J. CHEM. SOC. FARADAY TRANS., 1994, 90(18), 2725-2730 Aggregation, Hydrogen Bonding and Thermodynamic Studies on Boc-Val-Val-He-OMe Tripeptide Micelles in Chloroform R. Jayakumar, R. G. Jeevan and A. B. Mandal" Chemical Laboratory, Physical and Inorganic Chemistry Division, Central Leather Research Institute, Adyar, Madras 600020,India P. T. Manoharan" Department of Chemistry, Indian Institute of Technology, Madras 600036,India Evidence for micelle formation of Boc-Val-Val-lle-OMe (Boc = tert-butyloxycarbonyl) tripeptide (l),in chloroform has been obtained from IR and Raman scatter fluorescence spectroscopies. The critical micelle concentrations (c.m.c.s) of this peptide, obtained by these techniques, correlate well. It has been found that the micelle forma- tion of the peptide in chloroform is hindered by increasing temperature. The aggregation numbers of the peptide have also been determined to be almost independent of temperature.The AmGe, AmHe, AmSe and AmCp values have been estimated. Results from the above thermodynamic parameters indicate that the driving force for micellization of the tripeptide 1 in chloroform is entirely enthalpic in nature and the aggregates of the peptide in chloroform are ordered. The IR spectra of the peptide in the pre- and post-micellar regions were analysed; there is no change in the intensity of the intermolecular hydrogen-bonding pattern for the peptide in the mono- meric and micellar states. However, the intensity of the solvent-exposed -N-H stretching band increased as a function of peptide concentration after attaining c.m.c.The design of molecular subunits that can self-assemble'*2 into defined structures in dispersion or in the solid state is one of the rapidly growing areas of chemistry and biology. The molecular recognition of the peptides is particularly important during self-assembly processes which result in micelles, vesicles and liquid crystal^.^ Few pep tide^,^.^ most phospholipids and some biologically relevant molecules are known to exhibit such behaviour.6 These surface-active mol- ecules exhibit a wide variety of biological activity.' As membrane-bound receptors, proteins play several important roles in mediating their functions ; the aggregation of membrane-active peptides in apolar media is valuable in the modelling of some interactions.* Ordered aggregates of the peptides in apolar media are also useful for obtaining infor- mation about the physicochemical nature of the interactions operating during self-assembly proces~es.~ Therefore, this study aims to identify interactions that lead to the molecular assembly of peptide moieties.Peptide self-assembly is very similar to the modular assembly involved in protein folding in terms of compactness, the core of non-polar alkyl side- chains and internal architecture. This model explores the contribution to Gibbs energy arising from hydrogen-bonding, van der Waal's interactions, intrinsic conformational propen- sities and solvophobic interactions." The size and number of molecules in the aggregate may be controlled by manipulating the type and orientation of the non-covalent interactions between the monomers.' ' The strong and directional nature of hydrogen bonds between -NH-CO-groups in the peptides contributes to their widespread involvement in self-assembling systems.,,'' Especially in an apolar medium, the solvophobic nature of amide groups is an advantage because of their low solubility in apolar 1iq~ids.l~ This has led to a search for peptides which form persistent packing motifs and defined aggregation numbers. The micelle formation of ampipathic molecules in apolar solvents has