Extraction of amino acids to microemulsion - The Journal of Physical

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J. Phys. Chem. 1991, 95, 7925-7931

in this system. The marked difference in dithionite reducibility between (C7)*V+-PC and C,,MV+-DHP, amounting to 0.22-0.28 V, is again a consequence of stabilization of the cation in the anionic matrix. Finally, near-ultraviolet irradiation of frozen C,,MV'-DODAC suspensions has been shown to cause photobleaching of the viologen radical cation with stoichiometric formation of a new organic radical, assigned from its EPR splitting pattern as CH3CHCH2 on the surfactant alkyl chains.35 This photoinitiated hydrogen abstration reaction was barely detectable in PC and not observed in DHP vesicles. The relative ordering (DODAC > PC > DHP) parallels C,MV+/O reduction potentials (Table 11). This correlation (35) Sakaguchi, M.; Kevan, L. J . Phys. Chem. 1989, 93, 6039.

7925

between reactivity and thermodynamic potentials is probably indirect, although relatable to the relative interfacial stabilization of the C,MV+ cation. ESEM experiments have indicated that C,,MV+ ions are less exposed to solvent in DODAC than DHP, suggesting deeper bilayer penetration-hence, closer proximity to the alkyl chain reaction sites in DODAC. As with reduction potentials, this difference in microphase location has been attributed to repulsive interactions between the viologen cationic charge and the positively charged DODAC interface. Acknowledgment. Funding for this research was provided by the Office of Basic Energy Sciences, U.S. Department of Energy, under Grant DE-FG-06-87ER-13664; we are grateful for their sponsorship. We have also benefited from discussions with Prof. David K. Roe (Portland State University).

Extraction of Amino Acids to Microemuision Motonari Adachi,* Makoto Harada, Akihisa Shioi, and Yasuo Sato Institute of Atomic Energy, Kyoto University, Uji, Kyoto, 611 Japan (Received: February 13, 1991)

The mechanism of amino acid extraction to AOT/n-heptane microemulsion was elucidated from the experimental data on the amino acid distribution between aqueous and microemulsion phases and on the fluorescence spectra for tryptophan. The hydrophilic amino acids such as glycine are taken up in the water pools of the microemulsion globules. The amino acids with hydrophobic side chains, such as tryptophan, are mainly incorprated in the interfacial zone of the globule. The localization of the amino acid in the microemulsion is affected by the electric charge states and the hydrophobicity of amino acids. The partition coefficient of amino acid J in charged state i between the above two phases, Le., D,', is given by the sum of the for zwitterionic amino acids is related contribution of the water pool DJm and that of the interfacial zone DJIML.The DJfML to hydrophobicity indexes proposed by Nozaki-Tanford and Yunger et al. The electrostatic effect is evaluated from the DJ+ML/DJ+ML value for cationic amino acid and from the DJ-ML/DJ'ML value for the anionic one. Tryptophan acts as a cosurfactant when its loading to the microemulsion phase is high.

Introduction

Amino acids have common hydrophilic groups and various kinds of side chains of different hydrophobicities. Also, the charged states can be controlled by changing the pH in the aqueous phase. Due to these characteristics, the extraction of amino acids to microemulsion are important for elucidating the solubilization mechanism of the guest molecules, which are small in size compared with the microemulsion globules. The solubilization of several proteins to the globules is important for biological pro~essing,'-~ but its mechanism has not been well understood due to the complicated nature of proteins. The extraction mechanism of amino acids may also provide an insight into the solubilization mechanism of peptides and proteins. The extraction of amino acids to microemulsion globules has been extensively The main factors for the ex~

~~

( I ) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 949, 209. (2) Dekker, M.; Van't Riet, K.;Weijen, S . R.; Baitussen, J. W. A.; Laane, C.; Bijsterbosch, B. H. Chem. Eng. J . 1986, 33. B27. (3) Hatton. T. A. In Surfactant-Based Separation Process; Scamehorn. J. F., Harwell. J. H.. Eds.; Marcell Dekker: New York, 1989; Chapter 3. (4) Laane, C.; Dekker, M. In Surfacfants in Solution; Mittal, K. L., Ed.; Plenum: New York, 1989; Vol. 9, p I . (5) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Khnelnitski, Y. L.; Berezin, I. V. Eur. J . Biochem. 1986, 155, 453. (6) Luisi, P. L.; Steinmann-Hofmann, B. Method Enzymol. 1987, 136, 188.

