Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 563

_____________________________ _____________________________

Protein-Surfactant Interactions

BY

ANK VALSTAR

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2000

Dissertation for the Degree of Doctor of Philosophy in Physical Chemistry presented atUppsala University in 2000

Abstract

Valstar, A. 2000. Protein-Surfactant Interactions. Acta Universitatis Upsaliensis.Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 563. 50 pp. Uppsala. ISBN 91-554-4795-3

Protein-surfactant interactions in aqueous media have been investigated. The globularproteins lysozyme and bovine serum albumin (BSA) served as model proteins. Several ionicand non-ionic surfactants were used.

Fluorescence probe measurements showed that at low sodium dodecyl sulfate(SDS) concentration (< 0.1 M) one micelle-like SDS cluster is bound to lysozyme. Fromdynamic light scattering (DLS) results it was observed that lysozyme in the complex doesnot correspond to the fully unfolded protein. At high SDS concentration (> 0.1 M) onecompact and one more extended lysozyme-SDS complex coexist.

The influence of surfactant alkyl chain length and headgroup on BSA-surfactantcomplex formation was investigated. In these studies, binding isotherms were determined bynuclear magnetic resonance (NMR), DLS was used to measure the hydrodynamic radii ofthe complexes and the size of the micelle-like aggregates on BSA was determined usingfluorescence probe methods.

It was observed from fluorescence measurements that the number of bound SDSmolecules does not depend on the presence of the disulfide bridges. Reduced proteins wrapmore efficiently around the micelle-like structures, resulting in somewhat smallercomplexes, as observed with DLS.

Concentrated BSA-SDS solutions and the corresponding heat-set gels wereinvestigated using DLS and fluorescence probe methods. Correlation lengths in the gel weredetermined and it was concluded that SDS forms micelle-like aggregates on BSA inconcentrated solution and gel phase. The gel region in the ternary phase diagram BSA-SDS-3.1 mM NaN3 has been determined at room temperature.

Ank Valstar, Department of Physical Chemistry, Uppsala University, Box 532, SE-751 21Uppsala, Sweden

Ank Valstar 2000

ISSN 1104-232XISBN 91-554-4795-3

Printed in Sweden by Akademitryck AB, Edsbruk 2000

To my mother

For Lars

List of Papers

This thesis is based on the papers listed below. They are referred to in thesummary by their Roman numerals.

I The Lysozyme-Sodium Dodecyl Sulfate System Studied byDynamic and Static Light ScatteringAnk Valstar, Wyn Brown, and Mats AlmgrenLangmuir 1999, 15, 2635

II The Interaction of Bovine Serum Albumin with SurfactantsStudied by Light ScatteringAnk Valstar, Mats Almgren, Wyn Brown and Marilena VasilescuLangmuir 2000, 16, 922

III Interactions of Globular Proteins with Surfactants Studiedwith Fluorescence Probe MethodsMarilena Vasilescu Daniel Angelesu, Mats Almgren, andAnk ValstarLangmuir 1999, 8, 2635

IV Binding of Sodium n-Alkyl Sulfates to Bovine Serum AlbuminStudied by Magnetic Resonance and Light ScatteringCécile Vigouroux, Ank Valstar, Mats Almgren, and Peter Stilbsin manuscript

V Heat-Set Bovine Serum Albumin-Sodium Dodecyl Sulfate GelsStudied by Fluorescence Probe Methods and Light ScatteringAnk Valstar, Marilena Vasilescu, and Mats Almgrenin manuscript

Reprints were made with permission of the publisher.

Contents

1 Introduction 71.1 Self-Assembly of Surfactants 91.2 Proteins 9

1.2.1 Protein Structure 91.2.2 Unfolding of Proteins 101.2.3 Lysozyme 111.2.4 BSA 12

1.3 Protein-Surfactant Interactions 121.3.1 Binding Isotherms 121.3.2 Structure of Protein-Surfactant Complexes 15

2 Experimental Techniques 172.1 Light Scattering 17

2.1.1 Light Scattering Apparatus 182.1.2 Dynamic Light Scattering 192.1.3 Static Light Scattering 212.1.4 Light Scattering on Gels 22

2.2 Fluorescence Probe Methods 232.3 Nuclear Magnetic Resonance 25

3 Results and Discussion 273.1 The Lysozyme-SDS System 273.2 The BSA-SDS System 293.3 BSA-Surfactant; The Influence of the Surfactant Headgroup

on Complex Formation 343.4 BSA-Surfactant; The Influence of the Surfactant Chain

Length on Complex Formation 363.5 Reduced Protein-SDS Systems 373.6 Ternary Phase Diagram: BSA-SDS-3.1 mM NaN3 383.7 Heat-Set BSA-SDS Gels 39

3.7.1 Heat-Induced Aggregation of BSA-SDS Complexes 403.7.2 Structure of Heat-Set BSA-SDS Gels 41

4 Concluding Remarks and Future Outlook 44

References 45

Acknowledgements 50

7

1. Introduction

The relevance of studies on protein-surfactant interactions1 comes fromthe manifold applications of protein-surfactant systems: e.g., from the foodindustry, pharmaceutical industry and analytical biochemistry.

