A pulse radiolysis study of the reactions of 3-hydroxykynurenine and kynurenine with oxidizing and reducing radicals

l~iochimic4 et Biophysica Acta, 1158 (1993) 75-82 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4165/93/$06.00

75

BBAGEN 23831

A pulse radiolysis study of the reactions of 3-hydroxykynurenine and kynurenine with oxidizing and reducing radicals S t e p h e n J. A t h e r t o n a, J a m e s D i l l o n b a n d E l i z a b e t h R. G a i l l a r d a a Center for Fast Kinetics Research, University of Texas at Austin, Austin, TX (USA) and b Department of Ophthalmology, Columbia University, New York, N Y (USA)

(Received 26 October 1993)

Key words: Kynurenines; Pulse radiolysis; Oxidation; Reduction Pulse radiolysis has been used to study the reactions of 3-hydroxykynurenine and kynurenine with solvated electrons, superoxide radicals, hydroxyl radicals and azide radicals. Both 3-hydroxykynurenine and kynurenine react with solvated electrons with diffusion controlled rate constants (k -- 2.5.10 l° M - i s - 1 and 2.3.101° M - i s - 1 , respectively). Neither compound was observed to react with superoxide radicals under our experimental conditions, an upper limit of 1.2.105 M - i s - ~ for the rate constant of this reaction was estimated for both compounds. However, we do observe that a stable product of autooxidation of 3-hydroxykynurenine reacts with superoxide radicals and we calculate a lower limit for the rate of this reaction of 5.8.106 M - i s -1. Reactions of 3-hydroxykynurenine and kynurenine with hydroxyl radicals proceed with diffusion controlled rate constants (1.2" 101° M - i s - 1 and 1.3- 10 l° M - i s - 1, respectively). The measured values for the rate constants for reaction of 3-hydroxykynurenine and kynurenine with azide radicals are 2.1- 101° M - I s - 1 and 4.8- 109 M - I s - 1 , respectively. The differences in these rate constants are attributed to differences in the measured oxidation potentials for 3-hydroxykynurenine ( + 1.0 V vs. NHE) and kynurenine ( + 1.15 V vs. NHE).

Introduction

The tryptophan metabolite 3-hydroxykynurenine (HK) is found throughout the tissues of the human body. Recently, it has been suggested that its presence may be deleterious to biological systems. It is cytotoxic to neuronal cells [1], apparently causing oxidative stress via the formation of hydrogen peroxide, and has also been shown to cause bladder cancer [2]. In the human lens, its reactions have been associated with both aging [3] and cataractous processes [4]. Conversely, it has also been proposed to be an antioxidant, scavenging peroxy radicals in inflammatory diseases [5] and superoxide in the Malpighian tubes of insects [6].

NI-I2 OH

O

O

NH2 OH

OH HK

K

Correspondence to: J. Dillon, Department of Ophthalmology, Columbia University, 630 W. 168 St., New York, ICY 10032, USA; Fax 212-305-3173. Abbreviations: HK, 3-hydroxykynurenine; K, kynurenine.

Both kynurenine (K) and HK are found endogenously in the human lens where they are continuously absorbing radiation in the 300-400 nm region. Numerous studies have indicated that light induced damage to the lens occurs via an oxidative mechanism, which may involve derivatives of kynurenine. It has been shown previously that HK and K do not photosensitize either singlet oxygen or superoxide, suggesting that they may play a protecting role [7]. Additionally it has been shown that the primary product of the absorption of light by kynurenine derivatives, the first excited singlet states, decay within tens of picoseconds to the ground state, with no optically observable products at longer times [8]. Again this may indicate a protective role of these species in the eye by effectively directing absorbed radiation into benign channels. Conversely, the kynurenine derivatives may react with oxidizing species derived from other channels, resulting in oxidative damage to the lens. Since HK is an ortho aminophenol, it might be expected to undergo complex oxidative processes. In fact under severe oxidative stress induced via the hydrogen peroxide-horseradish peroxidase system, HK was observed to form hydroxanthommatin and xanthommatin, the products of six and eight electron oxidations, respectively [9]. However, in biological systems