(7) Fletcher, P. D. I.; Parrott, D. In Structure and Reactivity in Reversed Micelles; Pileni, M. P., Ed.; Elsevier: Amsterdam, 1989; p 303. (8) Fendler, J.; Nome, F.; Nagyvary, J. J . Mol. Eool. 1975, 6 , 215.

traction are the electrostatic interaction between the charged guest molecules and the surfactant molecules with the reversed and the hydrophobicity of the guest molecules.8~1'*'2 The localization of the guest molecule to the globules is of primary importance for the extraction. The guest molecules may be distributed to three different environments of microemulsion: (1) the water pool in the globule, (2) the interface of the globule, and ( 3 ) the organic solvent continuum. Since amino acids are essentially insoluble in the nonpolar solvent, two locations, 1 and 2, stay as the candidates. Leodidis and HattonIz recently showed from phase-transfer experiment that the hydrophilic amino acids such as glycine and threonine are solubilized only in the water pool, whereas hydrophobic amino acids are taken up both in the water pool and in the interfacial zone. Their results were obtained only for amino acids in the zwitterion state, and direct evidence for the localization of the amino acids was not shown. The localization of amino acids with hydrophobic side chains in microemulsion must be closely connected with their charged states. There exists no experimental work concerned with the separate evaluation both of the hydrophobicity effect and of the electrostatic effect on the solubilization of amino acids. This work aims at this evaluation. Firstly, the distributions of amino acids (9) Dossena, P.; Rizzo, V.;Marchelli, R.; Castani, G.;Luisi. P. L. Biochim. Biophys. Acta 1976, 446. 493. (10) Boicclli, C. A.; Conti, F.; Giomoni, MN.; Giuliani. A. M. Chem. Phys. Lert. 1982, 89. 490. ( 1 1 ) Hatton, T. A. Ordered Media in Chemical Separations; ACS Symp. Ser., 342; American Chemical Society: Washington, DC, 1987; Chapter 9. (12) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990,94, 6400, 641 I .

0022-3654/91/2095-7925.$02.50/00 1991 American Chemical Society

7926 The Journal of Physical Chemistry, Vol. 95, No. 20, 1991

Adachi et al.

TABLE I: Molecular Structures, Ionization Constants, and Partition Coefficients of Amino Acids

glycine structure

tryptophan

COOH

I

H2N-CH2

phenylalanine

COOH

I I

HZN-CH y

leucine

COOH

COOH

I I

I I

H2N-CH

H2N-CH S'H2

2

H

PKI PK2 PK, D12+G DJ+ DJ* Di

COOH

COOH

I HzN-CH I

I

y42

CH2

y

/cy CH3

CH2

CH2

y

y

I I

CH3

2

I

2

2

H

2.34 9.60

2.38 9.39

2.13 8.62

2.36 9.60

0.07 0.02

8.3 0.29 0.025

2.0 0.10 0.010

1.7 0.035 0.008

0.0085

6-aminohexanoic acid

2

y

H A C H HC,cH5CH II I

arginine

4.08

2.17 9.04 12.48 1.2 0.28 0.048 0.010

10.9 0.16 0.015

0.008

aDJivaluesat I = 1 M. in different charge states to microemulsion are determined by using the usual phase-transfer method. Secondly, the localization of the amino acid with hydrophobic side chain to microemulsion globules is discussed, using fluorescence spectroscopy. Finally, the effects of charged state and hydrophobicity on the extraction of amino acids are separately evaluated with the help of the experimental results mentioned above.