One method used to investigate protein-surfactant interactions is thedetermination of binding isotherms at low protein concentration, which yieldsthe binding number, i.e., the number of surfactant molecules bound perprotein molecule as a function of the surfactant concentration. The techniquesused in these binding studies include equilibrium dialysis, ultrafiltration/ultracentrifugation, potentiometry, ion-selective electrodes and surfacetension.

In addition to the information available from binding isotherms,knowledge of the structure of protein-surfactant complexes is important.Knowing the structure would, for instance, contribute to an understanding ofthe sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE)technique, which is routinely used to determine the molecular weight ofproteins. Proteins saturated with SDS are observed to have electrophoreticmobilities through polyacrylamide gels that are inversely proportional to thelogarithm of the molecular weight of the protein.2 The molecular weight isdetermined by comparison of the mobility of the unknown protein with thatof a set of standard marker proteins. Apparently, SDS bound to proteinsimposes comparable shapes and net-charge densities on the protein-SDScomplexes.

This thesis is an experimental study of protein-surfactant interactions.The water-soluble, globular proteins lysozyme and bovine serum albumin(BSA) served as model proteins. The lysozyme-SDS system (papers I and III)and several BSA-surfactant systems in dilute solution have been studied(papers II-IV). In these studies, nuclear magnetic resonance (NMR) was usedto determine the binding isotherms. By using dynamic light scattering (DLS)the hydrodynamic radius of the complexes along their binding isotherms weremeasured, and static light scattering (SLS) gave access to the number ofprotein molecules in the complex. Fluorescence probe measurements wereperformed to determine the size of the micelle-like aggregates formed on theprotein.

8

The effect of different surfactant headgroups on the complex formationwas studied by DLS and fluorescence (papers II and III). The effect of thesurfactant chain length on the complex formation was investigated by DLSand NMR (papers II and IV). The role of disulfide bridges on complexformation was also examined (papers I-III).

In paper V, the gel region in the ternary phase diagram BSA-SDS-3.1mM NaN3 at room temperature and constant pressure (1 atm) wasdetermined. Furthermore, concentrated BSA-SDS solutions and thecorresponding heat-set gels were investigated by DLS and fluorescence probemethods.

9

1.1 Self-Assembly of SurfactantsSurfactants are amphiphilic molecules consisting of a hydrophilic

headgroup and a hydrophobic tail. The headgroup may be ionic, zwitterionicor polar non-ionic and the tail may consist of one or two hydrocarbon chains.The solubility of surfactants in water is low since hydrocarbon-water contactsare unfavorable. This can be understood by considering the structure ofwater: the introduction of surfactant molecules into water requires a localordering of the water molecules that surround the hydrocarbon tails.However, in the formation of micelles or aggregates, which is a spontaneousprocess, the unfavorable hydrocarbon-water contacts are minimized and theentropy of the system is increased. This phenomenon is called thehydrophobic effect.3

Micelle formation is a cooperative process that occurs over a narrowrange of concentration, where the transition from the monomeric solution toa solution containing both monomers and micelles takes place. It is customaryto define a single concentration within this narrow range as a critical micelleconcentration (cmc). The cmc is considered the saturation concentration formonomers, and further increase of surfactant concentration leads to anincrease in the number of micellar aggregates, prior to any growth of theaggregates.4

Micelles can be characterized by the aggregation number, Nagg, and thehydrocarbon chain length sets the maximum radius for the spherical micelle.For a surfactant of hydrocarbon volume ν, optimal headgroup area a0 andcritical length lc, the packing parameter, ν/a0lc, describes the tendency forsurfactant molecules to assemble into different aggregates.5 For example,spherical micelles are formed if ν/a0lc <1/3.

1.2 ProteinsBelow, a brief introduction to protein structure and protein unfolding is

given, followed by a description of lysozyme and BSA, the globular proteinsused in this thesis.

1.2.1 Protein StructureProtein structure is described in terms of four levels.6 The primary

structure is the amino acid sequence and the location of any disulfide bridges.The secondary structure refers to regular local structure of linear segments of

10

polypeptide chains, e.g., the α-helix and the β-sheet. These regular structuresare cooperative in nature. In globular proteins, the α-helix consists of 10-15residues that are held together by hydrogen bonds formed between backbonecarbonyl oxygens and backbone amide hydrogens four residues ahead in theamino acid sequence. β-sheets consist of regularly folded β-strands (3-10residues in globular proteins), which are not stable by themselves but arestabilized by hydrogen bonding in the backbone.

The secondary structure elements fold into structural units, calleddomains, which comprise the tertiary structure. The folding of the secondarystructure elements into the tertiary structure is cooperative.7 Most proteinsconsist of several domains, which are often connected only by a singlesegment of polypeptide chain. The tertiary structure is maintained by fourtypes of interaction between side chain groups of amino acid residues:7 (1)hydrogen bonding, (2) ionic interactions between oppositely charged groups(salt bridges), (3) hydrophobic interactions, and (4) disulfide cross-linkages;these covalent links are much stronger than the noncovalent interactions (1-3). Disulfide bridges are said to increase the stability of the native state byreducing the number of unfolded conformations: the greater the number ofunfolded conformations of a protein, the higher the entropic cost of foldingthat protein into its single native state.8 The domains interact to varyingextents, but less extensively than do structural elements within domains.6

Proteins containing more than one polypeptide chain exhibitquaternary structure. Each polypeptide chain in such a protein is called asubunit (or monomer). Quarternary structure refers to the spatialarrangement of the subunits.