76 this mechanism may not be operative since the concentration of HK and various oxidants might be considerably lower. The present studies were performed to gather basic information about the reactions of HK and K with various highly reactive species, with a view to elucidating possible mechanisms for damage or protection in biological systems. Materials and Methods

Samples of K and HK were obtained from Sigma Chemical Company and were tested for purity by HPLC. Both compounds showed only one peak and were therefore used without further purification. All other chemicals were of the best available grade and used as received. Water was purified by passage through a Millipore filtration system. Since solutions of both K and HK were found to undergo oxidation when exposed to air, all solutions were freshly prepared immediately prior to the experiment. Solutions were prepared in 10 -2 M phosphate buffer (pH = 7.4), except for those used to study pH effects where the samples were made up in water and the pH was varied by addition of KOH or HCI. For these latter samples the pH was measured with a double junction epoxy body pH electrode and a Cole Parmer Chemcadet pH meter. Absorption spectra were recorded with a Hitachi U-3210 UV-VIS spectrophotometer. Cyclic voltammetry measurements were made on samples containing 10 -3 M K or HK in 10 -3 M phosphate buffer (pH = 7.4) with either 10 -2 M Na2SO 4 for oxidation or 10 -2 M KCI for reduction. Samples were bubbled continuously with N 2 throughout the measurements. The working electrode was a glassy carbon microelectrode, and a SCE reference was used together with a Pt counter electrode. Current vs. voltage scans were obtained using a Pine AFRDE4 bi-potentiostat and a Kipp and Zonen X-Y recorder. Pulse radiolysis was used to observe the reactions of K and HK with superoxide, hydrated electrons, hydroxyl radicals and azide radicals in aqueous solution. The primary species produced in the radiolysis of neutral water are solvated electrons (G = 2.6), OH" radicals (G = 2.7), and H atoms (G = 0.6), where the G values in parentheses are the number of radicals produced per 100 eV absorbed energy. Solutions may be made entirely reducing by addition of tert-butyl alcohol which serves to scavenge the hydroxyl radical (k = 6. 108 M - i s -1) and to a lesser extent the lower yield of hydrogen atoms (k = 105 M - i s - 1 ) , yielding a relatively unreactive radical [10]. Similarly, solutions may be made almost entirely oxidizing by saturation with N20, which reacts with ea-q to form OH" (k = 9.1 • 109 M - i s - l ) via

the following reaction: HzO N 2 0 + eaq

>OH'+ OH- +N 2

Addition of appropriate concentrations of sodium azide result in production of the azide radical, a well known one electron oxidant [11]. All dosimetry was performed using N20 saturated 10 -2 M KSCN solutions and assuming a G value of 6.2 and an extinction coefficient of 758 m2mo1-1 at 480 nm for (SCN)2-[12]. The experimental arrangement for pulse radiolysis has been described previously [13]. Solutions were bubbled for about 30 min with the appropriate gas prior to the experiment. Doses of between 1 and 15 Gy were delivered to samples contained in a 2.5 cm optical path length quartz flow cell. All optical densities were normalized to a dose of approx. 20 Gy for presentation. During the experiment, fresh sample was introduced into the cell after every pulse. Transient absorptions were monitored with a conventional xenon lamp, monochromator, photomultiplier tube arrangement. Data were recorded with a Biomation 8100 transient digitizer, and passed to an IBM compatible microcomputer for display, analysis, and hardcopy. Results Steady-state measurements p K a values. In order to identify the species in solution for the pulse radiolysis experiments we measured 0.80, ~0.60 c-

O

o0.40. 0~ -~0.20. 0,00

230

290 350 , 410 wavelength/nm

1.00

0°0.7b5 ~ ~ o C O

470

u

-o ~. 0 .50 in

0.25 0,00 230

290 350 , 410 wavelength/rim

470

Fig. 1. UV-visible absorption spectra of aqueous solutions containing 5' 10 -5 M H K as a function of pH. T h e spectra in (a) correspond t o the p H range 1.0 to 3.5 and those in (b) correspond to the p H range

7.0 to 11.0. The arrows indicate the changes in the absorptionbands upon increasingpH.

