Effect of succinylation (3-carboxypropionylation) on the conformation and immunological activity of ovalbumin.

The epsilon-amino groups of ovalbumin were modified with succinic anhydride; as many as 16 lysine residues were succinylated (3-carboxypropionylated). The five succinylated derivatives thus prepared were homogeneous with respect to the extent of chemical modification as shown by electrophoretic and immunological data. Succinylation of the amino groups altered electrophoretic mobility and isoionic pH of ovalbumin in the expected direction. U.v.-absorption and fluorescence spectra suggested changes in the microenvironment of the chromophores in the modified proteins. The difference-spectral results showed greater exposure of tyrosine and tryptophan residues in the succinylated ovalbumin. Increase in susceptibility to tryptic digestion, Stokes radius and intrinsic viscosity of native ovalbumin, which was observed on successive increase in the chemical modification, demonstrated a conformational change that was proportional to the extent of modification. The loss of immunological reactivity caused by chemical modification also indicated a conformational change in succinylated ovalbumin. The fact that the intrinsic viscosity of maximally modified ovalbumin was less than one-third of that for the completely denatured protein in 6M-guanidinium chloride suggested that the modified protein contained significant residual native structure. The latter presumably accommodates some antigenic determinants accounting for 37% residual immunological activity observed with maximally succinylated ovalbumin.     

Acetylation of amino groups and its effect on the conformation and immunological activity of obalbumin.

A procedure is described for the specific acetylation of the lysine residues of ovalbumin. Six acetylated ovalbumins varying in the degree of modification from 21 to 98% were prepared and were found to be homogeneous by polyacrylamide gel electrophoresis, immunodiffusion, and immunoelectrophresis. As expected, the anodic movement of ovalbumin increased and the isoionic point shifted to lower pH values with progressive acetylation of the protein. Measurements on ultraviolet absorption, fluorescence, tryptic digestion, intrinsic viscosity, gel filtration behavior, and immunological reactivity demonstrated that the native folded conformation of ovalbumin was appreciably altered by acetylation. However, even the maximally modified ovalbumin retained considerable residual structure consisting of regions of ordered structure containing antigenic determinants.     

Helix stability in succinylated and acetylated ovalbumins: effect of high pH, urea and guanidine hydrochloride

Previous studies (Batra, P.P., Roebuck, M.A. and Uetrecht, D. (1990) J. Protein Chem. 9, 37-44) showed that succinylation or acetylation of 75% of the lysine residues has little effect on the secondary structure of ovalbumin. The acylation of the remaining 25% lysine residues, which apparently are partially buried, results in a substantial loss of the helical structure. These conformational changes may be due not only to electrostatic repulsions introduced by succinylation or acetylation of the positively charged epsilon-amino groups but also to steric hindrance, since an increase in the ionic strength failed to reverse the loss of the helical structure. An increase in pH to 12.2 results in a complete helix-to-coil transition in the maximally succinylated ovalbumin (but not in the partially succinylated or in any of the acetylated ovalbumins including the maximally acetylated derivative), perhaps because it is most expanded and its molecular interior most accessible to solvent as succinylation replaces +1 charge of epsilon-amino group with a -1 charge so that a net of -2 charge per succinyl group is placed on the protein molecule. This helix-to-coil transition in the maximally succinylated ovalbumin induced by high pH is fully reversed by increasing the ionic strength, indicating that only electrostatic effects are responsible for this disruption. Studies have also shown that although there is no loss of the helical structure until after the 75% surface lysine residues have been acylated, the helical structure does become progressively destabilized with increasing degree of modification, a conclusion drawn from urea unfolding curves. This destabilization of the helical structure is due primarily to electrostatic effects, as an increase in the ionic strength led to an increase in the urea transition mid-point. Unlike urea, the guanidine hydrochloride unfolding curves indicate that the transition mid-point for the native protein, as well as for the maximally succinylated and acetylated derivatives, is about the same, perhaps because the denaturant itself acts as an electrolyte.     