1.2.2 Unfolding of ProteinsThe native structure is the three-dimensional arrangement of the

functional protein when it is located in its natural environment. Changes in theenvironment, e.g., a rise in temperature, variation of pH or addition ofdenaturants, may cause denaturation of the protein. When a protein isdenatured, the characteristic three-dimensional arrangement is disrupted (theprotein unfolds), and, consequently, its biological function is lost. Unfolding isan abrupt process that takes place within a limited range of conditions. Theabruptness of the unfolding transition indicates a cooperative process.

11

Depending on the denaturation process, the protein may unfold fully orpartially. Many proteins unfold fully in strong denaturants such as 6 Mguanidinium chloride or 8 M urea or at extremes of pH. These fully unfoldedproteins may be described as random-coil polypeptides.6 Reducing proteinsmay result in a partial unfolding. For example, Takeda et al.9 found that theproportions of the secondary structure of BSA change appreciably when itsdisulfide linkages are reduced. The helical content decreases from 66% to25%, the β-structure increases from 3% to 19% and the proportion ofrandom coil increases from 31% to 56%. The structural state of reducedlysozyme was found10 to be different from that of native lysozyme: helixstructure is lost and β-structure is gained upon reducing the disulfide bridges.(White10 mentioned, however, that circular dichroism (CD) measurementsperformed by others suggested the opposite: i.e., reduced lysozyme resemblesthe native conformation).

Proteins are partially unfolded by heat denaturation. For example, thenative conformation of BSA that is destroyed by heat is mainly the tertiarystructure; the secondary structure is only slightly changed.11 Also,Matsumoto et al.12 concluded that the size and shape of the protein are onlyslightly perturbed by heat denaturation. The heat-denatured protein is said tobe in a molten globule state.13 The precise structural features of this state arenot fully known, but some of the characteristics of proteins having the moltenglobule state are that the overall dimensions are only marginally greater thanthose of the fully folded state, and, furthermore, the average content ofsecondary structure is similar to that of the folded state.6

Ionic surfactants are said to initiate unfolding of the tertiary structure ofproteins.1 Changes in the secondary structure caused by binding of ionicsurfactants have also been reported.14 The surfactant-induced denaturationwill be discussed in section 1.3.

1.2.3 LysozymeLysozyme, an enzyme that dissolves certain bacteria by cleaving the

polysaccharide component of their cell walls, is a small protein with amolecular weight of 14 350 g mol-1 containing 129 amino acids and 4disulfide bridges. Its isoionic point is determined as 11.1 at ionic strength I= 0-0.1 M, and the isoelectric point as 11.0-11.35 at ionic strength I= 0.01-0.1M.15 Lysozyme has a helical content of 30%, and the content of β-sheet is

12

10%.16 The tertiary structure consists of two domains, separated by a cleftthat comprises the active site.

Lysozyme is an unusually stable protein:15 its thermal stability is highand characterized by a transition temperature of 77 0C at neutral pH anddilute salt, and no significant change of conformation over the pH range 1.2-11.3 in dilute salt at moderate temperatures has been found. Lysozyme isknown to form dimers in alkaline solution.17-19

1.2.4 BSABSA functions biologically as a carrier for fatty acid anions and other

simple amphiphiles in the bloodstream; association constants of the order of107-109 have been determined for long-chain fatty acids.20 BSA has amolecular weight of 66 411 g mol-1 (calculated from the amino acidcomposition) and consists of 583 amino acids in a single polypeptide chain.21

The protein contains 17 disulfide bridges and one free SH group, which cancause it to form covalently linked dimers.22 Formation of covalent oligomerstakes place when intermolecular disulfide bridges are formed by SH groupexchanging with saturated S-S bridges of another monomer.23 At the isoionicpoint (about pH 5.2) at which essentially all of the carboxylic acids aredeprotonated and the amino, guanidino and imidazole groups are protonated,the total charge consists of about 100 each of positive and negative charges.21

The isoelectric point in 0.15 M NaCl is about 4.7; bound chloride ions cause itto be lower than the isoionic point. BSA undergoes expansion below pH 4.3and above pH 10.5.24 The helical content is high, 68%, and the content of β-sheet is 18% (pH 7.4).25 The tertiary structure comprises three very similardomains.26

1.3 Protein-Surfactant Interactions

1.3.1 Binding IsothermsA binding isotherm (figure 1) shows the average number of surfactant

molecules bound per protein molecule (ν) as a function of the logarithm ofthe free surfactant concentration. In general,27 the isotherm of the binding ofionic surfactants to proteins displays four characteristic regions (withincreasing surfactant concentration): (I) specific binding, (II) non-cooperativebinding, (III) cooperative binding, and (IV) saturation.

