537
J . Phys. Chem. 1994,98, 537-544
4-Carboxybenzophenone-SensitizedPhotooxidation of Sulfur-Containing Amino Acids in Alkaline Aqueous Solutions. Secondary Photoreactions Kinetics Krzysztof Bobrowski,+-*Gordon L. Hug,'** Bronislaw Marciniak,*J and Halina Kozubekl Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556, and Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland Received: August 25, 1993; In Final Form: October 21, 1993"
Sulfur-containing amino acids and alanine were oxidized via photosensitization by 4-carboxybenzophenone (CB) in alkaline aqueous solutions. The mechanism of this reaction was examined using steady-state and laser flash photolysis techniques. The rate constants were determined for the quenching of the C B triplet state by five sulfur-containing amino acids and alanine and were found to be lo9 and 1.8 X lo8 M-* s-I, respectively. The observation of the (S:.S)+ radical cations of some of the amino acids showed that the quenching process involves a n electron transfer from the sulfur atom to the triplet state of CB. A slow process of formation of the ketyl radical anion occurring on the microsecond time scale was assigned to the one-electron reduction of C B by the a-aminoalkyl radicals that were formed earlier as a result of a n intramolecular electron transfer from the carboxyl group to the sulfur-centered radical cation followed by decarboxylation. For thiaproline, the pseudo-first-order rate constant, k b b , which characterizes the slow process of secondary ketyl radical anion formation, is linearly dependent on the C B concentration over the p H region 9.4-13.4. The slope of such a plot gives the bimolecular rate constant, krd, which is equal to the rate constant measured a t neutral pH. In contrast, for methionine similar plots show a marked departure from linearity which is exaggerated with increasing pH. At p H 13.4 the slow process is entirely suppressed. These results in basic solutions of methionine are associated with the presence of a new transient tentatively assigned to a n intramolecularly S:.N-bonded radical cation of methionine and with the acid-base equilibria involving sulfur- and nitrogen-centered radical cations. Supplementary measurements of C02 formation in steady-state photolysis experiments have revealed that with increasing p H there is a significant decrease in the value of the quantum yield of C02 in the solutions of methionine and a minor decrease of the quantum yield of C02 in the solutions of thiaproline. A detailed mechanism for the primary and the secondary photoreduction of C B is proposed and discussed.
-
-
Introduction
In recent studies132 it was shown that the interaction of triplet states of substituted benzophenones with sulfur-containing amino acids involves an electron-transfer (ET) mechanism. The mechanism in neutral aqueous solution1 was resolved into an initial electron transfer from the sulfur atom to the triplet state of 4-carboxybenzophenone (CB) followed by (1) the diffusion apart of the charge-transfer (CT-S) complex to form sulfur-centered radical cations and ketyl radical anions, (2) intramolecular proton transfer within the CT-S complex to form ketyl radicals and a-(alky1thio)alkyl radicals, and (3) back electron transfer to regenerate the reactants. A significant contribution of radical ions to the transient spectra showed that diffusion apart, not proton transfer, was a dominant pathway. The analogous mechanism was found to operate for quenching triplet states of substituted benzophenones by sulfur-containing amino acids in waterlacetonitrile solutions.2 Further evidence to support this mechanism was provided by correlations of the quenching rate constants with the free energy change for electron transfer (displayed as Rehm-Weller plots) applying both "quadratic" Marcus and "asymptotic" Agmon-Levine free energy relationships.2 In the present paper, this work is extended into the alkaline region since the nitrogen atom in the deprotonated amino group can potentially participate in the quenching mechanism, in competition with the sulfur atom. This question is addressed with respect to the mechanism proposed by Cohen and Ojanpera3 t On leave from the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. University of Notre Dame. 8 A. Mickiewicz University. Abstract published in Advance ACS Absrracts, December 15, 1993.
