Electron spin resonance studies on tryptophan photolysis in frozen micellar systems of anionic surfactants

J. Photochem. Photobiol. A: Chem., 75 (1993) 131-136

131

Electron spin resonance studies on tryptophan photolysis in frozen micellar systems of anionic surfactants E w a Szajdzinska-Pietek, J a n u s z B e d n a r e k a n d A n d r z e j P l o n k a The Institute of Applied Radiation Chemistry, Technical University of ~

Wroblewskiego 15, 93-590 ~

(Poland)

(Received February 1, 1993; accepted May ;_3, 1993)

Abstract U V photolysis of U~ptophan (Trp) was studied by the electron spin resonance method in frozen solutions of simple miceiles (SMs) and reversed miceUes (RMs) of anionic surfactants: sodium bis(2-ethylhexyl)sulphosuccinate, .~odium dodecylsulphate and ammonium perfluoropolyether carbonate. The primary species stabilized at 77 K in protiated systems are Trp "+, Trp" and alkyl-type radicals while, in the per'fluorinated RM system, only Trp "+ is observed. The yield of paramagnetic species is an order of magnitude higher in microheterogeneous systems than in homogeneous aqueous solutions. It is higher in RM than in SM samples, parallel to the fraction of Trp molecules bound to micellar interface. In the presence of oxygen the primary photoproducts transform into peroxy radicals on heating the sample above 100 K. In deaerated samples, Trp "+ survives up to temperatures of 190 K or higher (depending on the system).

I. l•troduction Tryptophan (Trp) is thought to play a crucial role in the light sensitivity of a number of peptides and proteins. Its photophysics and photochemistry have been subjected to numerous investigations (see refs. 1 and 2 for early reviews). Recently, besides homogeneous aqueous solutions, microheterogeneous systems such as simple micelles (SMs) and reversed micelles (RMs), thought to be simplified models of biological membranes and enzyme pockets, have been studied by absorption and emission spectroscopy [3-8]. The microenvironment structure, solute distribution between pseudophases, excitation, and energy transfer processes were of particular interest. In the pre,;ent electron spin resonance (ESR) study, U V photolysis of Trp in SM and R M solutions of various anionic surfactants was followed at cryogenic temperatures. As indicated previously [9], the micellar structure is retained on freezing such systems. So we could identify primary paramagnetic photoproducts and determine their yields and thermal stability in different aggregates. 2. Experimental details

Sodium bis(2-ethylhexyl)sulphosuccinate (AOT), and sodh~m dodecylsulphate (SDS) from 1010-6030/93/$6.00

Sigma, 1-octanol (99 + %, Gold Label) from Aidrich, n-heptane for high performance liquid chromatography from Fluka, and DL-tryptophan (Trp) from B D H Chemicals were used as received. Ammonium perfluoropolyether (PFPE) carbonate surfactant and PFPE oil were a gift from Professor Shulamith Schlick of the University of Detroit Mercy (Michigan, USA). Water was triply distilled. R M systems were prepared of the stock solutions of surfactants (150 mM AOT--n-heptane, 960 mM SDS-octanol [10] and 1100 mM PFPE (surfactant)-PFPE(oil) [11] by adding appropriate volumes of water to obtain desired water to surfactant molar ratios ¢o (5-50 for AOT, 16 for SDS, and 9.5 for PFPE). SM systems of 28 mM AOT [12] and 100 mM SDS in water were prepared. Trp was introduced in 0.3-0.5 mM concentration by adding appropriate volumes of its aqueous stock solution. All liquid components and resulting ~olutions were carefully deaerated by prolonged bubbling with argon. Controlling experiments were also done with systems degassed by freeze-pump-thaw method and with systems saturated with oxygen. Samples were flame sealed in 75 pi micropipettes (VWR, USA), aged overnight in dark and then quenched in liquid nitrogen. Irradiations were performed inside the ESR cavity using a KB-5703 slit illuminator (Cobrabid © 1993- Elsevier Sequoia. All rights reserved