Experimental Section Materials. Sodium bis(Zethylhexy1) sulfosuccinate (AOT) provided by Nacalai Tesque was used without further purification. It was confirmed from preliminary experiment on metal ion extraction that no difference was observed between the unpurified AOT and the AOT purified by Wong's method." Amino acids L-tryptophan (Trp), L-phenylalanine (Phe), L-arginine (Arg), L-leucine (Leu), glycine (Gly), and 6-amino-n-hexanoic acid (6Aha) also provided by Nacalai Tesque were used as supplied. Sodium chloride and n-heptane of reagent grade were used in the experiments. Methods. Equilibrium experiments were performed at 25.0 "C by bringing the AOT in n-heptane solution in contact with the aqueous phase, which contained the amino acid, sodium chloride for adjusting the ionic strength, and HCI or NaOH for adjusting pH. A small amount of potassium phosphate buffer solution (0.2 M potassium hydrogen orthophosphate aqueous solution of pH = 8.2 adjusted by NaOH) was added to the original aqueous solution (the volume ratio O.OS/lO) in order to perform the experiments at pH 7. After equilibration, the amino acids in both aqueous and organic phases were analyzed. The concentrations of Trp in both phases and of Phe in the aqueous phase were determined by UV spectroscopy (Shimadzu Co., Ltd., UV-ZOOS).Gly, Leu, Arg, and 6Aha in the aqueous phase were analyzed by the fluorescence method using flu~rescamine.'~Arg was also analyzed by spectroscopy following the Sakaguchi's method.Is The amino acids except Trp in the organic phase were analyzed after they were back-extracted to NaOH aqueous solution. The extraction of iodide ion 1- was also performed in the presence of NaCI. The iodide anion extracted to the organic phase was back-extracted to NaCl aqueous solution and was analyzed by the UV spectrometry at 226 nm. The amounts of water extracted to the organic phase were determined by the Karl-Fisher method. Fluorescence spectra due to L-tryptophan were measured with the spectrofluorophotometer (Shimadzu Co., Ltd., RF-540). The

-

(13) Wong, M.;Thomas, J. K.; Nowak, T. J . Am. Chem. SOC.1977, 99, 4730. (14) Bohlen, P.; Stein, S.;Dairman,

W.;Udenfriend, S. Arch. Biochem. Biophys. 1973, I55, 213. (1 5 ) Okumura. N. Chemisrry OjProreinr; Akabori, S.. et al., Eds.; Kyoritu Shuppan: Tokyo, 1969; p 286 (in Japanese).

1

I

I

I

I

1

R = 0 . 1 6 ( [ v ] / [ A m ] ) +1.2nm

I

e 1.0

12.7 7 0 0.2 7 0 0.1 7 A

"0

10

20

30

I

0.5

40

w=[w/l[ A m 1 (-1 Figure 1. Relationship between the radius of the W/O microemulsion globule and the w value. excitation wavelength was 280 nm. Sodium nitrite was used as the fluoerescence quencher. The size and shape of the microemulsion globules in the organic phase were determined by the small-angle X-ray scattering method (SAXS, Rigaku Electronic Co., Ltd., SAXS goniometer E7).

Extraction of Water to the W/O Microemulsion Phase We measured the concentration of water extracted to the microemulsion phase, which was in equilibrium with the aqueous bulk phase. The water concentration less its saturated concentration in pure n-heptane, was proportional - to the AOT concentration in the microemulsion phase, [AOT]. The water- mole ratio in the microemulsion phase, w to-AOT [AOT]), depended on the ionic strength I alone, over wide ranges of the pH in the aqueous phase, 1 . I C pH < 11.5. The SAXS spectrum of the microemulsion phase except the range of large wavenumbers was well interpreted by assuming that mono-dispersed spherical globules existed in the phase. The radius of the globule was determined from the Guinier plot of the SAXS intensity as shown in Figure 1. The radius R is a function of the w value over a wide range of the ionic strength I, 0.1 C I < 5 M, and of the pH values I C pH C 12.7: R = 0 . 1 6 ~+ 1.2 nm (1)

[m],

(=[m]/

The radius in low ranges of pH (pH < 1 ) deviates a little from cq I. The experimental results almost agree with the results by

The Journal of Physical Chemistry, Vol. 95, No. 20, 1991 7927

Extraction of Amino Acids to Microemulsion

N 1M

DTrp+=8. 3

0.1

bpq

1 " " " " " " " ' l

7

ArgC+=1*2

1

L-Arginine

IC)

:

Glycine (a)

10.07

-

-

I

I

I

=2.38

0.1

~0.29

P

A 1=0.2M

U

a

0 I=lM

[ATTfl=O.lM

P K ~

0.01 0

2

6

4

8

1

0

1

2

1

9

PH

PH

PH

Figure 2. pH dependency of amino acid distribution between the microemulsion and aqueous phases: (a) glycine, (b) L-tryptophan, (c) L-arginine. Chen et a1.,I6 which was obtained from the small-angle neutron scattering for salt-free AOT solution.