for the quenching of excited triplet states of CB by methionine and related compounds. For methionine, two CT complexes have been propo~ed:~ a CT-S complex [>C*-O--.+*SC*-O-.-+*NI]. These two forms exist in equilibrium, and only the CT-N complex may lose COZ directly to form a-aminoalkyl radicals. In the current photochemical study, CB triplets were quenched by alanine (a non-sulfur-containingamino acid) and by five sulfurcontaining amino acids: thiaproline, S-(carboxymethyl)cysteine, methionine,a-methylmethionine, and homomethionine (see Chart 1). Electron transfer from the sulfur atom to the triplet state of CB to form the CT-S complex is shown to be a principal reaction. A slow process of formation of the ketyl radical anion occurring on the microsecond time scale is observed. An analogous process, observed in neutral aqueous solutions,' has been assigned to the one-electron reduction of CB by the a-aminoalkyl radicals that were formed earlier in the quenching event as a result of an intramolecular electron transfer from the carboxyl group to the sulfur-centered radical cation followed by decarboxylation. For methionine the pseudo-first-order rate constant, k b , for the secondary reduction is shown to exhibit a sublinear dependence on the CB concentration. On the other hand for thiaproline, kbb is linearly dependent on the CB concentration over the pH region 10.9-1 3.3, and the slope of such a plot gives the bimolecular rate constant, krd, which is equal to that measured at neutral pH.1 The results in basic solutions of methionine are associated with the presence of a new transient (which is tentatively assigned to an intramolecularly S:.N-bonded radical cation) and with acidbase equilibria involving sulfur- and nitrogen-centered radical cations. Further evidence to support the proposed reaction mechanism is provided by supplementary measurements of C02 formation in complementary steady-state photolysis experiments. On the basis of the above observations, a reaction scheme is
0022-3654/94/2098-0537$04.50/0 0 1994 American Chemical Society
538 The Journal of Physical Chemistry, Vol. 98, No. 2, 1994 CHART 1 HN-CH-COO-
I
YHl -0GC-CH2-S-CH2-CH-COO-
I
HiC,
,CHI S
thiaproline
1
S-(carboxymethyl)cysteine
methionine
3
a-methylmethionine
homomethionine
5
alanine
2
4
6
SCHEME 1 1-13)
A
>c’
I
(7)
-0
(12)
[OH-]
proposed for the primary and the secondary photoreduction (Scheme 1, vide infra) of CB in alkaline aqueous solutions.
Experimental Section 4-Carboxybenzophenone (Aldrich) and the sulfur-containing amino acids wereobtained from Sigma as the best available grades and were used without further purification. A sample of homomethionine was a generous gift from Professor K.-D. Asmus (HMI, Germany). Alanine was purchased from Aldrich and was used as received. Water was purified by a Millipore Milli-Q system. The concentrations of sulfur-containing amino acids in the laser flash photolysis experiments were in the range 8 X 10-510-3 M (in the quenching experiments) and 2 X 10-2-10-1 M (when recording spectra in the time range following complete quenching of the 4-carboxybenzophenone triplet). In quenching experiments the 4-carboxybenzophenone concentration was 2 X lo-’ M in water. During monitoring of the secondary process, its concentration was in the range of 4 X 10-4to 4 X 10-3 M. All solutions were deoxygenated by bubbling high-purity argon through them. The pH of the solutions were adjusted by adding sodium hydroxide. The nanosecond laser flash photolysis apparatus has been described in detail el~ewhere.~ Laser excitation either at 337.1 nm (operated at 1-3 mJ, pulse width 8 ns) from a Molectron UV-400nitrogen laser or a t 308 nm (operated at 4-6 mJ, pulse width 20 ns) from Lambda-Physik EMG 101 MSC (Xe/HCl) excimer laser was used in a right-angle geometry with respect to the monitoring light beam. Rectangular quartz cells (0.5 X 1