E. Szajdzinska-Pietck et ai. I ESR of Trp photolysis

132

Z D , Opole, Poland) equipped with a HBO-200 high pressure mercury lamp (Osram) and 250-390 nm bandpass filter having the maximum transmission at 330 nm (UFS-2, Russia). Samples w e r e irradiated for 20-30 min, the time after which no remarkable increase in the total photoproducts yield was observed in our experimental set-up. E S R spectra w e r e recorded at 100 k H z magnetic field modulation with a Bruker E R 200D-SRC X-band spectrometer equipped with an E R 4102ST rectangular cavity. T h e spectrometer was on line with the Bruker ESP 3220-200SH system for data acquisition and processing. A n MgO crystal doped with Cr ~+ ( g = 1.9796) was used as an external g factor scale standard. A h o m e - m a d e gas flow cryostat was used in the annealing experiments. Samples w e r e heated outside the E S R cavity for 3 min at a given t e m p e r a t u r e and recooled rapidly in liquid nitrogen to record the E S R spectra. Transfers from and into the E S R D E W A R were d o n e with care to avoid extra sample heating.

2mW

Results

E S R spectra obtained at 77 K after U V irradiation of the examined micellar systems ([Tip] =0.3 m M ) are presented in Fig. 1. The spectrum of Trp stock solution in w a t e r (25 mM) is also included for comparison. T h e data are independent of the method of s a m p l e deaeration. The observed signals are entirely due to Trp photolysis. They are absent in the systems containing no Trp and their yields are drastically reduced in the samples irradiated with the use of 300-400 nm bandpass filter (UFS-6, Russia), for which transmission in the range of the Trp absorption band is negligibly low. It is clearly seen from Fig. 1 that the presence of micellar aggregates brings about an e n h a n c e d stabilization of the paramagnetie photoproducts. The E S R signal intensity for the homogeneous system is an order of magnitude lower, in spite of the fact that the Trp concentration was higher by an order of magnitude. F o r h o m o g e n e o u s sampies containing 0.5 m M Trp, we could hardly record any spectrum. A weak signal was d e t e c t e d after the addition of 5 m M SDS, which is less than the critical micelle concentration, but its intensity was still an order of magnitude lower than in micellar systems. For both SM and R M systems containing A O T or SDS complex, multicomponent spectra consisting of a multiplet and some other signals

Ir:

/

AOTB

M

l cr ÷

~

,~z'--~-BM w=5 O j

AOTS M , ~ ~

SOS S

~

PFPEFIM w=9.5

xt0 I 3250

3.

ZwO l -~W

I

a30O

I

a350

I

I

3400 3250

I

I

330D

3350

!

a400

IG] Fig. 1. ESR spectra obtained at 77 K after UV irradiation of frozen solutions of anionic surfactants containing Trp.

overlapped in the central region are observed at a microwave power of 2 m W (cf. Fig. 1). Lowering of the microwave power to 2 x 1 0 -4 m W allows us to discern the easy saturable singlet of about 6 G peak-to-peak linewidth (PPW) at g =2.0027 +0.0002, superimposed on a broader signal (cf. Fig. 1). For the R M system of P F P E , only a singlet at g = 2 . 0 0 3 4 + 0 . 0 0 0 2 having a P P W of about 18 G is present, independently of the microwave power (cf. spectra in Fig. 1). The signal is similar to that observed in a homogeneous system (g=2.0040+0.0002; PPW, about 22 G). T h e total spin concentration is evidently higher in R M than in SM samples. Furthermore, it increases by about 30% with decrease in water content from (0----50 to (a= 5 in R M solutions of A O T , which seems a meaningful difference in view of the experimental uncertainty not higher than 20%. The same trend can be noticed for the intensity of the narrow (PPW, 6 G) singlet (cf. low power spectra in Fig. 1) as well as for the multiplet intensity (cf. low field lines of the high power spectra in Fig. 1). T h e results of thermal annealing expermaents d e p e n d on the m e t h o d of sample deaeration. In

