Reactivity of ozone towards micelle incorporated unsaturated compounds

Journal of Free Radicals in Biology & Medicime. Vol. I. I~. 327-330. 1985 Primed in Ihc USA. All rights reserved.

0748-5514/85 $3.00+ .O0 © 1986 Pergamon Press I.J.d,

REACTIVITY OF OZONE TOWARDS MICELLE INCORPORATED UNSATURATED COMPOUNDS DANIEL ARAVENA and E. A. LISSl Depanamento Qufmica, Universidadde Santiago, Casilla 5659, Correo 2, Santiago, Chile (Received 19 June 1985;Revised 27 September 1985;Accepted 15 October 1985)

Abstraetmlncorporation of reactive substrates (i.e., iipids) to membranes could protect them towards ozone mediated chain oxidation processes. We have measured the effect of incorporation of unsaturated compounds (stilhene or alkyl methacrylate) in sodium dodecyl sulfate micelles on their reactivity towards ozone. It is found that incorporation of substrate in the micellar pseudophase barely modifies their reactivity towards ozone. The same conclusion is reached when stilhene is incorporated to di-octadecyl-dimethylammonium chloride vesicles. These results would indicate that incorporation of a reactive olefinic compound to a lipidic microphase does not provide "per se" an efficient protection towards its attack by ozone. Keywords--Ozone, Sodium dodecyl sulfate, Di-octadecyl dimethylammonium chloride transstilbene, 4',4'-diaminostilhene-2-2'-sulfonate

substrates. As a first approximation to this very complex subject, we have carded out a comparative study of the reactivity of olefinic double bonds in water (pH = 6.5) and in micellar solutions of sodium dodecyl sulfate (SDS). The results obtained are reported in this article.

INTRODUCTION Ozone is a powerful oxidant able to initiate free radical type lipoperoxidation processes. ~ Two types of molecules have been of primary concern in biochemical studies of ozone: polyunsaturated fatty acids and amino acids, either free or in polypeptides or enzymes. Kinetic data regarding the reactivity of unsaturated compounds towards ozone have been reported either in aprotic solvents, 2 or in aqueous media, 3 and only a very limited number of studies have been devoted to ellucidate the influence of the medium upon the interaction rate between organic molecules and ozone. 3"4The results obtained can not then be extrapolated to in vivo since they were obtained in homogeneous microenvironments and the cellular geometry and inhomogeneity undoubtedly determines the accessibility of ozone to a potential substrate. 4 In particular, the fact that the reactive sites of the polyunsaturated fatty acids are localized within the membrane interior have been considered to reduce their accessibility to the ozone avoiding in this way peroxidation of the lipids, which is a devastating event for membrane organization.I Nevertheless, to the best of our knowledge, no study has been devoted to quantitatively investigate the effect that inclusion of a substrate to a lipidic microphase can have upon its reactivity towards ozone, in spite of the fact that these data are needed to allow a quantitative analysis of the interaction between ozone and membrane incorporated

MATERIALS AND METHODS

Trans-stilbene (S) and 4,4'-diaminostilbene-2,2'suifonate (DAS) consumption was followed spectrophotometrically. Due to the low concentrations of S and DAS employed, the substrate concentration was evaluated from the fluorescence intensity measured at 365 and 445 nM for S and DAS respectively. Excitations were carded out at 320 nM (S) and 340 nM (DAS). No changes in the fluorescence spectra shapes were observed when the compounds were ozonized alone or in the presence of olefins. The ozonolysis was carried out by bubbling an oxygen-ozone gas stream through the solution. Typical ozone concentrations in the oxygen stream were in the 50 ppm range. All experiments were carried out employing water containing l0 -4 M allyl alcohol as solvent. The allyl alcohol was used to avoid S or DAS consumption by the residual ozone remaining in the solution when the flow is stopped. Under these conditions the S and DAS consumption follows first order kinetics. All measurements were carded out at room 327

328

D. ARAVENAand E. A. LlssI

temperature (20 -4- 2°C). Most experiments were performed at pH -~ 6.5 in order to minimize hydroxyl radical formation) SDS (Merck or Fluka, puriss) were employed as received. Dioctadecyl-dimethyl ammonium chloride (DODAC) (Herga Ind. Chem. Brazil) was recrystallized from hot acetone by methanol addition. DODAC vesicles were prepared by sonication. RESULTS AND DISCUSSION

The results obtained will be interpreted in terms of scheme 1:

.

M + O3 *') products

(1)

O3(solvent) to, products

(2)

03 + X

kx) products

(3)

where M is a substrate molecule (either S or DAS) in the aqueous phase or incorporated in the micelles, and X is an added unsaturated compound (either in the aqueous or micellar pseudophase). In this scheme, the pseudo ~mimolecular process (Eq. 2) comprises all processes that are determined by the solvent and, in miceilar solutions, can depend upon the suffactant concentration. In absence of X, the consumption of DAS follows first order kinetics both in water and in SDS solutions (see Fig. 1). Furthermore, closely similar slopes (k°,p) were obtained when DAS concentration was changed

2 < C3 t..t

" v

1

e.