0 6Aha A Phe

pH Dependency of Amino Acid Distribution In this work, the distribution coefficients between the aqueous and the organic phases were measured for six kinds of amino acids summarized in Table 1. The experiments were performed a t a low loading ratio of the amino acid to AOT. Figure 2 shows the effects of the pH value in the aqueous phase on the distribution coefficients Dj which is defined by

DJ = [Jl/[Jl

3 A Leu 0 Cly J

23 W

(2)

c R

where [J] and [I] represent the concentrations of species J in the aqueous and organic phases, respectively. The Dj values for Gly and Trp steeply change around the pKI and pK2values. For singly charged Gly and Trp, a maximum value DJ+and a plateau value Dj* were observed in the ranges pH < pKi, and pKi < pH < pK2, followed by the presence of minimum value DJ-in pH > pK2. A similar result was reported by Hatton et al.," but they dismissed the presence of the minimum value. The above results show that three kinds of charged species are extracted into the organic phase:

+ H+ =

R-CH(NH,+)-COO-(J*)

R-CH(NH3+)-COOH(J+); R-CH(NH,)-COO-(J-)

K1 (3) K2 (4)

For Arg, there exist four characteristic partition coefficients, the maximum DAr? for the divalent cation, two plateau values, DAg+ for the univalent cation and DArs*for the zwitterion, and the minimum DAr- for the univalent anion. The steep change in the partition coeftfcient around pK, shown in Figure 2c is attributed to the formation of the divalent Arg. The distribution coefficient Dj for the amino acid J is expressed in terms of the maximum, plateau, and minimum values, DJ2+, DJ+,Dj* and Dj-: Dj

+

= h2'Dj2+ f,+Dj+

+ f,*Dj* + f,-Dj-

(5)

Heref,' represents the fraction that the amino acid J is in charge state i in the aqueous bulk phase. T h e n ' values are evaluated from the ionization constants shown in Table 1. The plateau value DJ* for zwitterion was determined rather strictly as shown in Figure 2. In the range pH < pK2, the Dj+ value for univalent cations can be determined from eq 6. The Dj -f,*DJ* = Dj'f,'

1

0 f,+

+ H+ = R-CH(NH3+)-COO-(Ji);

L

(6)

(16) Chen, S. H.; Kolarchyk, M. In Physics of Amphiphiles; Micelles. Vescicles and Micrwmulsiom; Deriorgio et al., Eds.; International School of Physics, Enrico-Fermi CourseeXC: North Holland, 1985; p 768.

P

(-1

Figure 3. Determination of DJ' from DJ and DJ'.

plots of ( D j -f,*DJ*) againstfJ+ are shown in Figure 3. The slopes of the straight lines provide the DJ+values. The D{ ( i = 2+, f,-) values determined from the similar procedures are summarized in Table I . The solid curves in Figure 2, which are calculated from eq 5 with the resultant Dj' values, fit well the experimental results. The DJ+values are strongly dependent on the species of the amino acids, whereas the Dj- is independent of the species except for Trp and Phe. The hydrophobicity of the side chains of amino acids would play an important role in the

+,

DJ+.

Effects of Ionic Strength on Amino Acid Partitioning The effects of the ionic strength I on the partition coefficients Dj-,DJi, and DJ+are shown in Figure 4. In Figure 4a, the Djvalues for the amino acids, Gly, 6Aha, Leu, and Phe, agree with each other within experimental precision when I < 1 M. In this work, we also performed the extraction of iodide ion I-. The Djfor the amino acids also agree with the Di- value. Since iodide ion I- is entrapped only in the water pool of the microemulsion globule, the amino acids with negative charge are also estimated to be taken up only in the water pool. and DPk-deviate from those of the other amino acids with the increase in 1. This suggests that Trp and Phe are taken up both in the water pool and in the monolayer of the microemulsion globule, the fraction of Trp dissolved in the water pool increasing with the increase in w .

7928 The Journal of Physical Chemistry, Vol. 95, No. 20, 1991

Adachi et al. loo

30 0.1

1.

I

I

I

I

I I l l 1

I

I

1

I

I

'0

L-LJ

0.0030.,

+ 6Aha

0.3

1

I

3

0.01

0.3

0.1

(kmol/m3)

I

3

1

(kmollm3)

Figure 4. Effects of ionic strength I on amino acid partitioning: (a) partition coefficients for anionic amino acids and for iodide ion I-; (b) partition coefficients for zwitterionic amino acids; (c) partition coefficients for cationic amino acids and for Na+ and K+.