F,. 5zajdzinska.Pietek et al. I ESR of Tap photolysis

the samples degassed by the freeze-pump--thaw method, only a gradual decrease in the ESR signal intensity with increasing temperature was observed. On the contrary, in samples saturated with oxygen, complete transformation of the initial ESR spectrum to that of the peroxy radical is observed on heating above 100 K. Figure 2 shows the difference between the RM and SM systems of AOT deaerated by bubbling with argon. Heating of the RM system to about 130 K leads to the transformation of the entire initial spectrum to the axial symmetric signal characteristic for peroxy radical having the g tensor components g.--2.0065+0.0002 and gn= 2.0349 + 0.0002 (Fig. 2). Annealing of the samples at higher temperatures causes decay of peroxy radicals and they have entirely disappeared at temperatures above 190 K. These transformations are seen at both high and low microwave power levels. In the SM system, however, only traces of the peroxy signal are observed (Fig. 2). In samples heated to 190 K the singlet of about 15 G PPW at g---2.0023 +0.0002 is the main constituent of the ESR spectra at both high and low microwave power levels. It decays at temperatures above 220 K. It is important to note that the final decay of

RHW=5 [Cp3÷

SM

133

the radical species occurs at temperatures somewhat higher in SM samples than in RM samples. Qualitatively similar data are obtained for the SDS system, but the respective decay temperatures are about 30 K higher than for the AOT system. Furthermore, the g tensor parallel component of the peroxy radical has a slightly higher value: gll-- 2.0352 + 0.0002. The peroxy radical is also observed in the argonbubbled RM system of PFPE, in spite of the fact that the initial spectrum is quite different in this case. The decay temperatures are similar to those for the RM system of SDS and the parallel g tensor component value is still higher: g~= 2.0363 + 0.0002. It is pertinent to know that the weak ESR signal in the homogeneous Trp-H20 system, bubbled with argon, decays with no change in shape on thermal annealing. The complex spectra of the AOT and SDS systems can be decomposed into three components which are presented in Fig. 3. The 6 G PPW singlet (Fig. 3(B)), double-dotted chain curve was

lCr,3,,.

B tg0

/ I Cr ÷

A 7

I 3250

I 3300

I 3350

I l 3400 3250 [G]

I 3300

I 3350

I 3400

Fig. 2. ESR spectra recorded at 77 K (microwave power, 2 roW) of RM and SM systems (deaerated by argon bubbling) of AOT after UV irradiation at 77 K and annealing at the indicated temperatures.

d I

]

I

3200

3250

3300

k.~ 3350

! 3400

I 3450 [G]

Fig. 3. Decomposition of (A) the initial ESR spectrum of the RM system of A O T into (B) the multiplet ( - - ) , 15 G PPW singlet ( - - - ) and 6 G PPW singlet ( - . . - ) . See text for details.

134

E. Sza]dz#uka-Pietek et at. I ESR of Trp photolysis

simulated using a mixed Gaussian-Lorentzian shape function and adjusted to reproduce the central part of the low power spectra in Fig. 1. The 15 G PPW singlet (Fig. 3(B)), single-dotted chain curve was similarly simulated and adjusted to the difference between the annealed SM and RM spectra in Fig. 2. Subtraction of the above two singlets from the initial spectrum leaves the multiplet with about 20 G hyperfine splitting. Its contribution to the total spectrum intensity in both SM and RM systems is significantly more than 50%.

4. Discussion Trp is hardly soluble in organic phases of the RM systems under study; so it may be assumed totally incorporated into the aggregates.Taking into account the available data on micellar sizes for A O T [13-16], for SDS [10] and for PFPE [11], one can estimate that the occupancies of probe molecules are less than unity for the Trp concentrations used by us in SDS and PFPE systems as well as in the AOT system with low co ratios. With increasing to the micclles become larger and their concentration diminishes, so that at to---50 a few Trp molecules are solubilized in one water pool. As indicated by the recent fluorescencequenching experiments [4], in small RMs (to up to about 20) the probe is entirely located at the interface. In SM systems, as shown by fluorescence-quenching experiments [3, 17] and measurements of diffusion coefficient [18], Trp is distributed between the miceilar and aqueous phases. On the basis of these results, only about half of the probe molecules were estimated to be bound to Zhe aggregates in the concentration range used in our experiments. The average micellar occupancy by Trp is less than unity and the most probable location site is the head-group region, since the probe is insoluble in alkanes. The observed enhanced stabilization of primary photolysis products in micellar systems may be interpreted in terms of retardation of back reactions if one of the products remains bound to the interface while the other diffuses away. The total yields of paramagnetic species are consistent with such an explanation. In RM systems they are about twice those in SM systems of AOT and SDS, similarly to the fraction of micellized Trp molecules. At the same time, the decrease in the total yield with increase in the ratio to in an RM system