77 the p K a values of both K and HK. Absorption spectra recorded for aqueous solutions containing 5 . 1 0 -5 M HK as a function of pH are presented in Fig. 1. The upper set of spectra (a) correspond to the pH range 1.0 to 3.5 and the lower set (b) correspond to the pH range 7.0 to 11.0. Similar changes in absorption are observed for K in the pH range 1.0 to 3.5, however, the changes at higher pH are considerably smaller. Optical densities at several different wavelengths for each set of spectra were plotted vs. pH and pKa's were obtained by calculating theoretical pK curves to best fit the data. The following values were obtained: HK, 1.9, 6.2, 8.5 and 9.6; K, 1.9, 5.1 and 8.5. Assignment of these values is not straight forward. However, on the basis of pK values for tyrosine, 2.20 (COOH), 9.21 (NH 2) and 10.46 (OH) [14], and anthranilic acid, 2.17 and 4.95 [15], it is probable that the two lower values for both K and HK correspond to proton loss from the carboxyl and anilino groups. Leggate and Dunn [15] have measured the pK values of a series of substituted anthranilic acids and find two p K values in the ranges 1-3 and 4-6. They consider a pH independent equilibrium between the zwitterion and neutral anthranilic acid at pH values between the two p K values. In addition they show that electron donating substituents (in particular 5-hydroxy) push the equilibrium toward the side of the zwitterion. The two higher values for HK are probably due to proton loss from the hydroxyl group and the side chain amino group. Since they are so close and are defined by small changes of optical density we do not attempt a definite assignment. On the basis of the above information we think the most likely assignments for our pK values are as follows: for HK, 1.9 (COOH), 6.2 (ArNH2) , 8.5 and 9.6 (NH 2 and OH); for K, 1.9 (COOH), 5.1 (ArNH 2) and 8.5 (NH2).

Cyclic voltammetry experiments We observe irreversible oxidation waves centered at + 1.0 V and + 1.2 V vs. NHE for HK and K respectively. It is not possible to accurately quantify the amount of current passed in these experiments because the observed signals are very weak and the concentration of HK and K (10 -3 M) used is at the solubility limit for these compounds in solutions of high ionic strength. The measured half wave oxidation potentials should be regarded as qualitative, given the degree of difficulty encountered in performing the experiments. However, it would be reasonable to expect K and HK to undergo oxidation at a potential similar to tyrosine which is reported to have a value of + 930 mV vs. NHE [16]. Furthermore, it would also be reasonable to expect that oxidation of HK would be facilitated by the increase in electron density compared to K, which is the trend observed in our experiments.

0.04.

(3

f

C

-

0.00

-

0 0 0

-0.04

J

300

l

560 I .i 600 700 Wavelength/nm

460

800

0.04

b Q)

~

0.00

1

- w

0 0

-0.04 • I

300

I

I

I

400

500

600

760

800

Wavelength/nm

Fig. 2. Observed transient absorption spectra obtained from pulse radiolysis of N2 saturated, aqueous solutionscontaining(a) 1.1.10-4 M HK, 2% by volume tert-butyl alcohol and 1.10-2 M phosphate buffer (pH = 7.4) and (b) 1.0"10-4 M K, 2% by volume ten-butyl alcohol and 1.10-2 M phosphate buffer (pH = 7.4). Both spectra were recordedat 12.1/~s after the pulse.

The most that we can say from these data is that oxidizing species with potentials greater than the above values will be capable of oxidizing HK and K, and that the oxidized species will be unstable on the electrochemical timescale (ms). No reduction wave was observed for either HK or K on scanning from 0 to - 1 V.

Time-resolved measurements Reaction with hydrated electrons and superoxide Fig. 2 shows the difference absorption spectra observed 12 /~s after pulse radiolysis of N 2 saturated aqueous solutions (pH = 7.4) containing ten-butyl alcohol (2% by volume) and 1.10 -4 M HK (figure 2a) and K (figure 2b). These absorptions are present immediately after the decay of the hydrated electron and are attributed to the product of hydrated electron attachment to HK and K. In N 2 saturated water (pH = 7.4), the hydrated electron was observed to decay with a first order rate constant of 6.9 + 0.7-104 s -1. This rate constant increases linearly with increasing concentrations of either HK or K, yielding rate constants for hydrated electron attachment to K and HK, of 2.5 + 0.2.101° M - t s ,1 and 2.3 + 0.2.10 l° M - i s -1 respectively. We have not studied the decay of these species in detail except to note that all absorbances decay to the prepulse baseline on a time"scale of ca. 1 ms.