Bobrowski et al. cm) with a path length of 0.5 cm for the monitoring beam were used. Typically 3-6 laser shots were averaged for each kinetic trace. Transient spectra were taken using a flow-cell apparatus. The transient absorbances a t preselected wavelengths were monitored by a detection system consisting of a double monochromator, a photomultiplier tube, and a pulsed xenon lamp of 1 kW as the monitoring source. The signal from the photomultiplier was processed by a 7912 AD Tektronix transient digitizer controlled by a LSI 11/2 microprocessor. Cutoff filters were used to avoid spurious response from second-order scattering from the monochromator gratings. All experiments were performed at 22 f 1 OC. All pulse radiolysis experiments were performed by applying 10-ns pulses of high-energy electrons from the Notre Dame 7 MeV A R C 0 LP-7 linear accelerator. Absorbed doses were on the order of 4-6 Gy (1 Gy = 1 J/kg). A description of the pulse radiolysis setup and data collection system is reported elsewhere.ss6 The experiments were carried out with a continuous flow of the sample. The steady-state photolysis experiments were carried out in a 1 X 1 cm rectangular UV cell on an optical bench irradiation system. The high-pressure mercury lamp (HBO 200) with a combination of a BC-4 glass filter (Mashpriborintog, U.S.S.R) and a 3 13-nm interference filter (Zeiss) were used for excitation. A solution of 2-hexanone in cyclohexane was used as an actinometer: and the intensity of incident light was determined to be 4.7 X einstein dm-3 s-l. Solutions containing CB (2 X M) and amino acid (2 X M) at an appropriate pH, adjusted with concentrated sodium hydroxide solution, were purged with oxygen-free argon for 15 min and irradiated. The duration of the irradiation times were chosen to cause about 1025% CB conversion. The changes in the CB concentrations during irradiation were determined spectroscopically. The quantitative analysis of C02 was achieved using a gas chromatographic head-space technique. The concentration of C 0 2was measured by analysis of the vapor phase in the reaction cell after acidification of the irradiated solutions with concentrated HCl. The gas chromatographic analysis was performed on a Chromatron GCHF 18.3 instrument with a thermal conductivity detector on a Porapak Q column. High-performance liquid chromatography (HPLC) analysis of methionine sulfoxide was performed on a Waters 600E instrument with a photodiode array detector using a reversed phase C18 column (Waters Nova Pak) eluted isocratically with 5% aqueous acetonitrile solution containing 0.1 M CH3COONH.+
Results Quenching Rate Constants of the CB Triplet by SulfurContaining Amino Acids and Alanine. Five sulfur-containing amino acids and alanine were used as quenchers of the 4-carboxybenzophenone (CB) triplet state in aqueous solutions at pH 11.5. Since only alkaline solutions were studied, it will be understood that the CB triplet state is always in its carboxylate anion form8 (pK, = 4.5 for CB)9 and that the amino acids are in their anionic form.1° The quenching rate constants, kq,were obtained from the experimentally measured, pseudo-first-order rate constants, kobr for the decay of the CB triplet, and by employing the formula (1) ko, = 7;’ + kJQ1 where 7 T is the lifetime of the CB triplet in the absence of a quencher. Typical experimental traces for triplet decay in the presence of about 5 X 10-4 M homomethionine and alanine are presented in the insets of Figure 1. The long-lived absorption has been attributed to the formation of the 4-carboxybenzophenone ketyl radical anion CB*-, that was established by comparison of the
The Journal of Physical Chemistry, Vol. 98, No. 2, 1994 539
Photooxidation of Sulfur-Containing Amino Acids Homomelhionine
35 0.005
30
1.0
in
25
Time
,
X
20
X
d
15
Y
0 10
Time
I
5
0
0.0
2.0
4.0
6.0
6.0
10.0
12.0
14.0
16.0
[Q] x 104, M
Figure 1. Plots according to eq I for the 4-carboxybenzophenone(CB) triplet quenching by (a) homomethionine, (b) S-(carboxymethyl)cysteine, and (c) alanine in aqueous solution at pH = 11.5. Inserts: experimental traces for the CB triplet decay at 540 nm in the presence of 5 X 10-4 M homomethionine (upper insert) and 4.8 X 1 V M alanine (lower insert).