Effect of lysine modification on the secondary structure of ovalbumin

In order to determine the effect of chemical modification of the -amino groups on the secondary structure of ovalbumin, we prepared six acetylated (17, 36, 54, 70, 82, and 98%) and four succinylated derivatives (25, 50, 72, and 97%) of the protein. Native ovalbumin and the acylated derivatives were homogeneous as revealed by the electrophoretic pattern. The UV-absorption and fluorescence spectra changed progressively with the extent of modification. However, circular dichroic (CD) studies indicated that acylation of 15 of the 20 lysine residues had little effect on the secondary structure of ovalbumin. Acylation of the remaining five lysine residues resulted in a fairly severe change in the secondary structure. The -helical content decreased from about 31% in the native state to 16.5% in the 97% succinylated ovalbumin and to 21.5% in the 98% acetylated derivative. A comparison of these data with the spectral and hydrodynamic data of Qasim and Salahuddin (1978) suggested that the secondary structure of ovalbumin is more resistant to acylation than is the tertiary structure and, thus, the tertiary and the secondary structures are, to some extent, mutually independent. Raising thepH to 11.2 did not alter the secondary structure of ovalbumin and increasing the ionic strength by more than 20-fold did not reverse the loss of helical structure in 97% succinylated protein. These two observations suggest that the change in secondary structure upon maximal acylation may not only involve electrostatic effects, but also certain other factors, such as steric hindrance due to the entering bulky groups.     

Chemical modification of buried lysine residues of bovine serum albumin and its influence on protein conformation and bilirubin binding.

Using a double modification technique about 20% of the lysine residues of bovine serum albumin (BSA) which are not easily accessible in the native protein have been modified. The technique involved approximately 80% modification of lysine residues of BSA with citraconic anhydride followed by chemical modification of the remaining lysine residues with acetic anhydride, succinic anhydride, potassium cyanate, or O-methylisourea. Finally, these preparations were decitraconylated under mild acidic conditions to yield acetylated, succinylated, carbomylated or guanidinated BSA. All of these preparations were found to be homogeneous with respect to charge and size. The spectral, hydrodynamic and bilirubin binding properties of these preparations are described. In contrast to most of the highly modified proteins these preparations with the exception of succinylated BSA are very similar to native BSA in their spectral and hydrodynamic properties. However, the equilibrium association constant (Ka) with bilirubin measured by fluorescence quenching was decreased by about 100-fold in acetylated, carbamylated and succinylated BSA, but only 3-fold in guanidinated BSA. Since conformationally acetylated and carbamylated BSAs are identical to guanidinated BSA we conclude that the decrease in Ka in these preparations is solely due to loss of positive charge on 'critical' lysine residues. The results support a binding model for BSA in which bilirubin binding site is buried and the protein undergoes a series of relaxational changes in conformation upon interaction with bilirubin.     

Effect of lysine modification on the conformation and indomethacin binding properties of human serum albumin.

In order to study the involvement of lysine residues of human serum albumin (HSA) in the binding of indomethacin, HSA was treated with different molar excess of acetic anhydride, succinic anhydride and O-methylisourea which resulted in differently modified preparations: 30%, 62% and 87% acetylated, 20%, 34%, 64% and 78% succinylated and 21%, 43% and 86% guanidinated HSAs. All the preparations were found to be homogeneous with respect to charge as well as size as judged by polyacrylamide gel electrophoresis and gel filtration on a Seralose-6B column. Hydrodynamic and circular dichroic results showed that pronounced conformational changes (both tertiary and secondary structures) were induced in the maximally acetylated (87%) and succinylated (78%) preparations. On the other hand, guanidinated preparations showed no expansion in the hydrodynamic volume. The percent decrease in alpha-helical content was 34% for 87% acetylated, 31% for 78% succinylated and 10% for 86% guanidinated HSAs. A significant increase in the values of Stokes radii and frictional ratios (from 3.43 nm and 1.29 for native HSA to 4.07 nm and 1.52 for 87% acetylated and 4.35 nm and 1.60 for 78% succinylated HSAs, respectively) was also noticed in these highly modified preparations. Fluorescence quench titration results obtained at pH 7.4 and ionic strength 0.15 showed that only 54.1% and 64.7% binding of indomethacin at 4:1 drug/protein molar ratio was retained by 87% acetylated and 78% succinylated HSAs, respectively, as compared to 91% retention in binding in 86% guanidinated preparation. No reversal in the binding of drug to 87% acetylated and 78% succinylated HSA preparations was observed on increasing the ionic strength to 1.0. Therefore, it seems that one or two critical lysine residue(s) that can form salt linkage with the carboxyl group of indomethacin, was (were) probably modified in these preparations. A small decrease in the binding of drug to the guanidinated preparation also confirms the involvement of positive charge, probably contributed by lysine residue(s), in the binding of indomethacin to HSA.     