2

4 time (minutes)

Fig. I. Values of in (F°/F) as a function of time obtained in the ozonolysis of DAS in 0. I M SDS. Each point is the average of six independent determinations.

between 8 x 10 -7 M and 1.2 x 10 -s M. This result implies that the average ozone concentration in the solution is not determined by DAS. The k°e~pmeasured employing DAS (a substrate that can be assumed to remain in the aqueous phase independently of the SDS concentration) is almost constant when the SDS changes from 6 x 10 -3 M ( n o micelles present) to 0.1 M. These data show that, in this range of surfactant concentration, the value of k,, can be considered to be independent of SDS concentration. At SDS concentrations below the CMC DAS is consumed 5.1 -+ 0.3 times faster than S. In 0.1 M S D S , where S can be considered to be completely incorporated to the micellar pseudophase: the ratio increases to 6.0 --- 0.3. These results imply that incorporation of S in the micelles reduces its reactivity towards ozone by less than 25%. In the presence of an added olefin, the value of k,,p decreases due to the increased ozone consumption. Under these conditions k,,

kexp°t ko + k,X

(4)

and hence it can be expected that

k°exp/kcxp = 1 + ~o (X)

(5)

Values of k,p under different conditions and employing different ozone scavengers were obtained and plotted according to Equation 5 in order to obtain kJ ko values. The data obtained conform to a simple law as that given in Equation 5 (see Fig. 2). Values of kJko obtained employing DAS for several compounds in SDS (0.1 M) and in water are given in Table 1. Some results obtained employing stilbene as substrate are also included in this table. These data indicate that the added compounds are trapping ozone and not OH radicals. This can be concluded from the similarity between the kx/ko values obtained at both pH and from the low efficiencies of acrylonitrile and benzyl alcohol, a compound that should behave as an efficient hydroxyl radical trap. In spite of this, it has practically no effect upon the DAS or S consumption rates. The data in Table 1 also quantitatively agree with previous results obtained regarding the reactivity of ozone in water. Pryor et al. data give km~,~/k,~,yk,~ = 8.2 x 103.4 The data in Table 1 lead to a value of 6.7 x 103 for the same ratio, in good agreement with Pryor et al. data. The data in Table ! show that both substrates (S, incorporated in the micellar pseudophase, and DAS remaining in the aqueous phase) are protected by the

Reactivity of ozone in microheterogeneous systems

similar decrease is observed for butene- 1,4-diol would indicate that most of the decrease is due to a change in ko implying that at high SDS concentrations the surfactant contributes to the ozone consumption. The data in Table 2 show then that incorporation of the substrate to the micellar pseudophase barely changes its capacity to trap ozone, and that this is true even at 0.6 M SDS where the miceiles have grown considerably generating more hydrophobic environments for the incorporated solutes. ~ The apparent bimolecular rate constant k, measured in the presence of micelles can be related to the intramicellar second order process by

/

3

o

S

"2

m

lentil

i S

i 12

ttatthe~ylate]

{raM}

Fig. 2. Values of k°Jk, as a function of butyl methacrylate concentration obtained in the ozonolysis of DAS in SDS: C) SDS 0. I M A SDS 0.15 M.

added olefins. For olefins that are water soluble (i.e., butene 1,4-diol) the effect of the olefin is similar for DAS and S. On the other hand, S in SDS micelles is considerably more protected by hydrophobic substrates. This result, that deserves further consideration, can be explained in terms of the high local concentration of ozone scavengers at the locus of the ozone-stiihene reaction. In order to further evaluate the effect of the solute distribution upon its capacity to interact with ozone, we have measured kx/ko values for three methacrylate esters of'different hydrophobicity as a function of SDS concentration. The data obtained is summarized in Table 2. In this table we have also included results obtained employing butene-1,4-diol, a solute expected to be in the aqueous phase irrespective of the SDS concentration. The data in Table 2 show that when no micelles are present, the three methacrylic esters have almost the same reactivity towards ozone. The presence of the surfactant produces only a moderate decrease in k~/k,, values at very high SDS concentration. The fact that a

Table I. Value of

k/ko

where Ko, is the ozone partition constant between the aqueous and micellar pseudophases. The near independence of kx with substrate incorporation in the micelles imply that either (kxhe/(kx) water and/to, are 1, or that there exists a compensation in the changes of kx and Ko~. This is likely since the reactivity of ozone towards olefinic compounds is larger in water than in less polar media, 2"5 while the solubility of ozone is smaller in water than in nonpolar solvents,l° suggesting a Ko3 larger than one. The lack of a micellar effect could be due to the open character of micelles, 11 and/or to the fact that most solutes are located at (or near) the micelle surface. In order to verify if a similar lack of protection also occurs in more organized systems, we have measured the relative consumption of S and D A S in a solution (I0 -2 M) of D O D A C vesicles. At this surfactant concentration, more than 9 0 % of the S molecules are incorporated to the vesicles. The fluorescence yield of S in this medium is nearly eight times larger than in water, which is indicative of a relatively deep incorporation to the lipidic microphase. In spite of this, D A S is consumed 7.5 -+ 0.5 times faster than S. Comparison of this value with that obtained in S A S micelles (6.0 0.3) indicates that the protection afforded by the more

in water and SDS (0. I M) measured employing DAS as substrate

Ozone Scavenger

Water (pH = 6.5)