The effect of I on the partition coefficient DJ* is shown in Figure 4b together with the 4H20 value, which is defined by the volume fraction of water in the microemukion phase. Dcly*and &ha* agree with the &.,10 value:

= D6Aha* = 6Hz0

(7) This relationship means that the concentrations of Gly and 6Aha in the water pools of the microemulsion globules agree with those in the aqueous bulk phase. Leodidis and Hatton'* also pointed out the same result. The DJ* values of a-amino acids with hydrophobic side chains are much larger than #H,? suggesting that the hydrophobicity of amino acids plays a role in the extraction of these amino acids. Dj+ is shown against f in Figure 4c together with the partition coefficients of K+ in aqueous NaCl solution and of Na+ in aqueous KCI solution. The partition coefficients of the hydrophilic amino acids DGly+and have the same f dependency as those of K+ and Na+. The difference between DK+and ha+ mainly arise from the difference in the hydrated ion radii of the metal ions."J* 6Aha has an isolated and mobile amino group, and Gly is an a-amino acid. The ionic radii of the cationic groups of the amino acids Gly and 6Aha differ from each other, and this results in the difference between DGlr+and D~A$+.The hydrophilic amino acids, Gly and 6Aha, seem to be solubilized only in the water pool of the microemulsion globule, because the DJ+values of these two amino acids change in a similar way to those of metal ions. The DJ+values of hydrophobic amino acids such as Trp, Phe, and Leu are much larger than DGI+,the hydrophobic group strongly interacting with nonpolar sofYvent or hydrocarbon tail group of AOT molecules. Fluorescence due to Tryptophan As mentioned previously, the & +,DTrp*,and values are much different from those of hydropxilic amino acids. To elucidate the reason, the fluorescence spectra due to Trp were measured adding the aqueous solution with Trp to AOT/n-heptane solution. The pH's in the aqueous solution were adjusted to fixed values. The results at pH 7 are shown in Figure 5. Curve 1 shows the fluorescence spectra of the aqueous bulk phase at pH = 7, which contained Trp by [Trp] = 2.33 X IOd M. A maximum value is observed at wavelength 354 nm. On the other hand, when the aqueous solution contains Trp and N a N 0 2 by [Trp] = 4.66 X M and [NaN02] = 0.5 M, the fluorescence completely

-

(17) Harada, M.:Shinbara, N.; Adachi, M.; Miyake, Y . J . Chem. Eng. Jpn. 1990, 23, 50. ( I 8) Leodidis, L. B.;Hatton, T. A. Langmuir 1989, 5 , 741.

300

400 Wavelength

450

(nm)

Figure 5. Fluorescence spectra of tryptophan at pH = 7. Curve 1: Trp in an aqueous solution of [Trp] = 2.33 X lod M; curve 2: Trp in an aqueous solution of [Trp] = 2.33 X IO4 M and [NaN02]= 0.5 M; curve 3: Trp in the microemulsion solubilizing 0.05 cm3of an aqueous solution of [Trp] = 4.66 X IO4 M into IO cm3n-heptane solution of [ m ] = 0. I M; curve 4: Trp in the microemulsion solubilizing 0.05 cm3of an aqueous solution of [Trp] = 4.66 X IO4 M and [NaN02]= 0.5 M into I O cm3of an n-heptane solution of [ m ] = 0.1 M; curve 5 : n-heptane solution of = 0.1 M.

vanishes due to the quenching action of NO2- ions as shown by the line 2. The spectra of Trp of the microemulsion phases with and without the quencher are also shown in Figure 5 as curves 3 and 4, respectively. Curve 5 is the blank spectrum, that is, the fluorescence of 0.1 M AOT solution in n-heptane. The fluorescence of Trp has a maximum at the wavelength 340 nm and still has high intensity even in the case with the quencher. Thus, the indole ring of Trp is protected from the attack of the quencher, the hydrophobic indole ring being embedded in the oil and the hydrophilic head part sticking to water. It is important to know how the fluorescence is affected by the w and pH values. Figure 6 shows the fluorescence spectra of the 7. The solid curves are microemulsions containing Trp at pH the experimental results obtained for quencher-free microemulsions. With the increase in w until w = 10.8, the quantum ef-

-

The Journal of Physical Chemistry, Vol. 95, No. 20, 1991 1929

Extraction of Amino Acids to Microemulsion 70

120

1

1

t

r

~

[NaOHl=O.Ol M 60

l

I

l

l

.....,.

..

.