78 Pulse radiolysis of oxygen saturated water, pH = 7.4, containing tert-butyl alcohol (2% by volume) results in the formation of O~- via electron scavenging by 0 2. The decay of absorption of 0 2, monitored at 290 nm, was unchanged upon addition of as much as 10-3M K or HK. The best first order fit to the 0 2 decay gives a rate constant of ca. 5.9- 102 s-1. Assuming that a 20% change in this rate constant would be outside experimental error, an upper limit for the rate of reaction of K or H K with 0 2 may be specified as 1.2. l0 s M - l s - - 1. H K can be autooxidized by bubbling a solution of H K with oxygen. The formation of the stable product of autooxidation, HK(OX), can be followed by UV-VIS absorption spectroscopy and results in the appearance of a broad, featureless absorption which extends into the visible region of the spectrum. Pulse radiolysis of an oxygen saturated solution of HK(OX), prepared by bubbling a solution of 10 -3 M HK, 10 -2 M phosphate buffer and 2% by volume tert-butyl alcohol with oxygen, leads to a loss of absorbance from 400 nm to 560 nm due to the reaction of HK(OX) with 0 2. Fig. 3 shows the difference absorption spectrum 360 g s after the pulse along with the time evolution of the absorption at 460 rim. The first order rate constant for loss of absorption at 460 nm is measured as 6.4- 103 s - 1. If we assume that all of the H K has been oxidized and use our 'best fit' first order decay rate constant for O~(5.9- 102 s - l ) , we calculate a rate constant for reaction of 0 2 with H K ( O X ) of 5.8.106 M - 1 S -1. The value of this reaction rate constant represents a lower limit for reaction of 0 2 with HK(OX), since we assume that all H K has been oxidized and also that the oxidation product, HK(OX), is not formed from more than one H K molecule.

Reaction with hydroxyl radicals and azide radicals. Fig. 4a shows the difference absorption spectra observed 120 ~s and 1.5 ms after pulse radiolysis of a N 2 0 saturated aqueous solution containing 1 . 1 0 -4 M

0.01 L

~ "m I

c"

Q)

o

~,/,.,

o 0.00 0 ~

o

o

o

o

--0.01

390

, 430

t 470

, 510

550

Wovelength/nrn Fig. 3. Observed transient absorption spectrum obtained from pulse radiolysis of an 02 saturated, aqueous solution containing autooxidized HK (10 -3 M HK, 2% by volume ten-butyl alcohol and 1.10 -2 M phosphate buffer (pH = 7.4)). The spectrum was recorded at 360 /~s after the pulse. The loss of absorption at 460 nm as a function of time (2 ms full scale) is shown in the inset.

0.06~

~-~.0.00 0

0 . 300 400

0 3 ~ 500 600

700

800

Wovelength/nm 0.04

b

~ O.OC 0

o -0.04

300

400

5()0 6()0 7()0 Wavelength/nm

800

Fig. 4. Observed transient absorption spectra obtained from pulse radiolysis of a N 2 0 saturated, aqueous solution containing (a) 1" 10 - 4 M HK, 1.10-2 M phosphate buffer (pH = 7.4) and b) 4'10 -5 M K, 1" 10 -2 M phosphate buffer (pH = 7.4). The spectra correspond to -(o)- 120/.~s and -(e)- 1.5 ms after the pulse for a) and -(o)- 61 /zs and -(e)- 765 ~s after the pulse for (b).