TABLE 1: Kinetic Data Regarding the Quenching of CB Triplets by Sulfur-containing Amino Acids and Alanine (&I k, x 10-9 ( ~ - 1s-*)o amino acid pH l l S b pH 6.V 1
2.6
2.1
2 3 4
0.75
0.81
S
2.7 2.9 2.6
6
0.18
2.6 2.6 2.9 S 6 X 1W
a Estimated errors (taken as twice the standard deviations from the least-squares fits) -5% for all amino acids except 6 (10%). This work. Reference 1.
observed transient absorption spectra with that from Inbar et a1.8 and Hurley et ale9(absorption maximum at 660 nm). The pseudo-first-order rate constants, kob, were calculated using eq I1 which takes
A - A" AO - A" into account a concomitant, underlying first-order growth of the photoproduct's absorption;ll in eq 11, AO, A, and A' are the absorbance changes at time 0, t, and infinity, respectively. Some typical plots based on eq I are presented in Figure 1, and the quenching rate constants obtained for all the sulfur-containing amino acids and alanine together with those obtained in neutral aqueous solutions' are summarized in Table 1. Secondary Reaction of the CB Ground State. At high amino acids concentrations (-10-1 M) for which the CB triplet quenching is complete within a short time (150 ns) after the laser pulse, the experimental traces clearly indicate two processes of ketyl radical anion formation. A fast component occurs on a nanosecond time scale within the time scale of triplet decay. It can be attributed to a process which starts with an electron transfer from the sulfur atom to the CB triplet to form the CT-S complex, followed by the diffusion apart of the ions, resulting ultimately in an efficient formation of sulfur-centered radical cations and ketyl radical anions. A slow process occurs on the microsecond time scale, and its rate constant depends on the concentration of CB in the ground state. Of the amino acids studied, this slow growth is absent only for S-(carboxymethy1)cysteine. As will be discussed later, the slow process of ketyl radical anion formation can be assigned to the one-electron reduction of CB in the ground state by the a-aminoalkyl radicals produced ina process analogous to the one proposed in neutral solutions,' Le., an intramolecular
In -= -k,t
1
2
3
4
0.5
1.0
1.5
2.0
[ce]x i o 3 , M Figure 2. The 4-carboxybenzophenone(CB) concentration depcndence of the pseudo-first-orderrate constant, for the reaction of CB ground state with a-aminoalkyl radicals derived from (a) thiaproline, (b) methionine,(c) alanineusinglaserflash photolysis in Ar-saturatedaqueous solutions at pH 11.5 containing 2 X M of the amino acid, (d) thiaproline, and (e) methionine using pulse radiolysis in N20-saturated solutions at pH 11.4containing 2 X M of the amino acid. [CB]x lo3, M
electron transfer from the carboxyl group to the sulfur-centered radical cation followed by decarboxylation. Contrary to theobservations at neutral pH,' pseudo-first-order rate constants, kbb, which characterize the slow process of ketyl radical anion formation, are not linearly dependent on the CBground-state concentration, except for the case of thiaproline. Figure 2 shows representative plots obtained for thiaproline, methionine, and alanine (2 X le2 M) and various concentrations of CB (4 X 10-4 to 4 X l t 3 M) at pH 11.5. As the data in Figure 2a show, for thiaproline kbb is linearly dependent on the CB concentration, and the slope of this plot gives the bimolecular rateconstant for the one-electronreduction of CB by a-aminoalkyl radicals produced from thiaproline. The calculated rate constant, krd = 9.5 X 108 M-I s-1, is