been debated in the past because the plot of optical properties vs. ampipathic concentration is not sharp enough to get a clear break point, and as a result this gives variable c.m.c.values. Recently' we have demonstrated that the Boc-Val-Val-Ile-OMe, 1, tripeptide, found in parallel p-sheets of triosephosphate isomerase,' forms micelles in an apolar medium like chloroform. Evidence for this was obtained by using UV-VIS, fluorescence and NMR spectroscopy' where clear break points were observed. Con- formational analysi~'~ of the tripeptide 1 in chloroform has also been carried out using the nuclear Overhauser effect (NOE). These NMR results15 suggested an extended struc- ture for the tripeptide 1 in chloroform. Once micelle formation and the conformation of the tri- peptide 1 in chloroform have been established,15 it is impor- tant to determine its aggregation number.*-14 We have determined aggregation numbers for Boc-Lys(Z)-Tyr-NH-NH, dipeptide and TFA -Tyr-Gly-Phe-Ala-OBz (TFA = trifluoroacetic acid) tetrapeptide micelles4 in aqueous solution and found them to be extremely low.However, the aggregation number of the tripeptide 1 in chloroform is sur- prisingly high, and its proper characterisation has tempted us to study this system further. Re~ently,'~ we have reported aggregation, hydrogen bonding and thermodynamic studies of aqueous tetrapeptide micelles in order to understand the secondary and tertiary structure of the peptides in the light of micelle formation. In this paper, we extend this study to the synthesis and characterisation of the tripeptide 1 aggregate in chloroform and the micellisation of 1.In this paper, we have employed two more independent techniques uiz., Raman scatter fluorescence and FTIR spectroscopy to substantiate our earlier c.m.c. results.' Experimental Materials and Methods Synthesis and Purification of Boc-Val-Val-Ile-OMe (1) Tripeptide All the amino acid derivatives were synthesised by standard procedures. Tripeptide 1 was prepared by a conventional solution phase method. All the intermediates were checked for purity by TLC on silica gel (solvent A: 2% MeOH in CHCl,; solvent B: 3% MeOH in CHCI,) and characterised by 90 MHz and 400 MHz 'H NMR. Specific procedures are described below. Boc-Vul-Zle-OMe. 4.34 g (20 mmol) of Boc-Val was dis- solved in CHCl, (20 ml) and cooled to -5 "C.Ile-OMe -HC1 was added, followed by 2.8 ml of triethylamine (TEA) (2.8 ml). 4.50 g (22 mmol) of N,N'-dicyclohexylcarbodiimide (DCC) was added in fractions for a period of 0.5 h. The reac- tion mixture was stirred at -5 "C for 3 h. After further stir- ring at room temperature overnight, the N,N'-dicyclohexylurea (DCU) was filtered and the filtrate was evaporated in vacuum. The residue was dissolved in CH,CO,Et (30 ml) and washed with 0.5 mol 1-' H,SO, (3 x 20 ml), 0.5 rnol 1-' Na,CO, (3 x 20 ml) and water (2 x 10 ml). Evaporation of CH,CO,Et yielded a solid mass which was dissolved in a minimum amount of CH,CN. The undissolved DCU was filtered and the filtrate was evaporated in vacuum, which yielded a solid mass, homogeneous on TLC (solvent A).Yield: 5 g, 72%. TFA . Val-lle-OMe. 3.44 g (10 mmol) of the above dipep- tide was treated with trifluoroacetic acid (TFA) (30 ml). The removal of the Boc group was monitored by TLC. After 1 h, TFA was evaporated with the help of a water aspirator. The residual TFA was removed in high vacuum. The residue was treated with anhydrous MeOH (3 ml) and evaporated in vacuum. This process was repeated twice to remove traces of TFA. The residue was suspended in water (30 ml), extracted with ether (20 ml) and the aqueous layer made alkaline with