H K at p H = 7.4. The early time spectrum is assigned to the products of reaction of OH" with H K and shows a bleaching around 370 nm, an absorption maximum at 410 and a broad band centered around 690 nm. The appearance of the absorption followed first order kinetics with a rate constant which was linearly dependent on the concentration of H K and yielded a bimolecular rate constant of 1.2 + 0.1.101° M - i s -1 for the reaction of O H with HK. Dosimetry measurements yielded a value of 70 + 7 m2mo1-1, independent of dose, for the extinction coefficient of the product of reaction of OH" with HK, at 690 nm. Similar experiments were performed with K. Fig. 4b shows difference absorption spectra 61 /zs and 765 jzs after pulse radiolysis of a N 2 0 saturated solution of 4 . 1 0 -5 M K at pH 7.4. The early time spectrum is assigned to the products of reaction OH" with K. The first order rate constant for the appearance of the absorption was linearly dependent on the concentration of K and yielded a bimolecular rate constant of 1.3 + 0.1 • 101° M - i s - 1 for reaction of K with O H " A n extinction coefficient of 32 + 4 m2mo1-1 for the product of reaction of OH" with K was measured at 690 nm and shown to be independent of dose. For both H K and K the products of reaction with OH" radicals were observed to decay via second order kinetics to give residual absorbing species which were stable to at least 1 s after the pulse. The difference

79 absorption spectra of these stable products are shown in the later time spectra in figures 4(a) and 4(b) for HK and K, respectively. The decay rate constants were essentially identical across the entire absorption spectrum indicating that only one species was responsible for the earlier time absorptions. Second order decay would be consistent with either disproportionation or dimerization of the initial reaction product. Assuming this to be the case and using the extinction coefficients above we measure bimolecular rate constants of 1.4 ± 0.2.10 s M-~s -1 and 3.4 + 0 . 4 . 1 0 a M-~s -~ for the decay of the products of OH" radical reaction with HK and K, respectively. Using dosimetry it is possible to correct the difference spectra of the products of OH" reaction with HK and K for the removal of the starting material. The corrected spectra for both HK and K are plotted in Fig. 5 along with the difference absorption spectra from which they were calculated. Extinction coefficients of 1.31 + 0.2" 102 m2mol -~ at 460 nm and 3.7 ± 0.4.102 m2mo1-1 at 340 nm were measured for HK and K, respectively. It is known that OH" can react with aromatic substrates through addition or hydrogen atom abstraction as well as one electron oxidation. In an attempt to determine which of these processes are occurring in the reaction of OH" with HK and K, we monitored the reaction between the azide radical and these substrates. The azide radical has been shown to be a selective one electron oxidant for many organic com-

0.12.

'~) 0.06 r-~

-6

o 0.00

-0.06 300

460

' 500

i 600

, 700

Wavelength/nm

°"141

/h,

800

b

{

~ o.o7÷i ~,

+h)0.00

0071 300

i'

-

,

400

,

500

.

J

.

600

.

.

.

760

Wavelength/nm

800

Fig. 5. Difference -(o)- and corrected -(e)- absorption spectra obtained from pulse radiolysis of a N20 saturated, aqueous solution containing (a) 3.8-10 - s M HK, 1.10 -2 M phosphate buffer (pH = 7.4) and (b) 4.0" 10-s M K, 1.10 -2 M phosphate buffer (pH = 7.4).

0.14,

0

t/)

0.07, ca

"6 o 0.00 n

0 -0.07 300

I

~

I

400

5 0

600

,I

700

Wavelength/nm

800

0.12.

b

•~ 0.08. t-

ca 0.04. O O

q

~a.O.O0' 0 -0.04

~ap u

--

300 460

560

_

660

760 800

Wavelength/nrn

Fig. 6. Difference -(o)- and corrected -(e)- absorption spectra obtained from pulse radiolysis of a N20 saturated, aqueous solution containing (a) 3.8.10 - s M HI(, 1.10 -2 M NaN 3 and 1.10 -2 M phosphate buffer (pH = 7.4) and (b) 1.9.10-SM K, 1-10 - 2 M NaN 3 and 1.10 -2 M phosphate buffer (pH = 7.4).