equal (within the experimental error) to that measured at neutral pH (6.8).' In contrast, the similar plots for methionine and alanineexhibit a marked departure from linearity (Figure 2, band c), Although, the kbb tends to increase with increasing CB concentration, it begins to approach a limiting value at high CB concentrations. Complementarypulse radiolysis experiments were performed at pH 11.4 in which the appropriate a-aminoalkyl radicals were generated and their reactions with the CB ground state were monitored. The formation of ketyl radical anions was observed, as in the case of flash photolysis, by monitoring of the absorption of ketyl radical anions at 650 nm. The kinetic behavior of the ketyl radical anions was the same in the pulse radiolysis experiments as in the flash photolysis experiments. In particular, the k,dvalue obtained for thiaproline (7.6 X 108 M-1 s-l, Figure 2d) is in good agreement with the result obtained from flash photolysis (vide supra), and k6b for methionine is not linearly dependent on the CB-ground-state concentration (Figure 2e). In order to resolve these intriguing results we have extended our studies over the pH range 9.4-13.4. Unlike thiaproline, the plots of k b b vs CB-ground-state concentration in methionine solutionsshow a marked departure from linearity with increasing 13.4 the slow process was entirely pH (Figure 3). At pH suppressed. This is illustrated in Figure 3 (insets A X ) , where kinetic traces showing the ketyl radical anion formation in the presence of methionine (2 X 1t2M) and 4-carboxybenzophenone (2.4 x 10-3 M) in aqueous solutionsat different pH, are presented as typical examples. TraasientAbmrptionSpectraof Intermediates. Flash excitation at 337 nm of argon-purged aqueous solutions of CB (2 X 10-3 M) in the presence of sulfur-containing amino acids and alanine at pH 11.5 resulted in the appearance of transients at 540, 570, and 650 nm. The transient at 540 nm is associated with the
-
-
-
The Journal of Physical Chemistry, Vol. 98, No. 2, 1994
Bobrowski et al.
.,
010
0 05
0.W
#
, 1
." -
2
-
I
I
3
i
pH 12.6
4
[CB] x IO3, M
Figure 3. The 4-carboxybenzophenone (CB) concentration dependence of the pseudo-first-orderrate constant kbt, for the reaction of CB ground state with a-aminoalkyl radicals derived from methionine using laser flash photolysis in Ar-saturated aqueous solutions containing 2 X lo-* M of methionine at different pH. Inserts: kinetic traces for the slow growth of ketyl radical anion formation at 650 nm in the flash photolysis of aqueous solutions containing 2 X 10-2M methionine and 2.4 X 10-3 M CB at pH: (A) 10.2, (B) 12.6, (C) 13.4.
triplet of CB, and the ones at 570 and 650 nm are assigned to ketyl radical (CBH') and ketyl radical anion (CB*-), respectively, on the basis of data previously published by Inbar et a1.8 and Hurley et aL9 Two additional intermediates could be identified from a composite spectrum taken where higher concentrations of methionine, a-methylmethionine, and homomethionine (10-I M) were used to rapidly quench more than 99% of the triplets at pH 11.5. The composite spectrum is reminiscent of the ketyl radical anion spectrum, but it has a short-wavelength shoulder in the region of 480 nm. The 480-nm spectrum was assigned to the intermolecular S:.S-bonded radical cation. Such electrontransfer intermediates were recently seen in the analogous system in neutral aqueous' and water/acetonitrile2 solutions. The detailed quantitative resolution of the observed spectra was done using a linear multiple regression12 of the form