Na,CO,. Extraction with CHCI, , followed by drying of the organic layer with Na2S0, and evaporation yielded H-Val- Ile-OMe as a sticky solid. Yield: 2.08 g, 85%. Boc-Val-Val-Ile-OMe.2.08 g (8.5 mmol) of the above free base was dissolved in dimethylformamide (DMF) (10 ml) and cooled in an ice bath. Boc-Val (1.84 g) and 1-hydroxybenzotriazole (HOBT) (1.19 g) were added. To this, 1.85 g (9 mmol) of DCC was added in small portions. The above mixture was stirred at 0°C for 6 h and then at room temperature for 12 h. The precipitated DCU was filtered off and the filtrate was evaporated in vacuum. The residue was dissolved in CH,CO,Et (100 ml). The CH,CO,Et solution was successively washed with 0.5 mol 1-' H,S04 (3 x 30 ml), 0.5 mol 1-' Na,CO, (3 x 30 ml) and water (2 x 30 ml). The organic layer was dried with anhydrous Na,SO, and evapo- rated under vacuum to yield the tripeptide 1as a white solid. The tripeptide 1was purified with solvent system B.Yield: 2.67 g, 80%. Tripeptide 1was fully characterised by 400 MHz 'H NMR (see Fig. 1). Spectroscopic Studies NMR spectra of the peptide were recorded on an JEOL 400 MHz spectrometer. Two-dimensional correlated spectra (COSY) were recorded using a Bruker WH-270 FT-NMR spectrometer. FTIR measurements were performed with a Nicolet 20 DXB spectrometer. The band positions are accu- rate to 0.1 cm-'. Spectral grade chloroform was used for the sample preparation. Raman scatter of chloroform in the absence and presence of the peptide was recorded on a Hitachi Model No. 650-40 fluorimeter using a band width of 5 nm on both excitation and emission monochromators. The c.m.c. of the peptide was determined by plotting the intensity of the Raman scatter of chloroform and the intensity of free NH us.peptide concentration during fluorescence and FTIR spectroscopic measurements, respectively. The c.m.c. determi- nation was also carried out using tert-butylphenol as an external probe. The concentration of the probe was kept low (2 xlo-' mol I-') during the fluorescence spectroscopic J. CHEM. SOC. FARADAY TRANS., 1994,VOL. 90 measurements so that the ratio between the probe and micel- lar concentration is $1. The details for the c.m.c. determi- nation, have been described previously.'* Prior to the measurements, all the solutions were thermostatted for a con- siderable length of time. The temperature fluctuation was in the range of & 0.05-0.1 "C.Determination of the Aggregation Number The aggregation number of the tripeptide 1 in chloroform was determined by measuring the quenching of a micelle- bound fluorescent probe by the binding of a quencher using the following expression :19,20 In(I,/Z) = N[Q]/(C,-c.m.c.) (1) where I, and Z are the emitted light intensities with quencher concentrations of zero and [Q], respectively. fl is the mean aggregation number of the peptide and C, is the total concen- tration of the peptide. Semi-Mg salt of 8-anilino-1-naphthal- enesulfonic acid (ANS) and cetylpyridinium chloride (CPC) were used as a fluorescent probe and quencher, respectively. The utility of ANS as a suitable probe with CPC quencher has already been examined by also performing the above experiment with a pyrene probe.The aggregation numbers obtained by employing these two probes are in good agree- ment with each other. The utility of ANS as a probe and the validity of eqn. (1) have been recently dis~ussed?~"*~' All the experiments were performed in the presence of HPLC grade chloroform and there were no trace amounts of water present in the system. The concentration of the probe was kept SUE-ciently low to prevent exciplex formation. Results and Discussion NMR studies are carried out to characterise tripeptide 1.The complete assigned spectra of tripeptide 1are shown in Fig. 1. Specific assignments of backbone C"H and NH protons are * Im 0 mB I~"'~"~.I~"'~''~~,."