pounds including a large number of aromatic systems [11]. The experimentally observed difference absorption spectrum after pulse radiolysis of a N 2 0 saturated aqueous solution containing 10 - 2 M NaN 3 and 10 -4 M HK (pH = 7.4) is presented in figure 6a. Fig. 6a also shows the spectrum after correcting for loss of ground state absorption through reaction with N~ The observed first order rate constants for the increase in absorption at ca. 410 nm and 690 nm as well as that for bleaching at ca. 370 nm are identical within experimental error and show a linear dependence on the concentration of HK. The bimolecular rate constant for reaction between HK and N~ is calculated as 2.1 + 0.2.101° M - i s -~ from a plot of the observed rate constant versus HK concentration. The extinction coefficient at 690 nm and the bimolecular decay rate constant for the species formed from reaction of IlK with N~, are the same within experimental error as those measured for the species formed from reaction of HK and OH" radicals. These experiments were repeated using K as the substrate and the observed difference absorption spec. trum is given in Fig. 6b. The appearance of the transient absorptions and bleaching are again observed to proceed with identical rate constants, however, these rates were found to depend on the concentration of NaN 3. This can be understood if the azide radical and

80

20,0. -~ 15.0. ,~ I0.0. ~

5.0. 0.0

i

0

500

I

1000 [N3-]/[K]

I

1500

2000

Fig. 7. D e p e n d e n c e of the rate constant kob s on the concentrations of N a N 3 and K. Units of the y-axis are M - I s - 1.

oxidized K are in equilibrium under the conditions of our experiments, as shown below. kl N~+K.

k_i

"N3+K +

Using cyclic voltametry we measured an oxidation wave centered at + 1.2 V for K, which is sufficiently close to the reported oxidation potential of azide, + 1.32 V [17] for equilibration of these radicals to be expected. The equilibrium constant may be calculated from measurements of the rate of approach to equilibrium, kobs, via Eqn. 1 [18]: kob s / [ K ] = k 1 + k_ t[N3 ] / [ K ]

(1)

We have measured the rate of formation of oxidized kynurenine, kobs, at 420 nm as a function of [N3]/[K] and the data are shown in Fig. 7, plotted according to equation 1. From the intercept and slope of this plot we calculate k I = 4.8- 109 M - ] s -t and k_l = 6.9.106 M - i s - t , which gives us an equilibrium constant, Keq, of 695. From the value of the oxidation potential for azide and this equlibrium constant we calculate + 1.15 V for the oxidation potential of K [18], which is close to the value measured by cyclic voltametry. Also using this equilibrium constant we are able to correct the difference absorption spectrum of oxidized K for loss of ground state absorption, this is shown in Fig. 6b. As was the case for HK the bimolecular decay decay rate constant for this species is the same within experimental error as that for decay of the species formed by reaction of OH" with K. Discussion

From our measured pK a values for K and HK the species present at a pH of 7.4 are primarily zwitterions which have zero net charge. Both HK and K react at essentially diffusion controlled rates with hydrated electrons to give electron adducts. Although cyclic voltammetry showed no reduction on scanning to - 1 V, the reduction potential of the hydrated electron is - 2.8 V [t0], which is clearly sufficient to reduce either

K or HK. Reduction does not lead to stable species absorbing in the visible region of the spectrum. Thus reduction of these species in the lens would not directly lead to opacification. Neither HK or K react at any appreciable rate with O~- as has been observed for numerous other amino acids (for example, tyrosine + 0 2 , k < 10.0 M-~s-1; tryptophan + O~-, k < 24.0 M - i s -1 [19]). Thus our resuits show that it is unlikely that HK could have a protective effect as an antioxidant via direct scavenging of O~ radicals. However, we observe that the initial stable product of autooxidation of HK does react with O~- (lower limit for k is 5.6" 106 M - i s - l ) , and it is possible that this autooxidation product could be responsible for protection from the deleterious effects of O~-. Thus the presence of HK in tissue may indirectly lead to protection from O~. The measured rate constants for reaction of O H radicals with HK and K are in accord with those reported in the literature for similar compounds (tyrosine, 1.3.101° M - i s -1 [20]; acetophenone, 5.9.109 M - ] s - l [21]). The value of the rate constant for reaction of N 3 with HK is diffusion controlled while that for K is approx. 50% slower. We attribute this difference to the difference in oxidation potentials of the two compounds ( + 1.0 V for HK, + 1.15 V for K) since the reported value for azide is + 1.32 V [17], very close to that of K. The products of reaction of OH" with HK and K are potentially complex, since O H radicals may react by addition, one electron oxidation, or hydrogen abstraction. It appears, however, from the observed transient absorption spectra and kinetic analysis of the decay of absorption, that reaction of HK and K with the OH" radical produces only one observable species. Furthermore, reaction with both OH' and N 3 lead to the same intermediate. This can be understood on the basis of previous studies of similar compounds. The hydroxyl radical reacts with aniline [22] via direct hydrogen atom abstraction from the amino group to form the anilino radical (36%) as well as by addition to the aromatic ring to form an ortho adduct (54%) and a small amount (10%) of the para adduct. However, the ortho adduct rapidly dehydrates (k = 1.5 • 105 s -~) to form the anilino radical thus increasing the total yield of anilino radical to 90%. We suggest a similar process for K, resulting in the rapid transformation of the majority of the initial products into the anilino radical, which is the species we observe after the reaction of OH" with K. In the case of HK there is also the possibility of initial formation of the phenoxyl radical, however, this species can also rapidly interconvert to the anilino radical. Addition of OH' ortho to the hydroxyl group could also dehydrate to give the phenoxyl radical, followed by the anilino radical. Literature values of the