.->. 4d

u)

E

2

-

50

too

C

8

5

x

f 2

,.’ in aq.

phase

40

.-x C

2al

30

ii

80

-E C

>” ‘3 20

-m

3E

xf

Q)

e

10

60

0

3 L

0 300

al

Wavelength

.->

400

350

4d

(nm)

al

40

Figure 6. Effect of w on the fluorescence spcctra of zwitterionic trytophan. Solid curves: Microemulsion solubilizing water in n-heptane solution containing 0.1 M AOT, 2 X lod M Trp, and 0.08 M H20. Dashed-dotted curves: Microemulsion solubilizing 0.5 M NaN02 aqueous solution into n-heptane solution containing 0.1 M AOT, 2 X lod M Trp. and 0.08 M H20. Dotted curve: aqueous solution containing Trp by [Trp] = 2 X IOd M.

ficiency of the fluorescence decreases, and the wavelength at maximum intensity, A,, shifts to higher values. With further increase in w, the quantum efficiency increases in turn, and the A, continues to shift to higher value. The fluorescence spectrum of the aqueous solution with Trp is also shown by the dotted curve as a reference. Since the A,, values for the microemulsions shift toward that of the aqueous phase, the local environment around the indole ring of Trp is estimated to vary from nonpolar to polar character as the w values increases. Similar results on the shift of the A,, have been reported by C ~ w g i l l for ’ ~ Trp in various solvents with different polarity. The dashed-and-dotted curves in Figure 6 are the experimental results obtained for the microemulsion solubilizing aqueous NaNO, solution. These fluorescence intensities take almost same values as in the quencher-free case when the w value is small, whereas, in higher w ranges, the intensities become much lower than those in the quencher-free cases. The A, values are confined within 340 nm even at high w values. From these results, the following conclusions are derived: ( I ) With w < 3, the zwitterionic Trp is localized in the position that the fluorescence due to Trp is protected from the attack of the quencher. (2) The zwitterionic Trp is trapped in the interfacial zone when w is small. With the increase in w , Trp partially dissolves in the water pools of the microemulsion globules. Figure 7 shows the fluorescence spectra of the microemulsions solubilizing 0.01 M NaOH aqueous solutions with or without NaN02. The spectrum of Trp of the aqueous bulk solution with NaOH is also shown as the dotted curve in the figure. In aqueous solution of high pH, high fluorescence intensity of Trp was observed, as reported by Cowgill.20 The fluorescence intensities in the quencher-free case (solid curves) at w = 0.8-2.8 agree with 7,and decrease as the w values rises to 10.8, the those at pH A, shifting toward higher values. With further increase in w , the intensity increases in turn, especially in the range of high wavelength, and the spectra at w = 20 and 30 have shoulders near the AI“,x for the aqueous solution, indicating that a large amount of anionic Trp dissolves in the water pools of the microemulsion globules.

-

(19) Cowgill, (20) cowgill,

R. W.Biochim. Biophys. Acta 1967, 133, 6. R. W. Arch. Biochem. Biophys. 1963, 100. 36.

20

n 300

400

350

Wavelength

(nm)

Figure 7. Effect of w on fluorescence spectra for anionic tryptophan. Solid curves: Microemulsion solubilizing 0.01 M NaOH aqueous solution in n-heptane solution containing 0.1 M AOT, 2 X IOd M Trp, and 0.08 M H20. Dashed-dotted curves: Microemulsion solubilizing 0.5 M

NaN02/0.01 M NaOH aqueous solution into n-heptane solution containing 0.1 M AOT, 2 X IO” M Trp, and 0.08 M H20. Dotted curve: 0.01 M NaOH aqueous solution containing Trp, [Trp] = 2 X lod M.

In the case with the quencher (see dashed-dotted curves), the is confined near 342 nm. The difference between the intensities with and without the quencher increases compared with the case of pH 7. These results also support that the Trp in anionic state prefers to dissolve in the water pool of the globule compared with the Trp without net charge. Figure 8 shows the fluorescence spectra of the microemulsions which solubilized 0.1 M HCI aqueous solutions with or without 0.5 M NaN0,. The A, in the quencher-free case remains near 335 nm even in higher w ranges. It is, therefore, concluded that cationic Trp is taken up more tightly in the interfacial zone of the microemulsion globule than the Trp in anionic and zwitterionic states. A,

-

Partition Coefficients With reference to the results on fluorescence, the partition coefficient Dj‘ can be divided into two parts: DJi = DJiML + DJ IWP (8) Here, DJiMLis the partition coefficient for the amino acid entrapped in the monolayer of the microemulsion globules and DJNP for the amino acid extracted in the water pool. The DJiMLvalue can be evaluated from eq 8 with the observed DJi, if the DJiwp value is known. Dj-wpcan be represented by the solid curve in Figure Sa, because the D,-wp values are independent of the species of hy-

7930 The Journal of Physical Chemistry, Vol. 95, No. 20, 1991

Hydrophobicity scale by Yunger e t al.

l " " " " ' 1 [HCI]=O.l

701

Adachi et al.