......,......I..,I........,..6 5 4 3 2 1 6 Fig. 1 400MHz 'H NMR spectrum of 10mmol 1-'tripeptide 1 in CDC1, at 25°C. Two types of Boc-CH, signals (*) appear in the spectrum. The intensity pattern of these two signals alters in post- and pre-micellar states of the tripeptide. The complete analysis of this pattern will be discussed in the subsequent paper. J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 0 1 2 3 6 4 5 6 7 76543210 6 Fig. 2 270 MHz 'H NMR COSY spectrum of 10 mmol 1-' tri-peptide 1 in CDCl, at 25 "C made using a sequential assignment principle (based on a combination of COSY and difference NOE experiments). Examples of COSY spectra for tripeptide 1 with connecti- vities are shown in Fig. 2. The NOE spectra for tripeptide 1 have been reported" recently.The high-field doublet of the NH protons as seen in Fig. 1 is assigned to terminal NH Val because the urethane NH proton chemical shifts always occur at ca. 5.5 ppm in CDCl, solution.22 The CaH, CBH and CYH resonances of Val-1 are obtained by tracing its connecti- vities through cross peaks. The other Val-2, Ile-3 NH, and other connectivities, are obtained by assigning the high-field CBH resonance for Ile-3 as Ile CBH will appear at a higher field than CBH of Val because of the extra -CH2- substi-tution in Iie.23 The c.m.c. values of peptide 1, obtained by UV-VIS, fluo- rescence, NMR, IR and Raman spectroscopies are in good agreement with each other. The Raman scatter fluorescence spectra of chloroform in the absence and presence of various concentrations of peptide are shown in Fig.3. The c.m.c. and aggregation number of the tripeptide 1 at various tem-peratures are depicted in Table 1. The aggregation number of the tripeptide is found to be quite high and almost indepen- dent of temperature (cf: Table 1).The fluorescence intensity of ANS in chloroform increases on interaction with tripeptide micelles suggesting, even in apolar media, that ANS binds to the peptide in the region (either inside or in the interfacial region of the micelle~~~) of lower polarity than the chloro- form alone. The fluorescence emission spectra of ANS in chloroform in the absence and presence of various concentra- tions of tripeptide are shown in Fig.4. Note that there is no r 390 415 465 490 515 540 A/n rn Fig. 3 Raman scatter fluorescence spectra of chloroform at various concentrations of tripeptide 1 at 20°C. Curves 0-6: 0, 0.5, 1, 1.5, 2, 3 and 4 mmol 1-' tripeptide 1, respectively; A,, = 365 nm, A,, = 438 nm. appreciable shift in the ANS emission in the presence of the tripeptide owing to'7324 the decrease in polarity as well as the unaltered microviscosity of the environment.' 7*24 The NH stretching region of the IR spectra of the tri- peptide 1 shows two absorption peaks (cf: Fig. 5): one at 3448 cm-' is attributed to the presence of a solvated N-H and the other at 3300 cm-' may be due to intermolecularly H- bonded N-H groups.25 The IR spectra show that intermo- lecular hydrogen bonding occurs even at pre-micellar levels 40 P 4 400 500 600 700 A/n m Fig.4 Fluorescence spectra of ANS in chloroform at various con- centrations of tripeptide 1 at 20°C; [ANSI = lo-' mol 1-' (fixed)A,, = 346 nm, A,, = 478 nm. Curves 0-4: 0,2, 2.5, 3 and 4 mmol 1-' in tripeptide, respectively. Table 1 C.m.c., aggregation number (m)and some thermodynamic parameters for the tripeptide 1 in chloroform solution at various tem- peratures T/"C ~.m.c./lO-~mol 1-' m