of A O T may suggest that, in multiply occupied large micelles, not all Trp molecules are bound to the interface. Some of them are likely to reside inside the water pool and to behave as those dissolved in the bulk aqueous phase of the SM system. A similar model of solubilization loci has been given recently for another water-soluble probe, pyrenesulphonate [19]. However, it is pertinent to note that for hydrophobic chromophores the yield of photoionization was also found to diminish with increasing to ratio and the result was explained in terms of higher water disorganization at the interface of small RMs [20]. The broad ESR singlets observed as the only lines in the homogeneous system (PPW, 22 G; g = 2.0040 + 0.0002) and in the RM system of PFPE (PPW, 18 G, g=2.0034+0.0002), as well as that obtained by decomposition of the total spectra of P O T or SDS systems (PPW, 15 G; g = 2.0023 + 0.0002) may be assigned to the radical cation Trp "+ produced by photoionization: Trp-

hv

- Trp "+ + e -

(1)

A similar assignment has been made previously for various frozen matrices containing Trp [21-24] and the reported PPW values are in the range 17-25 G, while g=2.0021-2.0048. Apparently, the spectral parameters depend on the system composition and microenvironment of the radical cation. In the absence of micelles the back reaction Trp "+ + e -

, Trp*

(2)

readily occurs, and only a small fraction of the initially formed Trp "+ is stabilized. In micellar systems, photoejccted electrons are repelled from the negatively charged head-group region and thus separated from the radical cations [25]. In RM systems a fraction of the electrons might be trapped insid~ water pools. However, we could hardly see any colour in the samples examined and their bleaching with visible light did not produce any significant changes in the ESR signal. Since the water used in our study was slightly acidic (natural pH), one could assume that e - ( a q ) decayed at 77 K in the reaction with protons in both the SM and the RM systems. Unfortunately, preliminary experiments with alkaline solutions have shown neither trapped electrons nor any significant changes in the ESR spectra. The narrow ESR singlet (PPW, 6 G; g--2.0027+0.002) observed in the micellar systems of AOT or SDS can be assigned to the tryptophyl radical, Trp', produced by N - H bond fission in the indol ring and subsequent rearrangement, lead-

E. Szajdzlnska.Pietek et al. I ESR of Trp photolysis

ing to localization of the unpaired spin density on the C(8) and/or C(9) atom: Trp*

~ Trp'+ H

(3)

A similar interpretation has been given for the sit~glet spectrum obtained by Pailthorpe and Nicholls [22] in a Trp--ethanol matrix at 110 K (PPW, 9.6 G; g--2.004) and by Moan and Kaalhus [21] in aqueous matrices containing Trp "+ after thermal annealing at ca. 150 K (PPW, 13-14 G; g--2.004). Comparison of our data with those reported earlier reveals that for both Trp "+ and Trp" the linewidths in miceUar systems arc smaller than in homogeneous systems. Most probably, it reflects binding of the paramagnetic species to the headgroup region of micelles. The line narrowing by about 1 G has been earlier reported for Trp" bound to the peptide chain of Gramicidin D [22]. It is relevant to note that quite the opposite assignment of the narrow and broad ESR singlets has been given recently by Mahmoud [26] and Mahmoud and Melo [27] to explain the results of photophysical studies of froaen Trp solutions containing antioxidant spermine. However, our interpretation, consistent with the earlier works (see above), seems more justified if the unpaired electron of Trp "+ is delocalized over the indoi rings, while in Trp" it is localized on the C(8) and/ or the C(9) atom. The different ESR spectra obtained in this work for various systems can be well accounted for by the postulated assignment of the broad and narrow singlets. For concentrated Trp stock solution in water, one cannot expect marked formation of Trp" since the intersystem crossing leading to triplet states is quenched owing to Trp dimerization [25]. Accordingly, we do not observe a narrow singlet at low microwave power (see Fig. 1). A similar result is obtained for the RM system of PFPE, in spite of the much lower Trp concentration. We thus infer that efficient stabilization of Trp" at 77 K is possible only if H atoms formed in reaction (3) are readily scavenged by other molecules of the system. This is the case in micellar systems composed of protiated surfactant and oil where we do observe narrow singlets of Trp'. At the same time the multiplet is recorded corresponding to an alkyl-type radical [28] formed in the reaction RH+H