81 reduction potentials of phenoxyl ( P h O , H + / P h O H ) and anilino radicals are 1.352 V [23] and 1.030 V [24], respectively which may indicate that for H K the anilino radical is the more stable form. For the purposes of this discussion, however, we consider the two radical species to be in equilibrium. The spontaneous rate of dehydration of OH" adducts of substituted benzenes has been shown to increase with increasing electron density of the aromatic system [25]. The reported rate constants for dehydration of the OH" adduct of phenol (1" 103 s - I ) [26] and aniline (1.5" 10 5 s -1) [22] illustrate this point since an amino group is a better electron donor than an hydroxyl group. The presence of both an hydroxyl and an amino group in HK would further increase the rate constant for dehydration. We suggest that the reaction of OH" with K results in the formation of anilino radicals, whilst reaction with HK results in the formation of anilino and phenoxyl radicals which are in equilibrium. Possibly other minor products are formed which we are unable to resolve. Oxidation of H K or K with the azide radical may also result in the formation of an anilino radical. The radical cation formed from electron abstraction by N~ probably deprotonates rapidly. The PKaS of similar radical cations indicate this (tryptophan p K a = 4.3 [27], indole pKa = 4.9 [28]). Also, it has been shown that removal of an electron from 4-aminophenol is followed by rapid deprotonation from either the hydroxyl or amino group to yield a neutral radical [29]. The decays of the radicals derived from H K and K follow second order kinetics suggesting dimerization and disproportionation as possible mechanisms for their deactivation. Disproportionation of the radical derived from K is highly unlikely since there are no energetically favorable products available. In addition the major product formed from reaction of OH" with aniline is hydrazobenzene, resulting from dimerization of the anilino radicals [22]. We therefore favor dimerization for the decay path for the kynurenine radical as shown below [ R = COCH 2CH(NH ~-)COO - ].

R

2

,

R

NH2

H2N

NH R

H

NH2 The radical derived from H K may decay either by dimerization, in reactions analogous to those for K

shown above, or disproportionation, as shown below [ R = COCH2CH(NH~-)COO-]. Although our data does not allow us to differentiate between these two pathways, we note that the product of disproportionation has been proposed to be an intermediate in the autooxidation of H K catalyzed by H 2 0 2 and horseradish peroxidase [9]. Also an analogous disproportionation process has been suggested to occur in the decay of the 5,6-dihydroxyindole semiquinone [30].

Our results show that oxidation of H K and K by hydroxyl radicals results in the formation of a stable species with absorptions in the visible region. If these reactions occurred in the lens then certainly opacification would result, however, since the formation of this species is via a second order reaction of the initial oxidation product, it is highly unlikely that sufficiently high concentrations could be formed for this reaction to occur. It is more likely that the anilino radical would react with other lens-components, possibly leading to direct or indirect damage to the lens. Acknowledgments

This work was supported by a grant from the National Eye Institute (1 RO1 EYO8883). The Center for Fast Kinetics Research is supported jointly by the Biomedical Research Technology Program of the Division of Research Resources of the National Institutes of Health (RROO886) and the University of Texas at Austin. We thank Billy Naumann for operation of the accelerator. References