A,Ge/kJ mol-' A,H*/kJ mol-' A,Se/J K-' mol-' ACJJ K-' mol-' 5 l.l"Vb 140 -15.7 -19.3 -12.9 15 1.5".b 138 -15.6 -20.7 -17.7 25 2.0a.b.c.d 137 -15.4 -22.1 -22.5 -123 2.1e 30 2.5"*b 136 -15.1 -22.9 -25.7 40 3.1"*b 135 -15.0 -24.4 -30.0 " From IR spectroscopy. From Raman scatter fluorescence.From fluorescence spectroscopy. From NMR spectroscopy. From UV-VIS spectroscopy. J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 3500 3400 3300 wavenumber/cm-Amide A regions of IR spectra of tripeptide 1 in chloroform at various concentrationsat 20 "C. Curve 1-5: 1, 3, 4, 6 and 8 mmol 1-of tripeptide 1, respectively. of the tripeptide 1 in chloroform (Fig. 5). However, on micel-lization, there is a steep increase in the intensity of the solvat-ed NH peak suggesting that the onset of micellization accumulates solvated NHs in the peptide aggregate. This is quite surprising because the solvophobic -NH-CO-is supposed to hydrogen bond intermolecularly in a low-dielectric medium.26 The increase in fluorescence intensity of ANS in the presence of tripeptide 1, and the increase in the intensity of the solvated NH stretching band of the tripeptide, suggest that, for this peptide, the solvophobic group might be the isopropyl group of the peptide.Mean peptide aggregation numbers, A, are calculated from the slopes of the plot of ln(Zo/Z)us. [Q] (cf: Fig. 6). Neglecting activity effects and using a biphasic micellar the standard Gibbs energy change for micelle for-mation, A,Ge, of the peptide has been calculated from the following equation : A, Ge = RT In c.m.c. = A, He -TA, Se (2) The standard enthalpy change for micelle formation, A, He, is determined from the slope of the plot of In c.m.c. us. T (Fig.:::I 1.O h% 0.8 0.4 0.2 0 0 2 4 6 8 10 12 [O]/l 0-5 mol dm-3 Fig.6 Results of ANS quenching experiments: ln(Zo/I) us. concen-tration of N-cetylpyridinium chloride for micellar solutions of tri-peptide 1 at 25°C. [Tripeptide] = 12 x mol 1-' (fixed) and [ANSI = moll-' (fixed) in chloroform solutions. Aex = 346 nm; Aem = 478 nm. v E -6.4' v S --6.0' 273 283 293 303 313 T/K Fig. 7 In c.m.c. us. T 7) using the following equation:28 d In c.m.c.A,H~= -RT~ dT (3) To calculate all the thermodynamic parameters, the standard states are chosen as the hypothetical solutions at unit molar concentration. In recent p~blications~*'~we have considered aggregation number as one of the thermodynamic variable~~*'~.~~'in the calculation of the above thermodyna-mic parameters because the aggregation number of such pep-tides is sufficiently low.However, the aggregation number has not been taken into consideration as a thermodynamic variable in the present work because the tripeptide 1 in chlo-roform possesses quite high aggregation numbers which are almost independent of temperature. The A, Ge, A, H0 and AmSe values for the tripeptide 1at various temperatures are given in Table 1. The standard heat capacity change for the micellization, ACF, obtained from the slope of A,He us. T is also given in Table 1. It is assumed that a major factor driving the surfactant molecules into aggregation in water is a positive entropy change, presumably associated with the breakdown of the structured water which surrounds the hydrocarbon chain in the unassociated species.This interpre-tation is relevant to the formamide system in which some structuring by dissolved hydrocarbon also occurs. According to Evans et al.29athe above interpretation is erroneous or, at least, misleading because at high temperatures water loses most of its structural properties and the formation of struc-tured water in the walls of the hydrocarbon cavities is no longer