On the bicontinuous microstructure induced by a guest protein in a typical AOT microemulsion

COLLOIDS AND ELSEVIER

Colloids and Surfaces A: Physicochemicaland Engineering Aspects 129-130 (1997) 327-338

A

SURFACES

On the bicontinuous microstructure induced by a guest protein in a typical AOT microemulsion Maura Monduzzi *, Francesca Caboi, Cesare Moriconi Dipartimento Scienze Chimiche, Universita' di Cagliari Via Ospedale 72, 09124 CagliarL Italy Received 17 September 1996; received in revised form 11 December 1996

Abstract

The ternary microemulsion AOT/water/isoctane was investigated in the presence of the human serum albumin (HSA) protein as solubilized guest molecule by NMR relaxation and self-diffusion measurements. The analysis of the 23Na NMR relaxation rates of the counterions and the interpretation of the 1H relaxation and self-diffusion data of water in terms of the usual spherical water droplet model gave a strong and direct evidence for important modifications of the water-in-oil droplet microstructure in the presence of HSA. With decreasing the water/surfactant (W/S) molar ratio, which implies a linear decrease of the water core, the system containing the protein shows a transition to a bicontinuous microstructure. Some protein residues are likely to contribute to the total interfacial area to retain the favored curvature of the AOT molecules located at the polar-apolar interface. At W/S larger than 25, which corresponds to an average radius of the water core larger than 2.8 nm, the relaxation parameters and the selfdiffusion coefficients are almost unaffected by the presence of HSA. Consequently, it can be suggested that HSA molecules are hosted in closed water domains, which are likely to assume an average spherical shape as well as the unfilled, AOT stabilised, water droplets. © 1997 Elsevier Science B.V.

Keywords: AOT microemulsions; Guest protein; Microstructure; NMR

1. Introduction

Microemulsions are thermodynamically stable mixtures of water and oil, stabilised by surfactant molecules. They commonly contain either droplets of water in oil or droplets of oil in water, or have a bicontinuous structure [1-7]. In most cases the oil-water interface is stabilised by a monolayer of surfactant. In some bicontinuous microemulsions surfactant bilayers have been also found. The characteristic size of the oil or water domain is generally in the nanometer range. A m o n g the various systems that form water-in-oil (w/o) micro* Corresponding author. Fax: 0039706758605; e-mail: [email protected] 0927-7757/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0927-7757 (97) 00050-2

emulsions, the anionic surfactant sodium bis(2-ethylhexyl) sulfosuccinate ( A O T ) has been widely investigated as a model system since it easily forms w/o microemulsions without a cosurfactant [ 1,3, 8,9]. A great deal of knowledge concerning the microstructure, the droplet size, the polydispersity, and the static and dynamic percolation phenomena, is actually available from m a n y different techniques, namely conductivity [10-12], stopped flow kinetics [13], fluorescence quenching [ 14], dielectric spectroscopy [ 15,16], scattering [17-22] and N M R [7,11,23-32] techniques. The latter two techniques have probably proven the most informative in relation to microstructure and dynamic features. Particularly the N M R results enabled one to

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M. Monduzzi et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 129-130 (1997) 327-338

clarify many aspects, including the conformational properties of the AOT molecules located at the interface and the dynamics related to the hydration of the polar heads and of the counterions inside the water core. A number of investigations pointed out that at least ten water molecules per surfactant are involved in the hydration of the polar groups, and then they are strongly affected in their dynamics [29, 30]. Although many detailed investigations have been undertaken, which are summarised in some interesting review articles [8,9,33], AOT is still a topical subject as a surfactant, because of several applications in pharmaceuticals, and as a stabilising agent of w/o microemulsions functioning as host systems for solubilized molecules [34-41]. Great interest has been devoted to guest molecules such as proteins or other small hydrophilic species. Several studies have pointed out that the solubilization process generally does not alter the functionality of a small molecule as well as that of a protein [9,13,42]. In addition, it is generally accepted that the w/o microstructure is not particularly affected. Depending on the chemical nature, the guest molecule can be located differently within the original water droplet structure. For instance, in a nice paper by Pileni [20], the analysis of the SAXR scattering data from the AOTwater(W)-isoctane(ISO) system in the presence of various solubilized guest molecules (ether, dioctylviologen, chymotrypsin and cytochrome c), demonstrated the occurrence of small-size perturbations of the original microstructure upon protein solubilization if the volume fraction of the hydrophilic solute is less than 5% of the water pool volume: otherwise a 'segregation effect' occurs, which gives rise to two populations of inverse AOT micelles. Obviously, if the water core volume is smaller than the hosted molecule dimensions, a rearrangement of the microstructure is expected, to enlarge the available size. The uptake of a hydrophilic macromolecule by a w/o droplet structure brings about a considerable enlargement of the radius of the filled droplets since they need more water per surfactant head than the unfilled ones. Hence, a larger water/surfactant molar ratio (W/S) results for the 'filled droplets'. Generally, to investigate the solubilization effects, very sensitive