-
where AA(XI) is the observed optical density change of the composite spectrum and ci(Aj) is the molar absorption coefficient of the ith species at the j t h wavelength of observation. N is the number of data points in the experimental transient spectrum and, in this work, was usually equal to 33 or 34. The elements of the set (a,)are the regression coefficients which in this case are each equal to ell,where ci is the concentration of the ith transient and 1 is the optical path length of the monitoring light. The component spectra and their molar absorption coefficients are as follows: the triplet absorption was collected in the current work in water at pH 11.5 with e535 taken to be 6250 M-I cm-l for aqueous solution (ref 8 and revised in ref 9 based on an improved13 measurement of the actinometer, benzophenone in benzene, of e = 7220 M-* cm-I); the intermolecular S:.S-bonded radical cation of methionine spectrum14 was extended to the long-wavelength region with a fitted Gaussian tail; and thespectra of the ketyl radical anion and the ketyl radical of CB were taken from ref 8 with the ketyl radical anion and the ketyl radical spectra renormalized to e650 = 7660 M-l cm-I and €570 = 5200 M-I cm-l, respe~tively.~J~ With only three transients in the spectral mixture of eq 111, there was a deviation in the shortwavelength region C430 nm which is evident in the difference between spectra 4a and 4b in Figure 4. This discrepancy in the 400-nm region is present even in slightly basic solutions, Le., near
u x I 5 m s m 7 W
u x I 5 m s m 7 W
1 w 5 m ( y x I m ,
Wavelength (nm)
Wavelength (nm)
Wave(eqth (nm)
Figure4. Resolution of the spectralcomponentsin the transientabsorption spectra following the quenching of 4-carboxybenzophenone triplet in aqueoussolutionsof 4-carboxybenzophenone(4 X I t 3M) and methionine (0.1 M) at pH 11.15 taken -20 ns (a), (b); -280 ns (c) after the flash. From the regressions analysis concentrationsof transients are (a) (4.84 f 0.83) X lo", (33.4 & OS) X 10-6, (2.45 & 1.23) X 10-6,and (19.2 & 1.7) X lo" M for (S:.S)+radical cation, ketylradicalanion,ketyl radical, and S;.N-bonded radical cation, respectively; (b) (11.5 & 1.4) X 10-6, (33.6 & 1.1) X l v , and (-2.28 2.62) X 10-6 M for (S:.S)+ radical cation,ketyl radical anion,and ketyl radical, respectively; note that [S:.Nbonded radical cation]was assumed 0; (c) (45.9 0.5) X 10-6M for ketyl radical anion.
*
*
CHART 2
the pK.of the amino group, and can be explained by the formation of a new intermediate which we suggest is an S:.N-bonded radical cation.Is Formation of an S:.N-bond requires interaction of the unpaired electron on sulfur with the free electron pair on nitrogen and is assisted by a suitable steric arrangement (five-membered ring)16 (Chart 2). The S:.N-bonded radical cation derived from L-methionine ethyl ester spectrum was taken from ref 15 and renormalized with c385 = 4800 M-I cm-1. The quantitative agreement in the spectral analysis in Figure 4a after taking into account a contribution of the S.