Acylation of L-asparaginase with total retention of enzymatic activity

Acylation of L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) with complete retention of catalytic activity was achieved. Several parameters of the acylation method, based on the binding of palmitoyl residues to epsilon-NH2 groups of protein, were optimized. The correlation between the acylation degree of L-asparaginase and the retention of catalytic activity was established. For a palmitoyl chloride/protein molar ratio ranging from 50 to 900, a degree of modification of 10 to 30% and a retention of catalytic activity of 98 to 60% respectively, was observed. Hydrophobicity of 30% acylated protein was correlated with turbidity in water and octanol and was compared with the native protein. Acylated protein incorporated into liposomes, showed an increase in catalytic activity in intact form as compared to the native enzyme. By the introduction of a sequential acylation cycle, an improvement of the degree of modification with a maximal value at 50% was obtained. Total retention of catalytic activity was achieved by acylation in the presence of 8 mM L-asparagine in a reactional medium.     

The buoyant titration of native and carbamylated bovine serum mercaptalbumin.

The buoyant density titration curves of native and carbamylated bovine serum mercaptalbumin were measured throughout the pH range 5.3-12.7. Large increments in the buoyant density were observed above pH 10, with inflection pH values of 11.2 and 11.4 for native and carbamylated bovine serum mercaptalbumin, respectively. For the modified protein in which 25 out of 58 lysine residues were carbamylated, the buoyant densities were 0.048 g/ml higher at neutral pH and 0.024 g/ml higher at the extrapolated pH 13. The carbamyl groups apparently produce a larger residual density at pH 13 than they did in the case of ovalbumin. Homopolymer buoyant density titration data were demonstrated to be of value in calculating the contributions of titratable residues to the buoyant density of both proteins. The buoyant density increment at high pH was due largely to the deprotonation of the lysines as indicated by the diminished change in buoyant density between pH 10 and 12.7 for the modified protein. These density changes were attributable primarily to a gain of cesium ions. The limited modification of the lysine residues under mild reaction conditions and the rather high intrinsic dissociation constant of tyrosine residues in mercaptalbumin may indicate a preferential modification of easily accessible lysine residues. Phenolic deprotonation is facilitated by the neutralization of normally charged lysine residues and demonstrates ionic interactions between internal lysines and certain carboxyl and tyrosine residues thereby stabilizing the native state of the protein.     

A correlation between changes in conformation and molecular properties of bovine serum albumin upon succinylation

Using succinic anhydride, six succinylated derivatives of bovine serum albumin having percent modification in the range of 23-87% were prepared and their physicochemical and immunological properties were studied. Measurements of Stokes radius, frictional ratio, UV spectra, solvent perturbation, solubility, and immunological cross-reactivity against anti-bovine serum albumin antiserum revealed that the protein undergoes gradual changes in its native conformation with increase in the degree of succinylation. These changes were less marked below 50% modification but became pronounced above 50% modification. However, even the maximally modified preparation (87%) contained a significant amount of folded structure. Interestingly, though the measurements of various molecular properties revealed significant changes in 23-49% modified preparations, the solubility parameters for these preparations which were obtained at high ionic strength were indistinguishable from those of the native protein. The various results taken together suggest that at lower degrees of chemical modification, the conformational changes were produced mainly because of an increase in electrostatic free energy, whereas at higher degrees of modification, steric hindrance in addition to the electrostatic factor seems to make a substantial contribution to the conformational changes in the modified proteins.     

Iionization of tyrosyl groups of ovalbumin under native and denaturing conditions.