1 Eastman,C.L. and Guilarte, T.R. (1989) Brain Res. 495, 225-231. 2 Bryan,G.T., Brown, R.R. and Price, J.M. (1964) Cancer Res. 24, 596-602. 3 Dillon,J., Garcia-Castineiras,S., Santiago,M.A., and Spector, A. (1984) Exp. Eye Res. 39, 95-104. 4 Tomoda, A., Yoneyama, Y., Yamaguchi, T., Kakinuma, K., Kawasaki,K. and Yonemura, D. (1987) FEBS Lett. 219, 472-476. 5 Christen, S., Peterhans, E. and Stocker, R. (1990) Proc. Natl. Acad. Sci. USA 87, 2506-2510. 6 Goshima, N., Wadano, A. and Miura, K. (1986) Biochem. Biophys. Res. Commun. 139, 666-672. 7 Krishna, C.M., Uppuluri, S., Riesz, P, Zigler Jr., J.S. and Balasubramanian, D. (1991) Photochem.Photobiol. 54, 51-58. 8 Dillon, J., Wang, R.-H. and Atherton, S.J. (1990) Photochem. Photobiol. 52, 849-854. 9 Ishii, T., Iwahashi, H., Sugata, R. and Kido, R. (1992) Arch. Biochem. Biophys.294, 616-622. 10 Buxton, G.V., Greenstock, C.L., Helman, W.P. and Ross, A.B. (1988) J. Phys. Chem. Ref. Data 17, 513-886.

82 11 Alfassi, Z.B. and Schuler, R.H. (1985) J. Phys. Chem. 89, 33593363. 12 Buxton G.V. (1982) in The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis (Baxendale J.H. and Busi F. eds.), pp. 267-287, Reidel, Dordrecht. 13 Rodgers, M.A.J., Foyt, D.C. and Zimek, Z.A. (1977) Radiat. Res. 75, 296-304. 14 Dawson, R.M.C., Elliott, D.C., Elliott, W.H. and Jones, K.M. (1987) Data for Biochemical Research, pp. 7 and 31, Oxford University Press, Oxford. 15 Leggate, P. and Dunn, G.E. (1965) Can. J. Chem. 43, 1158-1174. 16 Harriman, A. (1987) J. Phys. Chem. 91, 6102-6104. 17 Alfassi, Z.B., Harriman, A., Huie, R.E., Mosseri, S. and Neta, P. (1987) J. Phys. Chem. 91, 2120-2122. 18 Wardman, P. (1989) J. Phys. Chem. Ref. Data 18, 1637-1755. 19 Bielski, B.H.J. and Shine, G.G. (1979) Oxygen Free Radicals and Tissue Damage, Ciba Foundation Symposium 65 (New Series), pp. 43-56, Excerpta Medica, New York. 20 Solar, S., Solar, W. and Getoff, N. (1984) J. Phys. Chem. 88, 2091-2095.

21 Willson, R.L., Greenstock, C.L., Adams, G.E., Wageman, R. and Dorfman, L.M. (1971) Int. J. Radiat. Phys. Chem. 3, 211-220. 22 Solar, S., Solar, W. and Getoff, N. (1986) Radiat. Phys. Chem. 28, 229-234. 23 Surdhar, P.S. and Armstrong, D.A. (1987) J. Phys. Chem. 91, 6532-6537. 24 Huie, R.E. and Neta, P. (1986) J. Phys. Chem. 90, 1193-1198. 25 Steenken, S. (1987) J. Chem. Soc., Faraday Trans. 1 83, 113-124. 26 Land, E.J. and Ebert, M. (1967) Trans. Faraday Soc. 63, 11811190. 27 Posener, M.L., Adams, G.E., Wardman, P. and Cundall, R,B: (1976) J. Chem. Soc., Faraday Trans. 1 72, 2231-2239. 28 Shen, X., Lind, J. and Merenyi, G. (1987) J. Phys. Chem. 91, 4403-4406. 29 Steenken, S. and Neta, P. (1982) J. Phys. Chem. 86, 3661-3667. 30 Lambert, C., Chacon, J.N., Chedekel, M.R., Land, E.J., Riley, P.A., Thompson, A. and Truscott, T.G. (1989) Biochim. Biophys. Acta 993, 12-20.