techniques are needed, since a small number of 'modified' droplets is expected, and in addition changes are not very large. Often, however, no clear experimental evidence can be gathered for any of the suggested models of solubilization, and only a common chemical intuition would predict the preferred location of a guest molecule. It is well documented that N M R relaxation and self-diffusion techniques [7, 24, 28-32,41 ] give complementary information concerning the microstructure of surfactant systems. Generally the spinlattice relaxation rates increase significantly when the investigated nuclei are tightly bound and thus undergo a reduction of mobility. On the contrary, the molecular self-diffusion coefficients decrease significantly when motions occur over restricted domains. In this work, the ternary microemulsion AOT/water/isoctane [10] is reinvestigated along a water dilution line, close to the oil corner. The focus is on the variations of the water-in-oil microstructure induced by the presence of a solubilized globular protein, namely the human serum albumin (HSA). For this system, investigated by ESR and frequency domain fluorescence spectroscopy techniques [37,38], the analysis of the results suggested conformational changes of the protein, with significant variations of the rotational correlation times. The strong anisotropy of the molecular motion of the 3MAL-HSA complex (3MAL= 3-maleimidooroxyl is the ESR-probe), observed at low W/S, was interpreted in terms of conformational changes of the protein, mainly due to the limited number of water molecules, but due also to the interaction between the surfactant molecules and some protein binding sites [38]. For the AOT/W/ISO system, a detailed analysis of the 23Na N M R relaxation rates at 10 and 20°C by Halle [32] gave a rather clear picture of the dynamics of the counterions inside the water cores, whose diameters increase linearly with increasing W/S. In the present work, a similar approach along with the analysis of the water relaxation and self-diffusion data, at 25°C, will enable a deeper insight on the modifications of the water-in-oil droplet microstructure induced by HSA solubilization, which is predominantly expected within the water core.

M. Monduzzi et al. / Colloids Surfaces A." Physicochem. Eng. Aspects 129-130 (1997) 327-338

2. Experimental 2.1. Materials

Bis-(2-ethylhexyl) sodium sulfosuccinate (AOT), 2,2,4-trimethylpentane (ISO) and human serum albumin with a M W ~ 6 6 000 (essentially fatty acid free - A1887) (HSA), were purchased from Sigma-Aldrich. Before use, AOT was recrystallised from methanol and then freeze-dried, after solvent evaporation, and the oil ISO was dehydrated over molecular sieves (zeolite 3A, previously activated at 500°C) for 12 h. Samples for NMR measurements were prepared by weighing about 3 g of the stock solution with molar ratio AOT/ISO=0.0165 and then adding the proper amount of water to obtain the desired water/surfactant molar ratio ( W / S ) . HSA was dissolved in distilled water, without any buffer, to avoid possible interference in the NMR experiments and also to avoid changes of the ionic strength in the water pool. Then a suitable amount of this solution was added to the AOT/ISO mixture to obtain the HSA/AOT molar ratio of 5.8 x 10 -5, which was kept constant at any water dilution. The samples were transferred in the NMR tubes and accurately sealed to avoid oil evaporation. 2.2. M e t h o d s

~H NMR experiments were performed at 1.88 T on a Varian FT 80A spectrometer at the operating frequency of 80 MHz. The 23Na NMR relaxation measurements were performed at 7.05 T on a Varian VXR 300 spectrometer at the operating frequency of 79.35 MHz. The temperature was always kept constant with a precision _+0.5°C. Diffusion measurements were performed using the Fourier transform pulsed magnetic field gradient spin-echo (FT-PGSE) technique, as described by Stilbs [43,44]. The experiments were carried out by varying the gradient pulse length (6) while keeping the gradient strength (G) and the pulse interval (A) constant. The decay of the echo intensity with increasing the value of 6 is given by:

329

where D is the self-diffusion coefficient, Ao is the echo intensity in the absence of any gradient and ~, the magnetogyric ratio. For 1H self-diffusion experiments A = 70-140 ms (depending on the water content) and G = 2 gauss cm-1 were used. The self-diffusion coefficients were calculated by means of a two-parameter non-linear fit of the above equation to 10-14 different 6 values. The error in the measurements, as judged by repeated measurements, is estimated to be smaller than +5%. The 23Na and 1H NMR spin-lattice relaxation experiments were performed by the usual inversion recovery pulse sequence (delay-180°-z-90°-acquisi tion). The spin-lattice relaxation rates, R~, were obtained by a three-parameter non-linear fit of the partially relaxed NMR signal intensities obtained at 14-18 different z values: I(z) = A - B e x p ( - zR1)

The error in the measurements, as judged by repeated measurements, is estimated to be smaller than + 2%. The 2aNa NMR spin-spin relaxation rates (see below) were deduced from the deconvolution of the spectra recorded with a 90 ° pulse angle.