Allyl alcohol Butene-1,4-diol Fumaric acid Sorbic acid n-butyl methacrylate Styrene Acrylonitrile Benzyl alcohol Tryptophan

-190 80 600 --0.6 -4 x 10-~

'Value obtained employing stilbene.

329

SDS (pH = 6.5) i 10 190 160 500 190

(130)' ( 190)'

(7 x lO~)• (1.8 x I(P] ° -~1 ( < I P < 0 . 4 (<0.4)' --

SDS (pH --- 10.8) 120 160 80 900 190 400 2 ---

330

D. ARAVENAand E. A. LIsst Table 2. Values of kdko obtained at different SDS concentrations with DAS as substrate Scavenger n-butyl methacrylate

Methyl methacrylate

2-hydroxyethyl-methacrylate

K• 20.00(P

1.00(P

450"

Butene-l,4-diol

SDS (M)

%Micb

k~/k,: (M- * sec-')

6 x 10 -~ 0.02

-80

170 180

0.05

93

170

0.10 0.15 0.60 6 x I0 -3 0.02 0.05 O. I0 O. 15 6 x 10-3 0.02 0.05 0. l0 0.15 6 x l0 -~ 0.15 0.6

97 98 99

190 150 80 170 140 160 120 IO0 160 130 130 120 120 200 170 II0

--

17 41 60 70 -8 24 41 52

•Partition constant between the miceilar pseudophase and the aqueous solution. bPercentage of scavenger incorporated to the micellar pseudophase. ~Estimated error:. -+20. dDeterminad acconling to the method given in Ref. 7. 'Detenninad according to the method given in Ref. 8.

structured vesicles is only slightly larger than that of the more open micelles. From these results we can conclude that incorporation of a reactive substrate to a lipidic microphase does not provide "per se" protection towards its attack by ozone. AcknowledgementsmThanks are given to DICYT (University of Santiago) and to the Fondo Nacional de Ciencias (CONICYT) for financial support.

REFERENCES I. D. B. Menze!. The role of free radicals in the toxicity of air pollutants nitrogen oxides and ozone. In: Free Radicals in Biology Volume 2 (W. A. Pryor ed.), pp. 181-202, Academic Press, New York (1976). 2. W. A. Pryor, D. Giamalva and D. F. Church. Kinetics of ozonization i. Electron-deficient alkencs. J. Am. Chem. Soc. 105: 6858-6861 (1983). 3. J. Hoigne and H. Bader. Rate constants of reactions of ozone with organic and inorganic compounds in water. I. Nondissociating organic compounds. Water Res. 17:173-183 (1983).

4. W. A. Pryor, D. H. Giamolva and D. E Church. Kinetics of ozonization. 2. Aminoacids and model compounds in water and comparisons to rates in nonpolar solvents. J. Am. Chem. Soc. 106:7094-7100 (1984). 5. J. Hoigne and H. Bader. Ozonization of water. Role of hydroxyl radicals as oxidizing intermediates. Science 190:782-784 (1975): L, Well, B. Struif and K. E. Quentin. Reaction mechanism in the degradation of organic substances in water with ozone, Wasser 2:294-307 (1977). 6. J. C. Russel and D. G. Whitten, Hydrophobic-hydrophilic interactions in SDS micelles. Stilbenc-viologencomplex formation as a probe of the micelle interior. J. Am. Chem. Soc. 103: 32193220 (1981). 7. M. V. Encinas, E. Guzmin and E. A. Lissi. Intramicellar aromatic hydrocarbon fluorescence quenching by olefins. J. Phys. Chem. 87:4770-4772 (1983). 8. M. V. Encinas and E, A. Lissi. Evaluation of partition constants in compartmentalised systems from fluorescence quenching data. Chem. Phys. Letters, 91:55-57 (1982). 9. P. Llanos, J. Lang, C. Strazielle and R. Zana. Fluorescence probe study of oil-in-water microemulsions. I Effect of pentanol and dodecane or toluene on some properties of SDS micelles, J. Phys. Chem. 86:1019-1025 (1982). 10. S. D. Razunovskii and G. E. Zaikov. Solubility of ozone in various solvents, Izv. Akad. Nauk. SSSR, Ser. Khim. 686-691 (1971). I I. F. Menger. On the structure of micelles. Acc. Chem. Res. 12: I I I-I 17 (1979).