-.N-bonded species supports our earlier contention. After the decay of the (S:.S)+ radical cation, S:.Nbonded radical cation, and fast deprotonation of the ketyl radical, the ketyl radical anion is the only speciesobserved in the spectrum (Figure 4c). The formation of (S:.S)+ dimers and the absorption in the short-wavelength region (similar to the case of methionine presented in Figure 4) were also found for the CB-sensitized photooxidation of a-methylmethionine and homomethionine. In contrast, similar absorptions in solutions of thiaproline and S-(carboxymethy1)cysteine were not observed. The S:.N-bonded species cannot be formed in thiaproline since formation of the intramolecular S;.N-bond would require a thermodynamically unfavorable three- or four-membered ring structure. As shown in Figure 5a, the transient spectrum recorded in the solution of thiaproline within the time scale of the CB triplet decay (at the end of the flash) is due to the ketyl radical anion, CB*- (A, 650 nm), and the ketyl radical CBH' (A, = 570 nm), along with the contribution from the CB triplet (Amx = 540 nm). After the decay of the CB triplet and fast deprotonation of the ketyl radical, the ketyl radical anion is the only species observed in the spectrum (Figure 5b). (S:.S)+ Radical Cation Hydroxylation Kinetics. When the concentration of amino acids (3, 4, and 5) is high enough such that the (S:.S)+ dimers can be formed, the (S:.S)+ decay could be monitored at 480 nm. An interesting feature of the decay is
The Journal of Physical Chemistry, Vol. 98, No. 2, 1994 541
Photooxidation of Sulfur-Containing Amino Acids I
-
TABLE 2 Quantum Yield of Ketyl Radical Ani00 Formation
b1
c-*m
in the Photochemical Process and in the Secondary Photoreduction by Sulfur-Containing Amino Acids and Alanine amino @ICE.acid PH 11.2' DH6.86
Wavelength (nm)
1 2 3 4
Wavelength (nm)
Figure5 Resolutionofthespectralcomponentsinthetransient absorption spectra following the quenching of 4-carboxybenzophenonetriplet in aqueous solutionsof 4-carboxybenzophenone (2 X M) and thiaproline (10-1 M) at pH 11.2 taken (a) at the end of the flash; (b) -90 ns after the flash. From the regressionsanalysis concentrationsof transients are
*
*
(a) (7.55 0.95) X 10-6, (3.91 1.46) X 10-6,and (25.0 0.5) X 1od M for triplet, ketyl radical, and ketyl radical anion, respectively; (b) (47.6 0.7) X 10-6 M for ketyl radical anion.
b j b 30.0
/
r
002
II
5
6
0.73 0.93 0.72 0.71 0.30 0.96
0.80 (0.76-0.82) 0.33 ~0.32 (0.35-0.42) d
lim @Cb
4ICB.-
DH11.2' DH6.gb DH11.2' DH6.86 0.59 c
0.66 0.59 0.10 0.85
0.60 10.06 0.25 =0.13 50.07
1.29
0.40
1.40 0.82 0.58 0.45 0.42
c
1.81
c
c
1.38 1.30
a This work. Reference 1. Secondary photoreduction not observed. k, < 6 X lo5 M-* s-l.
(0) of the transients and final photoproducts as a result of the interactions between the triplet and the quencher is as important a mechanistic detail as are the magnitudes of the quenching rate constants. We have estimated the yields of the ketyl radical anions (CB*-) for concentrations of the amino acids when the CB triplet is almost totally quenched (>99%). The experimentswere carried out in matchedoptically flat cells with identicalabsorbance (due to CB) at either 337.1 or 308 nm. For 4-carboxybenzophenone and various amino acids, the absorption changes at the spectral maximum of CW- were measured and compared with an absorption change immediately after the flash at 535 nm due to the CB triplet. Taking values of the molar extinction coefficients = 6250 M-I cm-' for the CB triplet9." and 8660 = 7660 M-I cm-1 for CB'-,9J3 the yields (0) were calculated according to (IV)
In / Time
I
I
I
I
I
I
0.0
2.0
4.0
6.0
8.0
10.0
[OH-]x 103, M Figure 6. Plot of the observed pseudo-fint-order rate constant of the (S:.S)+radicalcation decay monitoredasa functionofOH-concentration. Inserts: kinetic tram for the decay of (S:.S)+ radical cation at 450 nm observed in the flash photolysis of aqueous solution of CB (2.4 X 10-3 M)and methionine (2 X 1 P 2M) at pH: (A) 11.76, (b) 11.12, and (C) 9.35.