1

M

-1

-2

0.3

-

I u

t

1

/

0.1 -I

57 D 0.03

0.01 0

Wavelength

(nm)

Figure 8. Effect of won fluorescence spectra for cationic tryptophan. Solid curves: Microemulsion solubilizing 0.1 M HCI aqueous solution in n-heptane solution containing 0.1 M AOT, 2 X IO" M Trp, and 0.08 M H20. Dashed-dotted curves: Microemulsion solubilizing 0.5 M NaN02/0.1 M HCI aqueous solution into n-heptane solution containing 0. I M AOT, 2 X 1 0" M Trp. and 0.08 M H20. Dotted curve: 0.1 M HCI aqueous solution containing Trp by [Trp] = 2 X IO" M.

1

3

2

4

5

Hydrophobicity scale by Nozaki-Tanford Figure 10. Relationship between DJ*MLand the hydrophobicity scale.

100 u

for -1

5n

10

.

cations

i

10-4

10-2

10-1

1

Figure 11. Effect of tryptophan loading ratio to AOT on the partition coefficients of Trp and on the w value.

$7

n

10-3

L= [VI/ [ A T 1

-1

1

DJ*ML is expected to be small because the net charge is nil. Since the electric potential at the interface of the microemulsion globule depends on the ionic strength alone, the Dj+ML/Dj*ML and DiML/Dj*ML values should be independent of the species of amino acids. As shown in Figure 9, the DJ+ML/DJ*ML and Dj-ML/Dj*ML values, which represent electrostatic effect, are determined by the I value alone. The DJaMLobtained previously is indeed dependent on the hydrophobicity of amino acid.2'-2s As shown in Figure 10, the DJaML is well correlated with the hydrophobicity indexes reported by Nozaki-Tanford2' and Yunger et al.,2s which represent the transfer free energy change for amino acids between water and oil phase. The index by Nozaki-Tanford was determined from the difference in the solubility of amino acids both in ethanol and dioxan from that in water, and that by Yunger et al. was determined from the partition coefficients of amino acids between water and octanol.

/anions

A A Trp

0.01

0.1

0 0 Phe X Leu 1

-

10

I (kmol/m3) Figure 9. Dependency of Dj+ML/Dj*ML, and DfML/DJ+ML on the ionic strength.

drophilic amino acids. Then, the DJ-ML values for Trp and Phe obtained from (DJ-- DJ-wp)are shown by the dashed-dotted lines in Figure 5a. D *WP can be taken as DGly*(or +H20) for all amino obtained from eq 8 is shown by the dashed-dotted acids. The eML in common to curves in Figure 5b. DJ+wpis assumed as DO!,,+ all a-amino acids. The error in this choice is insignificant because the Dd+ML'~ for hydrophobic amino acids are much higher than obtained from eq 8 is also shown by the DJ+w . The DJ+ML dashed-dotted curves in Figure 5c. The difference between DJ*ML,DJ+ML, and DJ-ML arises from the electrostatic interaction. The electrostatic contribution to

Effect of Tryptophan Loading We have discussed the partition coefficients observed at the condition that the concentrations of amino acid were maintained (21) Nozaki, N.;Tanford, C. J . Biol. Chem. 1971, 246, 2211. (22) Bull, H. B.; Breese, K. Arch. Biochem. Biophys. 1974, 161, 665. (23) Fauchere, J.-L.; Pliska. V. Eur. Med. Chem. 1983, 18, 369. (24) Wolfenden, R.; Anderson, L.; Cullis, P. M.;Southgate, C. C. B. Eiochemisfry 1981, 20. 849. ( 2 5 ) Yunger, L. M.;Cramer. R. D. Mol. Pharm. 1981, 20,602.