3. 2aNa NMR theoretical background 23Na is a quadrupolar nucleus with I = 3/2, and the nuclear spin relaxation is generally due to coupling between the nuclear quadrupole moment and the fluctuating electric field gradients at the nucleus [45]. Thus, the quadrupolar interaction determines both the spin-lattice (R0 and the spinspin (RE) relaxation rates. In w/o microemulsions the surfactant molecules form aggregate interfaces to stabilise the water cores. The Na + counterions inside the water pools reside close to the charged interface preferentially, but can be also located in the aqueous bulk. Assuming a rapid two-site exchange between bound (B) and free (F) sites, the relaxation rates are given by:

RI. 2 =(1 --pB)RF.2 +PBR~.2

(1)

where PB is the fraction of Na + ions in the bound

M. Monduzzi et aL / Colloids Surfaces A: Physicochem. Eng. Aspects 129-130 (1997) 327-338

330

state. In the free state the extreme narrowing condition generally applies, thus R1F= R 2F= R r. In the bound state, the relaxation data have been successfully described in terms of a two-step model [46]. Within this model, the fast motion (Jr), slightly anisotropic, related to the local motions of the observed nucleus, is assumed in the extreme narrowing regime (~ozf <<1), while the slow isotropic motion (js), related to the whole aggregate tumbling, falls in the slow motion regime (ogz~ >>1), and relaxation is bi-exponential. In that case the observed 23Na N M R resonance signal (related to the spin-spin relaxation) is given by the superposition of a broad Lorentzian line (fast component) with 60% of the total area and a narrow Lorentzian line (slow component) with 40% of the total area. Likewise, the spin-lattice relaxation rate consists of a slow (80%) and a fast (20%) relaxation component. Thus, according to various authors [47-50] the decays of the spinlattice and spin-spin magnetisation are given by: ME(t)-- ME(0)oc0.2 e x p ( - tRfB) + 0.8 e x p ( - tR]B )

(2)

MT(t) -- MT(0) oc0.6 exp(-- tRf2B) +0.4 exp(-- tR~zB)

(3)

with,

where n=0, 1, 2, whereas zf and z~ are the fast and slow correlation times for the bound species. As often observed, the decay of the longitudinal magnetization does not show any appreciable deviations from a single exponential if J(~o).,~J(2og), as it will be shown in our case. Thus a single R~B value can be obtained. On the contrary, since nonLorentzian bandshapes of the 23Na N M R signals [52] are always observed, a graphical deconvolution allows to estimate the fast and the slow R2B components. To this aim, assuming that the frequency shift between the two components is negligible, the experimental 23Na N M R spectrum can be fitted by the 4-parameter equation:

a

( b2+2+(2nv) 0.4b 2

0.6c

2

R f . = - ~ - Z J(o0

(4)

2n 2 R ] , = ~ - Z2J(2¢o)

(5)

(9)

where v is the frequency (Hz), a is the total area of the N M R signal, d is a baseline correction, whereas b = R~B and c=RfB. The unknown parameters g, Jr, J(0), J(~o) and J(2~o), contained in Eqs. (4)-(8), can be estimated provided that PB in Eq. (1) is known. Assuming the occurrence of spherical water droplets at any W/S molar ratio along the chosen water dilution line, the aqueous core radius is given by: Rd (nm) = 0.16 W/S+ 0.7

2X2

)

+c2+2+(2nv) 2 +d

(10)

and the fraction of counterions PB within the distance 6 = 0.5 nm from the interface can be calculated according to the method proposed in Ref. [32]: 4(1 _~)3

27z 2

Rfa --

Z2[J(0) + J(co)]

(6)

pB=l-

F+4-F(1-3)

2

(11)

5 27z 2

RS2B --

5

Z2[J(og)+ J(2og)]

(7)

where Z is the quadrupolar coupling constant. In terms of the two-step model, the various spectral densities J(nog) in Eqs. (4)-(7) are given by [51]:

J(mo) = J f + j s =2zf +

2~ 1 + (no~z~)2

(8)

where ( = 6/Rd and the dimensionless constant F is: F=

e2Rd

(12)

ae0 eRkB T where a = 0 . 6 0 n m 2 is the area per polar head, eR=78.5 is the relative permittivity of water at 298 K and the other symbols have the usual meaning.