possible. According to Evans et al.,29"it is sensible to attribute the micellization of the tripeptide in chloroform to a negative entropy change which is due to a transfer of the chloroform solvent into the peptide micelles. Table 1 shows that A,Se and AmHe values for the tripeptide are always negative in the temperature range of the investigation.The negative value of AmSOmay arise from the value of A, He, owing, to some extent, to the re-establishment of the hydro-gen bonds in the solvent. Therefore, the results indicate that the driving force for micellization of the peptide is entirely enthalpic in nature. This analysis has also been applied by Evans and Ninham29b to changes in protein conformation and to other biochemically important self-assembly processes. Comparing the results of thermodynamic experiments on model organic compounds, it is apparent that the heat capac-ity changes play a central role in characterising solvophobicinteraction^.^' The negative heat capacity change (see Table 1) is attributed to the disordering of solvent molecules around the exposed solvophilic groups.31 Therefore, the negative heat capacity change indicates disordering of chloro-form molecules around the tripeptide micelles.Convention-ally, solvophobic interactions provide a driving force for J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 micellization, with steric repulsion providing an opposing force.32 Therefore, our results suggest that the micelle forma- tion of the tripeptide l is hindered by the increase in tem- perature, as the c.m.c. value of the peptide increases with increasing temperature (cJ Table 1). In an apolar medium like chloroform, the main interaction in the peptide aggregation is believed to be intermolecular hydrogen-bond formation.33 However, a plot of a large number of literature values for AG* determined in chloroform vs.n (where n is -NH-CO-groups) lead to a straight line with a slope of -5 & 1 kJ mol- due to the stabilization of amide groups of the peptide on interaction with chloroform molecules. In the present case, the three amide groups interacting with chloro- form should result in a minimum of -15 3 kJ mol- ' of energy of stabilisation to the aggregate;34 the AmGe value obtained, -15.4 kJ mol-', is in agreement with this interpre- tation. Thus, chloroform associates with the amide moiety (of association constant, K = 1 dm3 mol-'). Therefore, the only interaction which makes A,,, Ge sufficiently negative is the enthalpic component which suggests the process is entirely enthalpic.If we attribute a negative AmS* to hydro- gen bonding with solvent molecules, then the process does not result in more ordered molecules around the exposed groups (which can interact with the chloroform molecules). Then such a process would also decrease ACF values,35 since release of chloroform solvation leads to a reduction in the number of heat-absorbing bonds. In the present case, the negative value of ACF implies that the chloroform is less ordered around the peptide micelles. Hence, the negative Am€€* may be attributed to the relatively well solvated peptide molecules in the micellar state. The experimental observations leading to these conclusions may be summarized by the following points. (i) The linewidths of the NH signals of the peptide do not change on micellization.' (ii) The ANS fluorescence emis- sions have no appreciable blue shift in the presence of the tripeptide indicating that there is not viscosity change in the interior of the micelles.