Extraction of Amino Acids to Microemulsion

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The Journal of Physical Chemistry, Vol. 95, No. 20, 1991 7931

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Figure 12. Effect of tryptophan loading on SAXS spectra: (a) pH = 0.5; solid curves, calculated assuming cylindrical globules; (b) pH = 7; solid curves, calculated assuming monodispersed spherical globules.

at a low level. Trp is a typical hydrophobic amino acid which is mainly taken up in the monolayer of the microemulsion globule. It is, therefore, interesting to examine how the extraction behavior and the structure of microemulsion globule change with the addition of Trp in high loading ratio of Trp to AOT. Figure 11 shows -the -relationship between the Trploading ratio to AOT L (=[Trp] [AOT]) and the Trp distribution coefficients at pH = 0.5 and pH = 7. The change in the w value with L is also shown in the figure. Trp is positively charged at pH = 0.5. In L < 0.05, a Trp molecule is, as a mean, surrounded by AOT molecules - in the monolayer, and the relationship DTrp+= 83[AOT] is obtained. In high Trp-loading ratios, DTrp+deviates from the above relation. In high Trp-loading ratios, the shape and size of the microemulsion globule would be drastically changed. The SAXS spectra are shown in Figure 12a for pH = 0.5. The Guinier plots provide no linear relationship. The spectra are well interpreted by the solid curves, which are obtained by assuming that the globule is of cylindrical shape with diameter d and length distribution function2" f l l ) a ( 1 / I 2 ) exp(-I/&), L, being the characteristic lengthsa The d and Lcvalues to fit the observed spectra are shown in the figure. The d values decreases and L, values increases as the Trp-loading ratio increases. The w values decreases when the shape of the globule changes from sphere to cylinder. For zwitterionic Trp at pH = 7, the relationship DTrp*= 2.9[AOT] is obtained in the ranges of low loading ratios as seen in Figure I I , whereas, -in high Trp-loading, &*, slightly increases to approach 4.O[AOT]. Typical examples of the SAXS spectra at pH 7 are shown in Figure 12b. The spectra are almost explained by assuming that monodispersed spheres are generated. The deviation of the observed spectra from the calculated solid curves at higher wavenumbers would be attributed to the polydispersity of the splierical globules.16 The diameter of the spherical globule became large with the increase in Trploading ratio. This corresponds to the trend of the increase in the w value at high Trp-loading ratio (see Figure 1 1 ) . The results mentioned above suggest that Trp molecules, which are incorporated in the monolayer of the globule, make the monolayer rigid, just like the condensation effect of cholesterol

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(26) Shioi, A.; Harada, M.; Matsumoto, K . J . Phys. Chem., in press. (27) Demel, R. A.; De Kruyff, B. Biochim. Biophys. Acra 1976,457, 109. (28) De Gennes, P. G.; Taupin, C. J . Phys. Chem. 1982, 86, 2294.

in the phospholipid membranea2' The persistence length of the interfaceB becomes large due to the increase in the rigidity. This causes the increase in the diameter of the microemulsion globule in the case of the zwitterionic state. When Trp has a cationic charge, the hydrophilic groups are packed tightly in the monolayer, due to the electrostatic effect between the cation and the anion. Then, the amount of water entrapped in a W/O microemulsion globule decreases. It can be expected from Turkevich's that a large rigidity constant for the splay deformation changes the spherical globule to the cylindrical one.

Conclusion The solubilization mechanism of small guest molecules to water-in-oil microemulsion was elucidated, by using several amino acids with various side chains as the guest molecules. Amino acid J of charge state i has its own partition coefficient DJi,and the overall distribution of J is given by the sum of these DJ'values. The DJ'values are specific to the species of amino acids. Hydrophilic amino acids are taken up only in the water pool of the microemulsion globules through the electrostatic interaction. The amino acids with strongly hydrophobic groups are mainly incorporated in the interfacal layer of the globule through the hydrophobic interaction. The partition coefficient Di is divided into two parts, Le., the contribution of amino acid entrapped in the monolayer of the globule DJiML and that taken up in the water pool DJIwp.DJtML for the zwitterion is directly related to the hydrophobicity of the amino acid J and is well correlated with the hydrophobicity indexes reported by Nozaki-Tanford and Yunger et al. The partition coefficient for tryptophan is affected by the loading ratio of tryptophan to AOT. The shape of the microemulsion globule changes from sphere to cylinder, when the loading of cationic tryptophan is large. Tryptophan acts as a cosurfactant. Acknowledgment. We thank Drs. S. Kato, M. Terashima, M. Tanigaki, and Y . Mori at the Faculty of Engineering, Kyoto University, and Dr. M. Shimada at the Wood Research Institute, Kyoto University, for their help in the amino acid analyses. We gratefully acknowledge financial support from a Grant-in-Aid for Fundamental Scientific Research (Fundamental Research B), Ministry of Education, Science and Culture. (29) Turkevich, L. A. In Physics of Complex and Supermolecular Fluids; Safran, S . A., Clark, N. A., Eds.; John Wiley: New York, 1987; p 241.