Phenolic titration of ovalbumin was performed in the pH range 7-12 at 30 degrees C and at three ionic strengths viz. 0.033, 0.133 and 0.200. The conformational integrity of ovalbumin was studied by viscosity measurements at different pH values in the pH range 7-12.4. At ionic strength 0.133 two phenolic groups titrated reversibly with pKint = 10.31, and w = 0.032 up to pH 11.25 under native conditions. The value of w expectedly decreased with increase in ionic strength. Two additional phenolic groups became available for reversible titration between pH 11.25 and 11.95 after some conformational change. Above pH 12, the phenolic titration became irreversible and all of the nine tyrosine residues were titrated at pH 13.3 Exposure of ovalbumin to alkaline pH (12.4) caused considerable disruption of the native protein conformation. The reduced viscosity increased from 4.2 ml/g at pH 7.0 to 16.8 ml/g at pH 12.4 under identical conditions of the protein concentration. All of the nine tyrosyl groups of ovalbumin were titrated normally (pKint = 9.9) in a mixture of 5 M guanidine hydrochloride and 1.2 M urea. However, even in this mixture electrostatic interaction, as measured by w was not completely abolished.     

Viscometric characterization of pennisetin from pearl millet

Pennisetin, the alcohol soluble storage protein of pearl millet (Pennisetum americanum), was isolated in a homogeneous state. The intrinsic viscosity [n] of this protein was found to be in the range of 16.5-17.7 ml/g in 70% (v/v) aqueous ethanol. The [eta] changed marginally when temperature was increased from 20 to 70 degrees C and also in the presence of 10 mM NaCl. The data indicated that pennisetin was a rigid, rod shaped asymmetric hydrodynamic particle with molecular dimensions in the range of 301 x 14.4 A - 317.7 x 14.2 A. During denaturation with guanidine hydrochloride (Gdn.HCl), the intrinsic viscosity of pennisetin increased from 16 to 25ml/g with a mid point at 3.6 M of the denaturant. The native protein structure was unfolded in 6 M Gdn.HCl as shown by the exposure of aromatic amino acid residues buried in the native state and this transition was found to be reversible. The intrinsic viscosity of pennisetin in 5.9 M Gdn.HCl corresponded to Mr 25,000 which was comparable to that determined by SDS-PAGE.     

The pH dependence of the reversible unfolding of ovalbumin A1 by guanidine hydrochloride.

The pH dependence of the reversible guanidine hydrochloride denaturation of the major fraction of ovalbumin (ovalbumin A1) was studied by a viscometric method in the pH range 1-7, at 25 degrees C and at six different denaturant concentrations (1.5-2.6 M). At any denaturant concentrationa reduction in pH favoured the transition from the native to the denatured state. The latter was essentially 'structureless', as revealed by the fact that the reduced viscosity of the acid and guanidine hydrochloride denatured state of ovalbumin A1 (obtained at different denaturant concentrations in acidic solutions) was measured (at a protein concentration of 3.8 mg/ml) to be 29.2 ml/g which is identical to that found in 6 M guanidine hydrochloride wherein the protein behaves as a cross-linked random coil. A quantitative analysis of the results on the pH dependence of the equilibrium constant for the denaturation process showed that on denaturation the intrinsic pK of two carboxyl groups in ovalbumin A1 went up from 3.1 in the native state to 4.4 in the denatured state of the protein.     

Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation

The influence of various levels of succinylation on the structure of the legumin from pea seed has been studied by the techniques of sedimentation velocity, viscometry, fluorescence and circular dichroism spectroscopy, as well as dynamic light scattering. The protein dissociates gradually into the 3S subunit forming a 7S intermediate. At a level of 75-80% succinylation, sudden unfolding of the protein occurs characterized by drastic changes in viscometric and spectroscopic properties. The fluorescence spectra point to the formation of a novel organized structure at a moderate degree of modification before the molecular unfolding takes place. The succinylated subunit was shown to have a sedimentation coefficient of 3.2S, a diffusion coefficient of 5.03 x 10(-7) cm2 . s-1 a Stokes' radius of 4.24 nm, a partial specific volume of 0.703 ml/g, an intrinsic viscosity of 0.13 dl/g, a molar mass of 52.2 kDa and a frictional ratio of 1.74.     