M. Monduzzi et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 129-130 (1997) 327 338

4. Results and discussion Fig. 1 shows the ternary phase diagram at 25°C of the A O T / W / I S O system [10], with the large L 2 region and the water dilution line, at the molar ratio S / 0 = 0 . 0 1 6 5 , here considered for the 2aNa N M R relaxation study of the A O T counterion and for the 1H N M R relaxation and self-diffusion of water. Samples with different water surfactant molar ratio (W/S) were examined in the absence (system A) and in the presence of the solubilized protein H S A (system B). The samples with HSA, have a constant molar ratio H S A / A O T = 5.8 × 10 -5. The addition of H S A did not modify the exterior aspect of the samples, which were always homogeneous, clear and isotropic liquids. The presence of H S A did not modify also the range of stability of the microemulsion along the chosen water dilution line. The composition of the samples is given in Table 1. At low surfactant concentration the w/o microemulsions of A O T have a microstructure of spherically shaped water-in-oil droplets, with a low degree of polydispersity. This type of microstructure should be retained also in the presence of water soluble guest molecules, as suggested by several authors. Therefore, the starting point of

ISO

40

60

40

20

W

AOT 80

60

40

Table 1 Compositions, 23Naspin lattice relaxation rates of counterions and related parameters W/S

Ro=0.16 W/S+0.7 (nm)

AOT/ISO = 0.0165 5.30 1.55 10.65 2.40 15.97 3.26 21.30 4.11 31.94 5.81 42.59 7.51 53.54 9.27 AOT/ISO = 0.0165 + HSA 5.00 1.50 10.00 2.30 15.00 3.10 20.00 3.90 30.00 5.50 40.00 7.10 50.00 8.70

PB

23Na Rlexp(s-l)

0.925 0.886 0.864 0.849 0.833 0.824 0.817

2415.5+29.7 498.5 _+2.4 322.8 +0.9 258.4+0.7 207.9+0.5 194.6+0.4 180.5_+0.3

0.928 0.889 0.867 0.853 0.836 0.826 0.819

1063.8+2.9 480.8_+2.6 367.6_+0.7 277.0_+0.9 221.7_+0.5 192.3+0.4 169.5_+0.2

the data treatment will consider a microstructural organisation of monodisperse spherical droplets with radius given by Eq. (10) where 0.16x W/S represents the water core radius to which the length of the A O T chain (around 0.7 nm in a partially extended conformation) is added to obtain the radius R d of the droplet, or, in other words, the radius of the electrokinetic unity. These data, obtained from the composition by Eq. (10) are reported in Table 1. 4.1. 23NaN M R relaxation

20

80

331

20

Fig. 1. AOT/W/ISO phase diagram at 25°C [10]. Only the L 2 microemulsion region is reported. The straight line shows the water dilution line, at the molar ratio AOT/ISO = 0.0165.

Table 1 reports the experimental 23Na spin-lattice relaxation rates along with the values of PB estimated by Eq. (11). Following the approach described in the previous section, the 23Na N M R experimental data were manipulated to extract the unknown parameters related to the dynamics of the counterions. The use of Eq. (91) to reproduce the experimental 23Na N M R spectrum allowed obtaining the quantities b=R~B and c=RfEB, assuming a Lorentzian shape of the two overlapped N M R signals. It should be mentioned that no dynamic shifts were considered since no appreciable asymmetry or distortion of the total signal bandshape is observed in any spectrum, even at

M. Monduzziet al. / ColloidsSurfacesA: Physicochem. Eng. Aspects129-130 (1997)327-338

332

the lowest W/S values. The R~B and RiB along with the Rip values are shown in Fig. 2(a) and (b), for the A and B systems respectively. The quantity Rip is obtained from the experimental Rlob, by subtracting the contribution of the free 23Na ions, whose spin-lattice relaxation rate RlF=17.5 s -~ was determined in a 0.1M NaC1 water solution at 25°C: R1B =

Rlobs - - ( 1 --pB)RIF

(13)

PB It should be noticed that the error associated with the R~B values is strictly related to the fitting goodness (r=0.99) which means errors comparable to those obtained for the R1B values (around+2%, see Table 1). The Rf~ values,

R$ r ($-1)

r

i

J

af

(sI) 4000

fl2B~

3000 F ',. 2500 I

R

"