(iii) The IR intensity of free NH increases upon micellization of the tripeptide in chloroform. (iv) The fluorescence emission intensity of the tert-butylphenol decreases upon micellization' of the tripeptide as the non-polar micellar interior decreases the fluorescence emission of the phenolic moiety.36 (v) The intensity of ANS fluorescence increases in the micellar environment, indicating a less polar environment of the micellar interior. (vi) The large aggregation number of the peptide and its constancy over the investigated temperature range are indicative of solvent-associated monomers in the micelle.(vii) The NOE observed is positive." If 140 molecules of the tripeptide 1 are associated, then the effective molecular weight of the aggre- gate will be ca. 62020. With this large aggregate molecular weight there will be an increase in correlation time which can result in negative NOE as oz, falls in a negative domain. The fact that we observe a positive NOE indicates that the motions of the monomeric peptides are not restricted and the rotational correlation times are short enough in the region oz, 1 at 270 MHz.~' Therefore, the tripeptide 1 micelles formed in chloroform consist of flexible solvated peptides of decreased enthalpy because of their interaction with chloro- form molecules.We are thankful to Dr. G. Thyagarajan, Director, CLRI, Madras for his keen interest in this work. Stimulating dis- cussions with Dr. T. Ramasami, Deputy Director and Head, Chemical Sciences Division are greatly appreciated. The support of the Research Council and the Regional Sophisti- cated Instrumentation Centre facilities, at the Indian Institute of Technology, Madras are gratefully acknowledged. We are grateful to the referees for their constructive suggestions and valuable comments. References 1 J. S. Lindsey, New J. Chem., 1991, 15, 153. 2 G. M. Whitesides, J. P. Mathias and C. T. Seto, Science, 1992, 254, 1312. 3 J. M. Lehn, Angew. Chem., Znt. Ed. Engl., 1990,29, 1304.4 (a) A. B. Mandal and R. Jayakumar, J. Chem. SOC., Chem. Commun., 1993, 237; (b) A. B. Mandal, A. Dhathathreyan, R. Jayakumar and T. Ramasami, J. Chem. SOC., Faraday Trans., 1993,89,3075. 5 (a) G. H. Beaven, W. B. Gratzer and H. G. Davies, Eur. J. Biochem., 1969, 11, 37; (b) W. B. Gratzer, E. Bailey and G. H. Beaven, Biochem. Biophys. Res. Commun., 1967,28,914; (c) D. J. Patel, Macromolecules, 1970, 3, 448; (d) L. C. Craig, J. D. Fisher and T. P. King, Biochemistry, 1965,4, 3 11. 6 S. Hoffmann, 2. Chem., 1987,27,395. 7 Chemistry and Biochemistry of Amino Acids, Peptides and Pro- teins, ed. B. Weinstein, Marcel Dekker, New York, 1974, vol. 3. 8 R. Kishore, S. Raghothama and P. Balaram, Biopolymers, 1987, 26, 873. 9 S. Bonazzi, M.Capabianco, M. M. De Morais, A. Garbesi, G. Gottarelli, P. Mariani, N. G. Ponziborsi, G. P. Spada and L. Tondelli, J. Am. Chem. SOC.,1991,113,5809. 10 K. A. Dill, Perspectives Biochem., 1991, 1. 11 J. D. Wright, in Molecular Crystals, Cambridge University Press, Cambridge, 1987. 12 A. J. Kisby, Adv. Phys. Org. Chem., 1980,17, 197. 13 A. J. Doig and D. H. Williams, J. Am. Chem. SOC., 1992, 114, 338. 14 H. Ihara, H. Hachisako, C. Hirayama and K. Yamada, J. Chem. SOC.,Chem. Commun., 1992, 1244. 15 R. Jayakumar, A. B. Mandal and P. T. Manoharan, J. Chem. SOC., Chem. Commun., 1993,853. 16 J. S. Richardson, Adv. Protein Chem., 1981,34, 168. 17 A. B. Mandal and R. Jayakumar, J. Chem. SOC.,Faraday Trans., 1994,90, 161. 