Effect of chemical modifications of myelin basic protein on its interaction with lipid interfaces and cell fusion ability

Summary The ability of native and chemically modified myelin basic protein to induce fusion of chicken erythrocytes and to interact with lipids in monolayers at the air-water interface and liposomes was studied. Chemical modifications of myelin basic protein were performed by acetylation and succinylation: the positive charges of the native protein were blocked to an extent of about 90–95%.Cellular aggregation and fusion of erythrocytes into multinucleated cells was induced by the native myelin basic protein. This effect was diminished for both acetylated and succinylated myelin basic protein. Native myelin basic protein penetrated appreciably in sulphatide-containing lipid monolayers while lower penetration occurred in monolayers of neutral lipids. Contrary to this, both chemically modified myelin basic proteins did not show any selectivity to penetrate into interfaces of neutral or negatively charged lipids. The intrinsic fluorescence of the native and chemically modified myelin basic proteins upon interacting with liposomes constituted by dipalmitoylphosphatidycholine, glycosphingolipids, egg phosphatidic acid or dipalmitoylphosphatidyl glycerol was studied. The interaction with liposomes of anionic lipids is accompanied by a blue shift of the maximum of the native protein emission fluorescence spectrum from 346 nm to 335 nm; no shift was observed with liposomes containing neutral lipids. The acetylated and succinylated myelin basic proteins did not show changes of their emission spectra upon interacting with any of the lipids studied. The results obtained in monolayers and the fluorescence shifts indicate a lack of correlation between the ability of the modified proteins to penetrate lipid interfaces and the microenvironment sensed by the tryptophan-containing domain.Abbreviations MBP myelin basic protein - DPPC dipalmitoyl phosphatidylcholine - DPPG dipalmitoyl phosphatidylglycerol - PA phosphatidic acid     

Effect of succinylation of antibodies on their conformation and interaction with the antigen

Using succinic anhydride, a succinylated derivative of anti-urease IgG having 49 ± 6% modification was prepared and its physicochemical and immunological properties were studied. IgG undergoes substantial changes in its native conformation on succinylation, which was mainly attributed to electrostatic destabilization of the native protein conformation. The modified IgG exhibited a decrease in its cross-reactivity with urease. This decrease is attributed to the conformational change in IgG upon succinylation and/or is due to the disruption of the lysine residues in the antigen-binding site of IgG upon succinylation, which may be involved in binding the antigen. IgG was able to bind to the specific antigen although its conformation was partially modified. Therefore, partial modification of the conformation of the antigen-binding site of IgG is permissible in order to bind to the antigen. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 12, pp. 1642–1647.     

Sequential comparison of peptides containing half-cystine residues from ovalbumins of six avian species

Hen ovalbumin contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryl groups (Cys11, Cys30, Cys367, and Cys382) in a single polypeptide chain of 385 amino acid residues. To investigate whether or not such a structure is shared by related avian species, the contents of disulfide-involved half-cystine residues and their positions in the primary structure of ovalbumins from five species were compared with those of hen ovalbumin. Ovalbumins were alkylated with a fluorescent dye, IAEDANS, under disulfide-reduced and disulfide-intact conditions and digested with a number of proteolytic enzymes. The sequences were deduced from peptides containing half-cystine residues labeled with the fluorescent dye. The results showed that the number of free cysteine sulfhydryl groups of ovalbumins was different among the species, three for guinea fowl and turkey (Cys11, Cys367, and Cys382); and two for Pekin duck, mallard duck, and Emden goose (Cys11 and Cys331). On the other hand, a single intrachain disulfide bond could be identified from ovalbumins of five species using a combination of peptide mapping and N-terminal amino acid sequencing analysis under reduced and non-reduced conditions, in which the intrachain disulfide bond was like that of hen ovalbumin (Cys73-Cys120). The results also indicated that the variations in amino acid sequences on these peptides containing half-cystine residues bear a close relationship with the phylogeny of the six species.     

Acetylation of Amino Groups and Its Effect on the Structure of Soybean Glycinin

Purified soybean glycinin (11S globulin) was acetylated at three degrees; low (21-51%), middle (60-81%) and high (90-92%) acetylation in lysine residues. With increasing the acetyl content, the β-structure gradually decreased and the random structure increased resulting in the exposure of tyrosine residue. These were determined from the results of optical rotatory dispersion, intrinsic viscosity, ultraviolet and fluorescence measurement. Gel filtration, ultracentrifugation and gel electrophoresis studies showed drastic conformational changes of highly acetylated 11S (over 90%), in which most of the modified protein (75%) polymerized, and the other dissociated into 3S protein. The close relation between the conformation of acetylated 11S and its emulsifying properties was discussed.     