13

Figure 1 Schematic plot of a binding isotherm.27

The specific binding is predominantly electrostatic:27 the headgroups ofthe surfactants bind to groups of opposite charge on the protein. A change inpH will cause a change in the net-charge of the protein and consequently inthe binding. In general, if the pH is lowered, the anionic binding isotherm isshifted to a lower surfactant concentration,28 the cationic binding isotherm toa higher concentration.29 Evidence for the selectivity of specific head groupshas been found: Reynolds et al.30 report that some of the binding sites ofBSA do not bind alkyl carboxylates (or do so with greatly diminishedaffinity), whereas both alkyl sulfates and sulfonates are strongly bound.

However, for specific binding to occur, the hydrocarbon chain lengthseems to be important: in a study of the binding of sodium n-alkyl sulfates tolysozyme (at pH 3.2)28 it was found that both sodium decyl and dodecylsulfates show specific binding, but not sodium octyl sulfate, which seems tointeract cooperatively only with lysozyme. Similar results were found for thebinding of n-alkyl trimethylammonium bromides to BSA:31 decyltrimethylammonium bromide does not exhibit a sharp transition from the specificbinding to the cooperative binding region, in contrast to the surfactants withlonger n-alkyl chains, i.e., dodecyl and tetradecyltrimethyl-ammoniumbromide. However, a study of the binding of sodium n-alkyl sulfates to

III

III

IV

log (c)

ν_

14

BSA32 showed the specific binding region for sodium dodecyl, decyl and alsofor octyl sulfates. It should be mentioned that BSA might be unusual in thisrespect, since it is capable of binding low numbers (3-10) of anionicamphiphiles with very high affinity. The number of specific binding sites wasfound to increase with hydrocarbon chain length for the interaction betweenBSA and homologous series of n-alkyl sulfates and sulfonates.30

Furthermore,28,31-33 the onset of specific binding occurs at a lower surfactantconcentration with increasing chain length.

Cooperative binding means that the binding affinity increases as moresurfactant is bound. The steep rise in the isotherm occurs over a narrowrange of free surfactant. Here, the formation of micelle-like structures ofsurfactants on the protein takes place, which is a cooperative process (likemicellization). It has been suggested that the protein unfolds in thecooperative binding region.34 Using spectroscopic techniques, e.g., circulardichroism or optical rotatory dispersion (ORD), the amount of secondarystructure along the binding isotherm can be determined. For example, a smallchange in secondary structure was found for BSA in solutions of severalanionic and cationic surfactants:14 all of these surfactants caused changes inthe helical proportion from 66% for native BSA to approximately 50% atsaturation. For the lysozyme-SDS system, however, no35 or little effect16,36

on the secondary structure of lysozyme was found. It is known, on the otherhand, that SDS may increase the helicity of proteins;35-38 this is obtained for,e.g., elastase, trypsin, and pepsin.

A wide variety of proteins (i.e., globular, fibrous, and membraneproteins) have been shown to bind identical amounts SDS at saturation: 1.4 gof SDS per gram of protein (i.e., one surfactant molecule per two amino acidresidues).39 This number is for proteins in which the disulfide bridges arereduced with reducing agents such as β-mercaptoethanol. Takagi et al.40

found membrane proteins to bind significantly higher amounts of SDS thanwater-soluble globular proteins, which was explained by the abundance ofhydrophobic amino acid residues in membrane proteins. The saturationbinding for anionic surfactants is pH-independent and seems to be controlledby the cooperative hydrophobic interactions.1 Cationic surfactants haverelatively low saturation binding as compared with anionic surfactants.1

The effect of ionic strength on the binding isotherms emphasizes thedivision into specific binding, which is largely electrostatic, and the

15

cooperative, hydrophobic binding. By increasing the ionic strength, theelectrostatic interactions are reduced and the specific binding region of theisotherm is shifted to higher free surfactant concentrations. The oppositeoccurs for the cooperative region: it is shifted to lower free surfactantconcentrations because the hydrophobic interactions are strengthened.34

Precipitation of the protein-anionic surfactant complex at pH valuesbelow the isoelectric point at low surfactant concentration is common and hasbeen reported for the lysozyme-SDS system (at pH 3.2),41 as well as for theBSA-SDS system (at pH 4.3).42 Below its isoelectric point a protein carries anet positive charge. On binding anionic surfactants the net positive charge ofthe complex is successively diminished, and the complex becomes less solubleand finally precipitates. The precipitate, however, redissolves at highersurfactant concentration due to an excess negative charge on the protein.

1.3.2 Structure of Protein-Surfactant ComplexesAs summarized by Guo et al.,43 different models have been proposed

to describe the saturated protein-SDS complex: (1) the “rod-like particlemodel,”39,44 which was proposed on the basis of viscosimetricmeasurements, describes the complex as a rigid rod with a cross-sectionalradius of about 18 Å and a length proportional to the protein molecularweight; (2) the “flexible helix model,”45 which is a theoretical model thatdescribes the complex as a flexible cylindrical micelle formed by the SDSmolecules, on the surface of which hydrophilic segments of the protein arebound; and (3) the “necklace model,”46 which is based on results from thefree-boundary electrophoresis technique and proposes an unfolded proteinwith SDS micelle-like clusters bound to it. In addition to this summary byGuo the “α-helix/random coil model”36 may be mentioned. This model isbased on CD measurements that indicate the structure of the protein ofprotein-SDS complexes is constituted of the α-helix and the random coil.However, the distribution of the SDS molecules in the complex is notspecified.