~ R'+H2

(4)

where R H stands for the protiated surfactant and/ or oil molecules. Abstraction of fluorine atom from PFPE does not occur, because of the higher dissociation energy of the C - F bond in comparison

135

with the C - H bond [29], and H atoms produced by reaction (3) probably react back with the tryptophyl radicals. Since the contribution of the multiplet to the total spectra of A O T or SDS samples is more than 50%, we conclude that the energy transfer process may also occur: Trp* + R H

~ Trp + R H *

(5)

which is followed by dissociation of excited surfactant and/or oil molecules leading to the formation of additional R" radicals: RH

RH*

~ R'+H

• R'+H2

(6)

Analogous processes were suggested previously to explain formation of alcohol radicals in homogeneous Trp systems containing ethanol [22, 24]. The observed decrease in the yields of Trp" and R" radicals with increasing ~oratio can be reconciled with the higher probability of Trp" production at single micellar occupancy by the probe (no dimer formation) and with the higher efficiency of reactions (4)-(6) is small micelles, where all Tip molecules are bound to the interface. Conversion of the low temperature ESR spectrum into the signal typical of peroxy radicals has been observed previously in homogeneous systems containing Trp and spermine at about 170 K [24]. This was ascribed to 0 2 - radical anion formation due to reaction of molecular oxygen with electrons initially trapped by spermine. In the present study, however, we could hardly observe trapped electrons. We thus infer that, on sample heating, molecular oxygen diffuses to the primary, photoproducts and reacts with them to give organic peroxy radicals. Structure differences between the systems cause changes in the g tensor parallel components of peroxy radicals on going from A O T to SDS and P F P E samples. At high 02 concentrations, as in oxygen-saturated samples, all primary radicals transform into peroxy radicals. In the absence of oxygen, as in vacuum-degassed samples, Trp "+ is more stable with increasing temperature than neutral radicals since it remains more firmly bound to the negatively charged micellar surface. All the results of thermal annealing experiments indicate that the formation of peroxy species occurs in the presence of molecular oxygen absorbed in the system and mobilized at temperatures above 100 K. The differences observed between various systems deaerated by various methods indicate the marked differences between the permeation of gases in microheterogeneous systems. This effect may be of great importance when modelling processes in biological systems.

136

E. 8zajdzlnska.Pietek et al. I ESR of Trp photolysis

The lower temperatures of final radical decay in the RM system of A O T in comparison with those of SDS or PFPE may be correlated with different melting points of the respective bulk phases, i.e. n-heptane and n-octanol or PFPE oil. This is consistent with the postulated location of Trp molecules and photoproduced radicals at the micellar interface, which seems to be highly penetrated by oil molecules. It seems that the final decay of reactive species involves radical transport through the bulk phase. Consequently, in SM systems, where the bulk phase is constituted of water, it occurs at higher temperatures than in RM systems, where the bulk phase is constituted of organic solvents. 5. Concluding remarks

U V photolysis of Trp occurred far more efficiently in anionic micellar solutions than in the homogeneous aqueous phase. This information is essential for the elucidation of photoprocesses occurring in biological systems. The yield of primary photoproducts increase.s with increasing fraction of Trp molecules bound to the micellar interface and thus is higher in the R M system than in the SM system of a given surfactant. In the presence of molecular oxygen the species stabilized at 77 K transform into peroxy radicals on thermal annealing of the samples above 100 K. In oxygen-free samples the radical cation of Trp is the most stable photoproduct. Perfluorinated R M systems are convenient media for ESR investigation of radical cations produced by photoionization of water soluble solutes since, contrary to protiated systems, other species (such as those resulting from bond fission of the probe and medium molecules) do not contribute noticeably to the low temperature spectra. References 1 D. Creed, Photochem. PhotobioL, 39 (1984) 537. 2 Yu. A. Vladimirov, D.I. Roshchupkin and E.E. Fesenko, Photochem Photobiol., 11 (1970) 227.

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