The role of histidyl residues in zinc-induced precipitation of alpha 2-macroglobulin-proteinase complexes

When alpha 2-macroglobulin (alpha 2M) is reacted with proteinases including trypsin, plasmin, alpha-thrombin, or with CH3NH2, each resulting alpha 2M derivative is precipitated by Zn2+ in a similar manner. By contrast, unreacted alpha 2M is not precipitated over the same Zn2+ concentration range. Zn2+-induced precipitation of alpha 2M-CH3NH2 or alpha 2M-trypsin is prevented by acylation of the protein employing the histidine-specific reagent diethylpyrocarbonate (DEP). The Zn2+-induced precipitation of alpha 2M-trypsin is prevented by acylation of the preformed alpha 2M-trypsin complex or by the reaction of acylated native alpha 2M with trypsin. Acylation of alpha 2M by treatment with DEP results in the modification of 13.5 histidyl residues per subunit of either native alpha 2M or alpha 2M-CH3NH2. Subsequent treatment with hydroxylamine reverses the modification of 10.5 histidyl residues per subunit in each protein preparation. These results indicate that histidyl residues are involved in the Zn2+-induced precipitation of alpha 2M-proteinase or alpha 2M-CH3NH2 complexes, and that these residues are accessible to extensive protein-metal interactions only after alpha 2M has undergone a major conformational change. These appear to be the same histidyl residues which, upon acylation by DEP, are responsible for recognition of alpha 2M-proteinase complexes by the acyl-low-density-lipoprotein cell surface receptor (S. V. Pizzo, P. A. Roche, S. R. Feldman, and S. L. Gonias (1986) Biochem. J. 238, 217-225).     

Digestibility of Acetylated and Succinylated Proteins by Pepsin-Pancreatin and Some Intracellular Peptidases

The molecular sizes of hydrolysates of acetylated and succinylated caseins by pepsin-pancreatin were examined by gel filtration on Sephadex G-15. There were more large peptides (average residual number > 15) in the hydrolysates of the acylated caseins than there were in the hydrolysate of unmodified casein. In these large peptides from the acylated caseins the contents of N^ε-acyl-lysines were high. The digestibility of N^ε-acetyl and N^ε-succinyl lysine bonds in peptides by aminopeptidases [(EC 3.4.11.1) and (EC 3.4.11.2)] and watermelon carboxypeptidase [model enzyme of cathepsin A (EC 3.4.16.1)] was examined using digest of acylated caseins by pepsin, trypsin and α-chymotrypsin and some synthetic peptides. All peptidases released either N^ε-acetyl or N^ε-succinyl-lysine from peptides.

The hydrolytic processes of acetylated and succinylated proteins before and after intestinal absorption are discussed.     

Interactions of native and modified cytochrome C with a negatively charged reverse micellar liquid interface

Native and chemically modified cytochrome C were dissolved in sodium bis(2-ethylhexyl) sulphosuccinate (AOT)-oil-buffer microemulsions. The native cytochrome C contains 19 lysine residues, these groups were modified by 1) acetic anhydride or 2) succinic anhydride. At pH 8.4 the native, acetylated and succinylated proteins carry +8, –3 and –12 elementary charges, respectively. The phase behaviour of the microemulsion systems was found to be highly dependent on the charge of the proteins. Compared to a protein free system the native protein induces a L-2 phase separation at lower temperatures. The acetylated protein has a small effect on the temperature for the phase transition, whereas in the case of succinylated cytochrome C the phase transition takes place at higher temperatures. When dissolved in AOT microemulsions, the native cytochrome C has a perturbed tertiary structure, as indicated by loss of the 695 nm absorption band, while both the modified proteins retain the same optical properties when dissolved in an AOT microemulsion as in a pure buffer solution. The pertubed structure of the native cytochrome C was further investigated by testing the stability of the reduced form of the protein dissolved in the microemulsion media. The native cytochrome is unstable at W > 10, whereas the two modified proteins were found to be stable at all W-values investigated. The average location of the three proteins was determined by pulse radiolysis. The quenching rate constant of the hydrated electron depends upon the location of the probe in the reverse micelle; the succinylated protein is localised in the aqueous core of the reverse micelles, but both the native and the acetylated forms were found to be localised close to or at the AOT interface.