Of the models mentioned above, the necklace model seems to be bestsupported by experimental results. As a result of a small-angle neutronscattering (SANS) study, Lundahl and co-workers47 concluded that protein-decorated, spherical micelles are formed, rather than a cylindrical structure asproposed earlier.45 Other SANS studies,48,49 as well as viscometry50 and

16

NMR,51 are also compatible with the necklace model. Turro et al.,52 in astudy combining fluorescence, electron spin resonance (ESR) and NMR,concluded that the unfolded protein wraps around the micelles (figure 2). Thenecklace model is similar to the structures reported for complexes formedbetween surfactants with polymers53 and polyelectrolytes,54 respectively.

Figure 2 Protein-SDS complex as described by the necklace model, where the unfoldedprotein wraps around micelle-like clusters. 52

17

2. Experimental Techniques

2.1 Light ScatteringDynamic light scattering (DLS) reveals information on the

hydrodynamic radius Rh (through the diffusion coefficient D0 at infinitedilution) and the hydrodynamic virial coefficient kd, which characterizesthermodynamic and hydrodynamic interactions between the particles. Staticlight scattering (SLS) gives information such as the weight-averagedmolecular weight and the second virial coefficient, A2. If the particles are largecompared with the wavelength, then the radius of gyration, Rg, may also bedetermined.

Light scattering is a non-invasive method that is well suited forstudying protein systems.55,56 Proteins are usually sufficiently large to bestrong scatterers at low concentrations, and their diffusion constants aregenerally appropriate to give rise to intensity autocorrelation functions thatcan accurately be measured. Conformational changes of protein upon bindingto substrates, as well as protein-lipid interactions, have been investigated usingDLS.56 In a few studies on protein-surfactant systems, DLS was used todetermine the change of Rh of the protein as a function of surfactantbinding.57,58 Furthermore, light scattering is a very suitable technique tostudy aggregation processes, since it is very sensitive to the presence ofaggregates, even at low concentrations.

Below is a brief discussion on how to determine the various physicalparameters from light scattering experiments. More information is to befound in several textbooks55,59,60.

18

2.1.1 Light Scattering ApparatusFigure 3 shows a schematic representation of the light scattering

apparatus. In this work, a frequency-stabilized Coherent Innova Ar ion laseremitting vertically polarized light at 488 nm was used.

Figure 3 The light scattering apparatus.

The laser beam is focused at the center of the scattering cell by entranceoptics. The detection optics with the analyzer (A) defining the polarization ofthe measured scattered light, are placed on a movable arm. The detectorconsists of a photomultiplier (PMT), followed by a pulse amplifierdiscriminator (PAD). The PMT amplifies the small single-photon signal; theincoming photon is converted into an electron, which is accelerated by anelectric field onto a metal sheet. The original electron is multiplied by a factorof 105-107. The PAD converts these electrons into a pulse of proper amplitudeand duration and rejects other small pulses not originating from the scattered

L

ComputerAutocorrelator

L

A

PMT

PAD

Thermostated bath

Scatteringcell

Laser

19

light. The autocorrelator computes the normalized time-averaged intensityautocorrelation function gT

2(t).

2.1.2 Dynamic Light ScatteringIn a DLS experiment the time-averaged intensity autocorrelation

function, gT2(t), is measured. In the vast majority of cases the time-averaged

correlation function gT2(t) is equal to its ensemble average (however, see

2.1.4). The Siegert relation relates the normalized ensemble-averaged intensityautocorrelation function, gE

2(t), to the normalized ensemble-averagedautocorrelation function of the electric field, gE

1(t):

g t g tE E2 1

21 1( ) ( ) ( )− = β

where β is a factor that accounts for deviations from ideality (β≤1).If only one relaxation process is present in the system (e.g., a

monodisperse solution), gE1(t) decays single-exponentially, which is expressed

by:

g t tt

D q tEm1

2 2( ) exp( ) exp exp( ) ( )= − =−

= −Γ

τ

where Γ is the relaxation rate of the process, τ the relaxation time (τ =Γ-1, isthe time required for the single-exponential correlation function to decrease toe-1 of its initial value), and Dm is the translational mutual diffusion coefficient.The parameter q is the scattering vector: q= (4πns/λ0) sin(θ/2) where ns is therefractive index of the sample, λ0 is the wavelength of the radiation in avacuum and θ is the scattering angle.

The translational mutual diffusion coefficient contains boththermodynamic and hydrodynamic factors:

DM

N f cmA ps T p

=−

( )( )

,

13

2φ ∂π∂

where M is the molecular weight (in kg mol-1), c is the concentration of thesolute (in kg m-3), φ is the volume fraction of the solute, ƒps is the particle-

20

solvent friction coefficient (in kg s-1) and (δπ/ δc)T,p is the osmoticcompressibility in (m2 s-2). If (δπ/ δc)T,p is written as a virial expansion [withthe osmotic pressure π= RT(c/M +A2c

2 + ...)], then:

DkT

fA Mcm

ps

=−

+ +( )( )... ( )

11 2 4

2

2

φ

where k is the Boltzmann constant, T the absolute temperature and A2 is thesecond virial coefficient. The effect of concentration on the friction coefficientmay be represented by a virial expansion, ƒps= ƒ0 (1 +kƒc +...), and eq. 4 using(1 +kƒc +...)-1 =(1- kƒc +..) as c→ 0 becomes:

DkT

fA M k c

D k c

m f

d

=−

+ − +( ) =

= + +( )

( )( ) ...