'

3000

2B

2000 I000

ooo,, ooo soo

0

0 ' 0

I0

' 20

~ 30 40 W/S

' 50

60

(a) as

af

(s -I )

(s-I) 2000 lSO0

1500 ZB

1000

IO00

500 0

500 L

0

10

20

30

w/s

40

50

60

(b)

Fig. 2. 23NaNMR relaxation rates for bound counterionsvs W/S. (11)Rm, ((3) R~B, (~) RiB.(a) SystemA (AOT/W/ISO), (b) systemB (AOT/W/ISO+ HSA).

instead, are affected by larger errors (10-15%, as estimated by repeated experiments on the same samples), since the values are strongly dependent by small misadjustments of the experimental baseline. The RtB and R~B values are very close in both systems and decrease with increasing W/S, whereas the Rf2Bvalues display a hollow trend which brings the ratio Rf2B/R~2B to increase significantly with increasing W/S. The trends are similar in the two systems, but the involved amounts are rather different with the values of system A much higher, particularly at W/S< 10, than those of system B. The high values of the 'bound rates' at low W/S are not surprising, since at least a W / S ~ 10 value is necessary to gain a fully hydration of the polar head and of the associated counterion [29]. Hence, if sufficient water molecules are not present to complete the first hydration shell of the ionic species, the sodium ions are expected to experience high distortions of the surrounding electric field gradients which, through the quadrupolar coupling constant, determine an increase of the quadrupolar relaxation rates. Indeed, the large differences between the two systems at low W/S suggest the possibility of a very different arrangement (location and mobility) of the 13Na ions, due to the presence of HSA in system B. The occurrence of possible binding sites within the protein structure induces higher mobility of sodium ions among different sites (fast exchange) thus decreasing the average local electric field gradients experienced by the nucleus during the NMR observation time (see below). From the 'bound' relaxation rates, the various J(nco) of Eq. (8) are calculated. These values are reported in Fig. 3a and b for the two systems. Clearly, the frequency independent correlation function J(0) determines the trend of the fast component RfB, whereas the substantial similarity between J(co) and J(2co), particularly at high W/S, is responsible of the single exponential decay of Rlobs, and determines the very small difference observed between the R1B and R~B rates. The use of Eqs. (3)-(8) allows the estimate of the unknown parameters z~, jr and Xfor the bound 23Na species. These results are shown in Fig. 4a-c. The slow correlation time z~ represents an effective correla-

M. Monduzzi et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 129-130 (1997) 327-338

J(n=)

i

T

I

n=O

333

$ r

~a (ns) S 10 .8

/

3000 4 2000

I 0 "s AOT/W/ISO +

3 10 ~ 2 10 ~

HSA ..o .!.....o~"

1000

1 10 ~

0

0

0

I0

20

30

40

S0 60 W/S

(a)

....

Y

0

I

I0

20

30

40

(a)

J(nco)

jf

1500

(s") 800

n=0

1000

i ~

S0 60 W/S

AOTIWIISO

600

n=1

400

500 n= 0

200 i

0

I0

i

20

30

40

i

S0W/S60

0

(b) Fig. 3.23Na NMR autocorrelation functions J(nco) vs W/S. ((3)

J(co), (0) J(2co), (0) J(0). (a) SystemA (AOT/W/ISO), (b) systemB (AOT/W/ISO+HSA). tion time determined by three different contributions, due to shape fluctuations, surface diffusion and droplet rotation, which are likely to have a relatively different time scale. Although a quantitative separation of the contributions cannot be actually achieved, this T~ is reasonably expected to be determined by the rotational contribution of the droplet mainly, since shape fluctuations and counterion surface diffusion generally occur over much faster time scales [32]. Hence, the slow motions (see Fig. 4a), which vary between 1.75 and 50 ns, can be related to the motion of the surfactant interface, that is the motion of the whole droplet. In the case of system A, z~ increases gradually with increasing W/S, in agreement with the increase of the droplet radius. In the case of

i

i

I0

20

30

40

(b)

50

WlS

60

jf

(s") 800

I ~

400600200 '

0

0

AOTAN/ISO "

'

"

I

i

I0

20

(c)

,

~

I

30

40

S0 60 WlS

Fig. 4. ( I ) System A and (O) system B. (a) 23Na NMR slow correlation times z~ vs W/S. (b) 2aNa NMR fast component Jf for the bound counterions vs W/S. (c) 23Na NMR Z for the

bound counterionsvs W/S.

