.; Schuler, R. M. J . Phys. Chem. 1983, 87, 3966. (17) Anbar, M.; Hart, E. J. J . Am. Chem. SOC.1964, 86, 5633.