... ( )

11 2

1 5

2

02

0

φ

where ƒ0 is the friction coefficient at infinite dilution, D0= kT /ƒ0 is thetranslational diffusion coefficient at infinite dilution and the hydrodynamicvirial coefficient, kd, may be expressed as:

k M A kd f= − −2 2 62 2ν ( )

where ν2 is the partial specific volume. For spheres with radius R the frictioncoefficient per particle at infinite dilution is ƒ0= 6πηR, with η the viscosity ofthe solvent. For particles with any shape the hydrodynamic radius Rh isdefined as the radius of a sphere with a solvation shell with the same D0:

DkT

Rh0 6

7=π η

( )

Equation 7 is the well-known Stokes-Einstein equation.Going from a monodisperse solution (eq. 2) to a solution containing

molecules of two different sizes, gE1(t) will be a double-exponential function. If

many relaxation processes are present, gE1(t) will be multiexponential and may

21

be expressed in terms of a distribution of relaxation times, A(τ) or as theLaplace transform of the distribution of relaxation rates G(Γ).

g t G t d A t dE1

00

8( ) ( ) exp( ) ( ) exp( / ) ln ( )= − = −∞∞

∫∫ Γ Γ Γ τ τ τ τ

In this work τA(τ) was obtained by regularized inverse Laplacetransformation (RILT) of the dynamic light scattering data using a constrainedregularization calculation algorithm called REPES.61

2.1.3 Static Light ScatteringIn a static light scattering experiment the time-averaged intensity is

measured. As it is experimentally difficult to measure absolute intensities, theRayleigh ratio, Rθ, of the excess intensity, Is =Isolution -Isolvent, is obtained byusing a reference (often toluene) with a known Rayleigh ratio Rθ,tol :62

R RI I

I

n

ntolsolution solvent

tol

solution

tolθ θ=

−

, ( )

2

9

where Rθ,tol is the Rayleigh ratio of toluene, Itol is the intensity scattered bytoluene, n and n tol are the refractive indices of the solution and toluene,respectively. If the interactions are weak and the dimensions of the scatteringsolute molecules are small compared with the wavelength of the incident light( i.e., diameter < λ/20), the Rayleigh ratio is related to the osmoticcompressibility:

Kc

R c RT MA c

T pθ

∂π∂

=

= + +

,

... ( )1 1

2 102

where the optical constant K= 4π2n02 (δn/δc)2/ λ0

4 NA, n0 is the refractive indexof the solvent and (δn/δc) is the refractive index increment. The SLS methodgives the weight-averaged molecular weight. For an uncharged macro-molecule the second virial coefficient, A2, depends on the volume of themolecule and on the nature of the solvent-solute interaction. For charged

22

macromolecules A2 depends on the same factor and, in addition, onelectrostatic interaction between the macromolecules (p 501 in reference 60).

Dialysis. SLS measurements on BSA-SDS and lysozyme-SDSsystems were performed to determine the number of protein molecules in thecomplex. Prior to the SLS measurements the protein-SDS solutions weredialyzed against a large volume of SDS solution in order to determine therefractive index increment ∂n/∂cprotein at constant chemical potential of theadded electrolytes. After dialysis, the multicomponent protein-SDS systemcan be looked upon as a two component system (i.e., the SDS-buffer solutionis regarded as the solvent for the protein), and the molecular weight of theprotein in the complex can be determined63,64 (papers I and II).

2.1.4 Light Scattering on GelsDLS on Gels. DLS measurements on heat-set protein-surfactant gels

were performed to obtain the correlation length ξ, which defines a meandistance between two points of entanglements.65 The cooperative diffusion orgel-mode diffusion is expressed as:

DkT

coop =6

11π ηξ

( )

This relation has been used frequently in polymer theory, for polymer gels.The protein-surfactant gels studied in this thesis are transparent. Thetransparency indicates a network of fine-stranded structures 13,66 formed bythe colloidal particles.

The colloidal particles are restricted by cross-links to particular regionsof the sample and are only able to execute limited Brownian motion about afixed average configuration.67 Different parts of a gel (i.e., different scatteringvolumes) are characterized by different average configurations. Thus, thetime-averaged gT

2(t) obtained from a single DLS measurement (a singlescattering volume) on a gel is not equal to its ensemble average and a gel is tobe considered as a non-ergodic medium.

The electric field of the light scattered by a gel can be written as thesum of two components,68 i.e., a fluctuating component and a constantcomponent. The fluctuating component is associated with the restricted

23

Brownian motions of the colloidal particles, and the constant field arises fromthe “frozen-in” density fluctuations or large-scale inhomogeneities.68

Different methods are described in the literature to obtain theensemble-averaged gE

1(t) of a gel.67,68 In this work the light scattering cellwas continuously rotated during the DLS measurement (1 rpm) to obtain theensemble-averaged gE

2(t). Rotating the sample places an upper limit on theobservable relaxation times. Slow modes, if present, will not be detected. Thisis a drawback of the method, which could be improved by rotating muchslower than 1 rpm. Also, one must be aware of the decay at τ ≈ 10-3 s, sincethis is not a feature of the system, but an artifact caused by rotation. The datawere analyzed using REPES61 and the subtraction technique69 (paper V).