18 (a)A. B. Mandal and B.U. Nair, J. Phys. Chem., 1991,95, 9008; (b)A. B. Mandal and B. U. Nair, J. Chem. SOC.,Faraday Trans., 1991,87, 133; (c) A. B. Mandal, B. U. Nair and D. Ramaswamy, Langmuir, 1988, 4, 736; (d) A. B. Mandal, B. U. Nair and D. Ramaswamy, Bull. Electrochem., 1988, 4, 565; (e) A. B. Mandal and S. P. Moulik, ACS Proc. in Solution Behavior of Surfactants-Theoretical and Applied Aspects, ed. K. L. Mittal and E. J. Fendler, Plenum Press, New York, 1982, vol. 1, pp. 521-541; (f) A. B. Mandal, S. Ray and S. P. Moulik, Indian J. Chem. A, 1980, 19, 620; (9) A. B. Mandal and B. U. Nair, in Advances in Measurement and Control of Colloidal Processes, ed. R. A. Williams and N. C. de Jaeger, Butterworth, London, 1991, ch. 2, pp. 136-149; (h) A. B. Mandal, D.V. Ramesh and S. C. Dhar, Eur. J. Biochem., 1987,169,617. 19 N. J. Turro and A. Yekta, J. Am. Chem. SOC., 1978,100,5951. 20 L. Luo, N. Boens, M. Vander Auweraer, F. C. de Schryver and A. Malliaris, J. Phys. Chem., 1989,93, 3244. 21 B. Geetha, A. B. Mandal and T. Ramasami, Macromolecules, 1993,26,4083. 22 (a) R. Kishore, S. Raghothama and P. Balaram, Biopolymers, 1987, 26, 873; (b) M. Iqbal and P. Balaram, J. Am. Chem. SOC., 1981, 103, 5548; (c) Y. V. Venkatachalapathi, B. V. V. Prasad and P. Balaram, Biochemistry, 1982, 21, 5502. 23 (a)A. Ravi, B. V. V. Prasad and P. Balaram, J. Am. Chem. SOC., 1983, 105, 105; (b) A. Ravi and P. Balaram, Tetrahedron, 1984, 40, 2577. 24 J. Slavik, Biochim. Biophys. Acta, 1982,694, 1. 25 (a) C.Toniolo, G. M. Bonola and S. Salardi, Znt. J. Biol. Macro- mol., 1981, 3, 377; (b) M. H. Baron, C. Beloze, C. Toniolo and G. D. Kasman, Biopolymers, 1978,17,2225. 26 (a) M. K. Gilson and B. H. Hanig, Nature (London), 1987, 330, 84; (b) S. Dao Pin, D. I. Liao and S. J. Remigton, Proc. Natl. Acad. Sci. USA, 1989,86,5361. 27 (a) A. B. Mandal, D. Mukherjee and D. Ramaswamy, Leather Sci.,1981, 28, 283; (b) A. B. Mandal, M. Kanthimathi, K. Govin- daraju and D. Ramaswamy, J. SOC. Leather Technol. Chem., 1983, 67, 147; (c) A. B. Mandal, J. Surface Sci. Technol., 1985, 1, 2730 J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 28 93; (d) W. B. Gratzer and G. H. Beaven, J. Phys. Chem., 1969, 73, 2270; (e) D. G. Hall and B. A. Pethica, in Nonionic Sur-factants, ed.M. J. Schick, Marcel Dekker, New York, 1967, ch. R. H.Ottewill, in Surfactants, ed. Th. H. Tadros, Academic 16, pp. 516-557. 33 34 Dekker, New York, 1967, vol. 1, pp. 478-515; (d) P.Mukerjee and K. Mysels, J. Am. Chem. SOC., 1955,77,2937. I. M. Klotz and J. S.Franzen, J. Am. Chem. SOC., 1962,84,3461. (a)H. J. Schneider, R.K. Juneja and S. Simora, Chem. Ber., 1989, 112; (b) H. J. Schneider, Angew. Chem., Znt. Ed. Engl., 1991, 30, 29 30 31 32 Press, London, 1984, ch. 1, pp. 1-7. (a)D. F. Evans, M. Allen, B. W. Ninham and A. Fouda, J. Solu-tion Chem., 1984, 13, 87; (b) D. F. Evans and B. Ninham, J. Phys. Chem., 1986,90,226. (a) J. M. Sturtvent, Proc. Natl. Acad. Sci. USA, 1977, 74, 2236; (b)R. L. Baldwin, Proc. Natl. Acad. Sci. USA, 1986,83, 8669. R. S. Spolar, J. Hu and T. M. Record, Proc. Natl. Acad. Sci., USA, 1989,86,8382. (a)C. Tanford, in The Hydrophobic Effect, Formation of Micelles and Biological Membranes, Wiley, New York, 1973; (b) J. H. Fendler and E. J. Fendler, in Catalysis in MicelZar and Macro- molecular Systems, Academic Press, New York, 1975; (c) P. Becher, in Nonionic Surfactants, ed. M. J. Schick, Marcel 35 36 37 1417. (a) M. H. Abraham, J. Am. Chem. SOC., 1982, 104, 2085; (b) N. Muller, Acc. Chem. Res., 1990,23,28. (a)J. E. Bailey, G. H. Beaven, D. A. Chignell and W. B. Gratzer, Eur. J. Biochem., 1968, 7, 5; (b)H. Greenspan, J. Birnbaum and J. Feitelson, Biochim. Biophys. Acta, 1966, 126, 13. (a)J. D. Glickson, S. L. Gordon, T. P. Pitner, D. G. Agnesh and R. Walker, Biochemistry, 1976, 15, 5721; (b) A. A. Bothner-By, in Magnetic Resonance in Biology, ed. R. G. Shulman, Academic Press, New York, 1979, p. 177. Paper 3/06942G; Received 22nd November, 1993