its dependence on the hydroxyl ion concentration. A similar influence of base on sulfur-centered radical cation decays was also found in the benzophenone-sensitized photooxidation of 13dithia~yclooctane.~~ This observation was rationalized in terms of a "neutralization" reaction either of >S*+radical cations and/ or (S;.S)+ by hydroxyl ions OH-. The rate constant for hydroxylation was determined by monitoring decays of (S:.S)+ radical cations at 450 nm obtained from the flash photolysis of CB (2.4 X 10-3 M) and methionine (2 X 10-2 M) in aqueous solutions varying in pH between 9.35 and 11.76. The higher ratio of molar absorption coefficients of (S;.S)+ vs ketyl radical anion absorption at 450 nm, compared to the corresponding ratio at 480 nm, made 450 nm a more favorable monitoring wavelength than the maximum of the (S;.S)+ radical cation at 480 nm. Appropriate values of pH were adjusted by adding NaOH. Typical examples of the influence of the pH on the decay kinetics of (S;.S)+ are presented in the insets of Figure 6. A plot of the observed rateconstant for the (S;.S)+ decaysvs theconcentration of OH- (Figure 6) allowed us to determine the second-order rate constant for hydroxylation of the (S;.S)+ radical cations in aqueous solution at room temperature, k = (4.30 f 0.14) X 109 M-1 s-1. The value obtained is in reasonable agreement with that measured in the pulse radiolysis studies of cyclo(methiony1methionine) peptide in aqueous solutions k[cyclo-Met-Met(S:.S)+ + OH-] = (2.6 f 0.3) X 109 M-1 s-l.l* KetylRadicalAdon (CB-) andCarbon Dioxide (CO,) Quantum Yields. Quantitative information regarding the quantum yields
where A A p is the product's absorption change extrapolated either (a) to the "end-of-pulse" value &4: under the conditions of nearly complete quenching or (b) to a plateau value AA;, corresponding to the total formation of the ketyl radical anion. These U p ' s correspond (a) to the formation of CW- in the photochemical process (0') and (b) to the formation in the secondary reaction (a"), respectively. PA: is an absorption change immediately after the flash due to the 4-carboxybenzophenone triplet at 535 nm, measured under the conditions of no quenching. Since the absorption spectra of the (S:.S)+ radical cation and the ketyl radical anion (CW-) overlap, the absorption change at A, = 650 nm ( U p ) was corrected to remove the contribution from the (S;.S)+ radical cation. The quantum yields of ketyl radical anions for all the amino acids studied are summarized and compared with the analogous quantum yields measured in neutral solutions1in Table 2. Since the overall yields of ketyl radical anion formation depend on pH (as shown in insets of Figure 3), we have measured quantum yields of ketyl radical anion formationin the photochemicalprocess (from the fast component), a', and the yield of ketyl radical anion formation in the secondary photoreduction (from the slow growth), a", over the pH range 9-13.5 for three representative amino acids: methionine, thiaproline, and alanine . All these data plus the calculated overall quantum yields of ketyl radical anion formation, @lotcrr, are summarized in Tables 3-5 As the plots in Figure 7 show, thevalues of ketyl radical anion formation in the secondary photoreduction depend on the pH. For methionine, 0" drops sharply from 0.66 (pH 11.3) to 0 (pH 13.2). In contrast over the same pH region, 0" changes from 0.54(pH 11.4) to0.17 (pH 13.15) forthiaproline,andfrom0.86 (pH 11.05) to 0.72 (pH 13.4) for alanine. Experimental limitations restricted the accuracy of the measurements of the quantum yields of CBH' and the (S:.S)+ radical cation. Reasons for these limitationsincludeveryfast ketyl radical (CBH') deprotonation kinetics19 and (S:.S)+ radical cation hydroxylation kinetics at high concentrations of hydroxyl ions
Bobrowski et al.
542 The Journal of Physical Chemistry, Vol. 98, No. 2, 1994
TABLE 3: Quantum Yields of C02 aad Ketyl Radical Anion Formation in the Photochemical hocees (eta-) and in the Secondary Photoreduction (Offca.-)by Methionine
@E!-
PH 6.8 9.2 10.4 11.3
@'CB.-
0.33" 0.56 0.66 0.71
0.25' 0.44 0.59 0.66
0.580 1 .oo 1.25 1.37
11.7 12.1 12.5 13.0 13.2
0.69 0.70 0.73 0.76 0.82
0.53 0.42 0.28 0.02 0.00
1.22 1.12 1.01 0.78 0.82
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0.200 0.6
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0.55b 0.49c 0.62d 0.53c
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