334

M. Monduzziet al. / ColloidsSurfacesA: Physicochem.Eng. Aspects129-130 (1997) 327-338

system B, the r[~ values fluctuate at low W/S, then increase for W/S>~30. The anomalous high z[3=50ns of system A, at W/S>50, particularly with respect to system B, arises from strongly interacting large droplets, since there is no reason to invoke a larger error on this value. In principle, the quadrupolar coupling constant should not change, but the Z values, shown in Fig. 4b, vary significantly at low W/S (<15), whereas at W/S>30 the values become almost constant around the average Z = 1 8 0 k H z . This behavior is clearly related to the non-complete hydration, particularly in system A, at W/S~5. The trend of Jf, reported in Fig. 4c, turns out to be related to the X values, as shown by Eq. (8). The subsequent calculation of the fast component of the correlation time zf of the bound species allows to verify that the condition of extreme narrowing (at our operating frequency 1/o9<2.1 ns), assumed for J f is not fully valid for all samples since zf ranging from 0.4 to 4 ns are calculated. This point will not be further developed since the calculated zf values exceed the condition of extreme narrowing only slightly. In conclusion, the 2aNa N M R data indicate that counterions at low W/S experience rather strong binding sites which create significant electric field gradients around the sodium ions, as demonstrated by the high values of the relaxation rates of the spectral densities, particularly JS(0), and of the values. In the range 5 < W/S<20 the presence of HSA, solubilized in the water core, induces a weakening of the binding sites, thus decreasing the asymmetries around the nuclei (Z decreases). Hence, the sodium ions in the bound state experience faster local motion (Jf decreases), although the overall isotropic slow motion is not significantly affected. In the presence of HSA, higher fluctuations of the bound counterions are allowed since a higher conformational freedom occurs at the interface. It can be stressed that the water spherical droplet microstructure is modified by HSA. On the other hand, if we take into account that HSA, in its globular shape, has a radius around 2.7 nm (including the hydration water molecules), it is rather obvious that the water droplets can accommodate the protein, without modifying their spherical shape, if the water core

becomes larger than 3 nm only (see the discussion below).

4.2. 1H N M R relaxation and self-diffusion of water Table 2 reports the spin-lattice relaxation rates and the self-diffusion coefficients of the 1H N M R signal of the water, obtained at 80 MHz, where the extreme narrowing condition applies up to Zc~2ns. The spin-lattice relaxation rate of the pure water at 25°C is R1°=0.325 s -1, with a re-orientational correlation time zc,.~ 3 ps. It is known that hydration water molecules are strongly bound, although they must be considered in fast exchange, on the N M R time scale, since a single signal is always observed. At low W/S, the water molecules are in the hydration shell of the ions only, and R1 values, more than one order of magnitude larger than the free water values, are observed. This is conveniently shown in Fig. 5 by the ratio R~/R1 ° vs W/S for systems A and B. As W/S increases the R t values approach R1 ° and the dynamic features of

Table 2 Compositions, 1H self-diffusioncoefficients(Dw)and spin lattice relaxation rates (R1) of water

W/S

Dw (10-1° m2 s-1)

AOT/ISO= 0.0165 5.00 3.400 7.50 2.699 10.00 1.600 12.50 1.499 15.00 1.391 20.00 1.069 25.00 0.968 30.00 0.782 40.00 0.754 50.00 0.458 AOT/ISO= 0.0165+ HSA 4.94 15.357 7.50 7.426 10.00 6.023 15.00 2.437 20.00 1.324 30.00 0.917 40.00 0.572 50.00 0.460 " From Eq. (14) assuming k=0.

Rcaal(nm)

IH R1 (s -1)

1.28 1.62 2.73 2.91 3.14 4.08 4.51 5.58 5.79 9.53

4.651 4.462 1.888 1.680 1.323 0.686 0.796 0.718 0.624 0.449

0.28 0.59 0.72 1.79 3.30 4.76 7.63 9.49

2.545 1.271 1.209 0.760 0.618 0.588 0.504 0.439

335

M. Monduzzi et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 129-130 (1997) 327-338

to the Stokes-Einstein relation:

15 qKq

R;/RI°

.

.

.

.

AOT/W/ISO I0

', ~ .

0

0

(14)

Dw = ( 1 - k~)kB T/6nrlRd

AOT/W/ISO + HSA

I

I

I

i

I

10

20

30

40

50

W/S

60

Fig. 5. Reduced 1H spin-lattice relaxation rates of water RI/R~° vs W/S at 25°C. R~°=0.325 s -1. (0) System A, and (©) system B. the bulk water are almost reproduced, in agreement with other investigations [29,53]. Indeed zc values around 4 ps are calculated at w / s = 50.