SLS on Gels. The scattered intensity I(t)q=cst, at a particular scatteringangle, was measured while the light scattering cell was continuously rotated (1rpm). The mean value equals the ensemble-averaged intensity ⟨I(q)⟩E. Toluenewas used as a reference and normalized intensities are calculated from ⟨I(q)⟩E /Itol.

2.2 Fluorescence Probe MethodsIn the study of aggregative adsorption of surfactants on polymers and

polyelectrolytes, fluorescence quenching has been very useful to determinethe size of the micelle-like aggregates formed by the surfactants. In this thesis(papers III and V) fluorescence quenching was applied to protein-surfactantsystems. Below is a short introduction to steady-state and dynamicfluorescence measurements.

Steady-State Fluorescence. The spectra were recorded on a SPEXFluorolog 16, combined with SPEX DM3000 software. The slits were 1.5(excitation) and 0.3 mm (emission). The probe was pyrene. The ratio of thefirst over the third vibronic band, I1/I3, in the fluorescence spectrum of pyreneis a measure of the polarity of the medium in the immediate vicinity of theprobe, i.e., a measure of the micropolarity. A decrease of the polarity in theenvironment of pyrene results in a decrease of I1/I3. For example, in a polarmedium such as water the I1/I3 ratio is 1.8; in an SDS micelle it is 1.2 (paperIII).

Aggregation numbers can be determined by the steady-statefluorescence quenching (SSFQ) method, based on Turro and Yekta’stheory.70 This method is based on the assumptions that the fluorescence is

24

entirely quenched in micelles with quenchers and that the quenchers arerandomly distributed among the micelles. For micelles of uniform size, thedistribution is Poissonian.71 The aggregation number, Nagg, is determinedusing:

ln ( )I

IN

Q

S cmcaggmic0 12

=

[ ][ ] −

where I0 and I are the fluorescence intensity without and with quencher, [S]is the total surfactant concentration, cmc is the concentration of freesurfactant not incorporated in the micellar aggregates, and [Q]mic is thequencher concentration in micelles.

Time-Resolved Fluorescence Quenching (TRFQ). The time-resolvedfluorescence measurements were performed using the single photon countingtechnique. The experimental set up used was the same as described inreference 72. Pyrene was used as the probe. The decay curves of thefluorescence intensity F(t) can be described by the simple Infelta73,74-Tachiya75-77 model:

ln( )

( )exp( ) ( )

F t

Fk t n k tq0

1 130

= − + − −( )

where k0 is the first order decay constant (k0=1/τ0), τ0 is the naturalfluorescence lifetime, n is the average number of quenchers per micelle, andkq is the first order rate constant for quenching in a micelle with onequencher. It is assumed that the probes and quenchers are Poisson-distributedover the (monodisperse) micelles and that they stay in the same micellesduring the observation time period. From the n value the aggregation numberis obtained:

N nS

Qaggmic

mic

=[ ]

[ ]( )14

where [S]mic is the surfactant concentration in micelles.

25

The rate constant kq decreases with decreasing mobility of probe andquencher and with increasing size of the aggregates. Bound aggregates (e.g.,in polyelectrolyte-surfactant systems) have smaller aggregation numbers thanfree micelles, still the quenching constant is much smaller in boundaggregates. This indicates a strong reduction of the mobilities of the probeand quencher.72

2.3 Nuclear Magnetic ResonanceThe use of NMR methods to study polymer-surfactant systems has

been reviewed recently.78 In this thesis (paper IV), 1H pulse gradient spinecho (PGSE) NMR multicomponent self-diffusion measurements wereperformed on BSA-surfactant systems. From these experiments, bindingisotherms could be determined (see below).

The experiments were performed on a Bruker AMX-300WBspectrometer using a 10 mm diffusion probe with self-shielded magnetic fieldgradient coils. The experiments were based on the three-pulse stimulated-echosequence (STE). The intensity Int of the NMR signal is described by:

Int Int D gs= − −

0

2 2 2

315exp ( )∆

δγ δ

where Int0 is the intensity of the resonance in the NMR spectrum in theabsence of external gradient pulses, ∆ is the diffusion delay time, γ is themagnetogyric ratio, g and δ are the gradient amplitude and duration timerespectively, and Ds is the self-diffusion coefficient. The data were processedwith the CORE program,79,80 and both diffusion coefficients of BSA andsurfactant for a given surfactant concentration were determinedsimultaneously.

Determination of the Binding Isotherm. The average diffusioncoefficient Dobs measured in a surfactant solution above cmc (no protein)equals:

D p D p D

p p

obs mon mon mic mic

mon mic

= +

+ = 1 16( )

26

where pmon and pmic are the fractions of surfactant molecules as monomers andin micelles, respectively, and Dmon and Dmic are the associated self-diffusioncoefficients. pmon was decuded from the free monomer concentration, that wascalculated for each total surfactant concentration81 (paper IV). Below cmc,with only monomer present, Dmon can be determined since Dobs= Dmon.Knowing pmon and Dmon, Dmic can be achieved.