It should be noticed that, in agreement with the 23Na N M R results, the water relaxation rates also decrease in the presence of the solubilized HSA at W / S < 2 0 . In terms of dynamic parameters, HSA causes a higher mobility of the water molecules before a complete hydration of the polar groups is reached. Indeed, the z¢=42.9ps, found at W / S ~ 5 for system A, halves almost in system B. Similar values for the two systems are observed above W/S = 20. If the N M R relaxation rates reflect the state of the water molecules mainly, i.e. the degree of re-orientational freedom, the water self-diffusion coefficients, being related to the free mean path, are strongly affected by the extension and shape of the domain over which molecules can move. In practice, the self-diffusion coefficients of a species can give a hint of the real microstructure. The selfdiffusion coefficient of the pure water, at 25°C, is D ° =2.29 x 10 -9 m 2 s -1 [54] which for an observation time in the range 0.07-0.14 s, as in an usual PGSE sequence, corresponds to a free mean path ( x ) ~ 1 5 - 2 5 ~ t m . When Brownian diffusion of water molecules occurs in the restricted domain of a dispersed system such as a spherical droplet, the water self-diffusion coefficient decreases according

where r/ is the viscosity of the medium (~/iso=0.5 cP, at 25°C) and Rd is from Eq. (10). The quantity ( 1 - k ¢ 0 represents the obstruction effect. In the case of hard spheres with no hydrodynamic interaction [55], k = 1.5-2 and ~=q~w+q~s is the obstruction volume of the droplets. The Dw values of the two systems differ substantially in the range 5 < W / S < 2 0 again, then above W / S = 20 they become rather close, as reported in Table 2, and as shown, more efficaciously, by the ratios Dw/D ° vs W/S in Fig. 6. Below W / S = 2 0 the reduced self-diffusion coefficients of system B are much higher than those expected for a diffusion in a restricted domain, such as a water droplet. Indeed, the observed values are more typical of bicontinuous domains where water is located in channels of small size, so that diffusion is freely allowed in a single direction only, but the free mean path is still large. The introduction of the Rd radii calculated by Eq. (10) in Eq. (14) allowed to stress that, from the comparison with the experimental Dw of system A, the k values in Eq. (14) are around zero at W / S ~ 10, around 2 at 10< W / S < 3 0 and slightly smaller at W / S > 30. It appears reasonable that, at low W/S, data can be interpreted without invoking any obstruction effect, whereas, at higher W/S, these effects are expected to become significant. D /D ° w

0.8

Q~

0.6

AOTIWlISO + HSA

0.4 6 0.2

o

AOTIWlISO

0 0

10

20

30

40

50

W/S

60

Fig. 6. Reduced XHself diffusioncoefficientsof water Dw/D° vs W/S at 25°C. D°=2.29m2s -1. (0) System A, and (©) system B.

336

M. Monduzziet al. / ColloidsSurfaces A: Physicochem.Eng. Aspects129-130 (1997) 32~338

However, in these systems, which are close to the oil corner, and the dispersed phase is never greater than q~= 0.12, the obstruction factor is likely to play a minor role [56], as shown in Fig. 6 by the calculated Dw/D ° (continuous line of system A, by Eq. (14) with k = 0 ) which fit the experimental data rather closely. Thus, it can be reasonably neglected. The trend observed for system B, at W/S <~20 provides strong evidence of a connected water network which tends to disconnect to spherical water droplets upon water addition, exactly as observed in other microemulsions based on DDAB or PFPE surfactants [57-59]. On the other hand if the Rd values are calculated from Eq. (14), within the approximation k = 0 , from the experimental Dw values, the best linear regression for the two systems are: Rd =0.167W/S+0.75 system A (r=0.97)

(15a)

Rd =0.225W/S- 1.18 system B (r=0.99)

(15b)

The good agreement between Eqs. (15a) and (10) is a further confirmation for the choice of the spherical droplet model to interpret the microstructure of system A. Obviously this does not imply that water droplets are monodisperse in size, have always spherical shape, and no exchange of molecules among different droplets does occur [14,60]: the monodisperse spherical shape is a suitable average microstructure, stable over the N M R time scale of a PGSE sequence. It should be noticed that the small discrepancies between the experimental data and the predictions from a spherical monodisperse droplet model have been generally ascribed to the occurrence of 10-20% of size polydispersity, which, however, is not expected to cause significant effects on N M R time scale events [32]. On the contrary, the linear equation from system B gives a meaningless negative intercept (that is the added surfactant chain length), as a consequence of the high D w values at low W/S, and a too high slope (that means a lower contribution of each polar head to the interfacial area), which is not consistent with the existence of closed droplets of any shape. In principle, it cannot be excluded that strong protein-surfactant interactions may lead to form an oil soluble complex. However, in our system, the occurrence of a

HSA-AOT complex, randomly dispersed in the oil domain, does not appear consistent with the experimental observations, since it would imply a redistribution of both AOT and W molecules in the system. In such a case, the average size of the water droplets is expected to decrease, thus leading to an increase of the relaxation rates (for counterions and water) and to a decrease of the water selfdiffusion coefficients. Moreover, with increasing W/S, a stable HSA-AOT complex, formed at low W/S, should not easily rebuild the w/o droplet microstructure consistent with the composition, as shown by the overlap of the relaxation and diffusion data, from both systems A and B, at W/S> 25. On the other hand, the water molecules associated with the hydrophilic groups of the protein-surfactant complex, if present, would produce high water self-diffusion coefficients at any composition. Turning the attention to the microstructure of the system at low W/S, it is evident that any type of bicontinuous arrangement would account for the observed relaxation rates and self-diffusion coefficients. Indeed, HSA is a highly hydrophilic protein with 550 amino acid residues among which there is a slight predominance of anionic charged groups, at the natural pH. Consequently, some charged groups of HSA are likely to be located at the interface, particularly at low W/S. Fig. 7 shows a schematic picture of a possible arrangement of the protein within a fused network of water droplets. This organization may be favored, with respect to a more usual bicontinuous microstructure, on the basis of two observations: (i) the easy transition to disconnected droplets upon water addition and (ii) HSA is likely to rebuild its

Fig. 7. Schematicpicture of a possiblearrangementof the protein HSA within a fusednetwork of water droplets at low W/S.

M. Monduzzi et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 129-130 (1997) 327-338

globular shape as suggested by ESR data. Indeed, the two correlation times, used to describe the re-orientation of the 3MAL-HSA complex in the AOT/W/ISO system [38], are very different at low W/S, become closer at W/S>25, and are almost equal at W/S>~50, thus indicating a possible transition of HSA from an unfolded to a folded, almost spherical, conformation with increasing water content.

5. Concluding remarks on the microstructure The NMR data can define a clear picture of the modifications of the water spherical droplets in the AOT/W/ISO microemulsion, due to the presence of HSA. Particularly, the self-diffusion coefficients of water provide a strong and direct evidence for a microstructural evolution from closed water domains at high W/S, to a water continuous network at low W/S, exactly as observed in the case of didodecyldimethyammonium bromide (DDAB) [57,58,61] or perfluoropolyether (PFPE) [59] ammonium carboxylate surfactants in ternary microemulsions. In view of the information deduced from the water self-diffusion data, the water R1 and the counterion relaxation parameters can be accordingly interpreted. A microstructure, where the protein is likely to lose its initial globular conformation in order to be accommodated within a network of fused droplets, can be suggested at low W/S. In this situation, a highly dynamic interface is expected since a contribution from the charged groups of the protein should not be excluded. Indeed, since the typical packing parameter reported for AOT is v/al=l.2, a phase separation rather than a decrease of the favored inverse curvature of the AOT molecules would have been expected upon addition of a fully hydrophilic macromolecule. Hence, the possibility of few protein groups acting as cosurfactant allows a decrease of the inverse curvature of the interface to produce a water connected network. This microstructure favors the redistribution of the Na + ions and of the water molecules, which thus move more freely in a larger (and more variable) domain. When sufficient water molecules enable the formation of suitably large

337

droplets to accommodate the protein, the microstructure of the system rearranges in order to achieve the most stable curvature of the AOT interface, while protein is allowed to re-build its fully hydrated globular shape. The reason for the protein rearrangement should rely on the weakness of van der Waals interactions between the hydrophobic groups of HSA and the oil and/or surfactant chains. The increase of conformational entropy due to protein-surfactant association phenomena at the interface must be relatively small in comparison with the stability of HSA in its folded shape. These latest considerations may represent a strong point to support the hypothesis of 'a fused network of water droplets' for accommodating HSA at low W/S. Finally it should be noted that whenever a surfactant molecule has a packing parameter close to unity, as in the case of AOT, and as observed for DDAB and a PFPE surfactant, the interplay between different microstructures, driven by small variations of the interfacial curvature, easily brings about important modifications of the internal polar and apolar domains.

Acknowledgment Italian MURST and CNR, as well as Consorzio per i Sistemi a Grande Interfase (CSGI, Firenze) and Sardinia Region (Public Health Dept.) are acknowledged for support.

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