In a solution containing both BSA and surfactant, the averagesurfactant diffusion coefficient D’obs is given by:

′ = + ′ ′ + ′ ′

+ ′ + ′ =

D p D p D p D

p p p

obs bound bound mon mon mic mic

bound mon mic 1 17( )

pbound is the fraction of surfactant molecules bound to the protein and Dbound isthe diffusion coefficient of surfactant bound to BSA, which is identical to thatof BSA. D’mon and D’mic can be estimated from the values of Dmon and Dmic,respectively (paper IV).

Eq. 17 is valid at high surfactant concentration, with free micellespresent. In this binding model, it was assumed that p’mon=pmon, since binding tothe protein mainly affects the micelle concentration. At very low surfactantconcentration, free micelles do not exist and the surfactant is either bound tothe protein or free in solution in monomeric form. In latter case the bindingmodel is similar to eq. 16: D’obs= pboundDbound+p’monD’mon with pbound+p’mon =1.Measuring D’obs and Dbound and using eqs. 16 and 17, gives access to pbound.The binding isotherm, which is combination of the results of the two models,is determined from the values of pbound.

27

3. Results and Discussion

This chapter is a summary of the most important findings of the papers I-V.

The components used and their abbreviations are as follows:BSA bovine serum albuminC12E8 polyoxyethylene 8 lauryl etherCTAB cetyl (C16) trimethyl ammonium bromideDTAB dodecyl (C12) trimetylammonium bromideLyz lysozymeSDS sodium dodecyl (C12) sulfateSDeS sodium decyl (C10) sulfateSOS sodium octyl (C8) sulfate

3.1 The Lysozyme-SDS SystemDLS: Determination of the Hydrodynamic Radius of the Complex.

The lysozyme-SDS system was studied at two pH values, i.e., 3.3 and 9.0, atwhich the net positive charges of lysozyme are 16 and 6, respectively.Measurements of the hydrodynamic radius of the lysozyme-SDS complexalong the binding isotherm were not possible because of precipitation. Amolar ratio of SDS/lysozyme lower than ≈ 55 results in a precipitation of thecomplex. However, the hydrodynamic radius of the complex at molar ratioSDS/lysozyme between ≈ 55 and ≈ 100 was determined (the intercept infigure 4 corresponds to the diffusion coefficient Dlyz=0, SDS=0 from which Rh wascalculated using eq. 7). The complex formed at pH 9.0 (≈ 2.6 nm) is found tobe somewhat smaller than that formed at pH 3.3 (≈3.2 nm). It was suggested,also based on differences in the second virial coefficient of the complex, A2C

(see below), that the conformation of lysozyme in the complex at the two pHvalues is different. Anyhow, the hydrodynamic radius changes from ≈1.8 nmfor lysozyme to ≈2.6 nm (pH 9.0) or ≈3.2 nm (pH 3.3) for the lysozyme-SDScomplex, such a small increase is not compatible with a fully unfolded andextended lysozyme molecule.

At high SDS concentrations (> 0.1 M) two diffusional species, bothcontaining lysozyme, were detected (figure 4). It is assumed that two differentcomplexes co-exist: complex 1 is similar to that obtained at low SDSconcentrations and has a compact structure, complex 2 is a larger complex in

28

which lysozyme probably has a more open, expanded structure, presumablycaused by the binding of a greater amount of SDS. The hypothesis of a moreexpanded lysozyme molecule corresponds well with the change in theobserved ∂n/∂clyz values (figure 3, paper I).

Figure 4 The extrapolated diffusion coefficient Dlyz=0 as a function of the SDSconcentration in different buffers. The diffusion coefficients of pure SDS are included forcomparison.

Fluorescence Measurements Probably one micelle-like SDS cluster isbound to lysozyme (paper III). From the value of the pyrene lifetime, it wasconcluded that lysozyme wraps partially (i.e., much less compared with thelonger and more flexible BSA molecule, see section 3.2) around the micelle-like SDS cluster.

SLS: Determination of A2C and the Number of Lysozyme Molecules inthe Complex. All lysozyme-SDS samples were dialyzed against a largevolume of SDS solution for 24 h prior to the SLS measurements. To

29

accelerate the equilibrium process an extra amount of SDS was added to thelysozyme-SDS inner solution (paper I). It was verified that equilibrium wasreached within 24 h by determining the ∂n/∂clyz values of solutions dialyzedfor one day, one week and three weeks, respectively. The ∂n/∂clyz values werefound to be constant after one day of dialysis.82 The number of lysozymemolecules per complex does not seem to depend on the SDS concentration.The average value is 1.4 ± 0.1 at pH 3.3 and 1.0 ± 0.2 at pH 9.0 (tables 1 and3, paper I). At pH 3.3 a complex containing one lysozyme molecule isexpected, because lysozyme is predominantly monomeric at low pH. Thesecond virial coefficients of the complex, A2C, at low SDS concentration, arefound to be substantially larger at pH 9.0 compared with the values obtainedat pH 3.3 (figure 2, paper I). The increase of A2C at high pH is probably dueto electrostatic interactions (if an equal amount of bound SDS is assumed, thehigher value will reflect the higher net negative charge of the complex) andalso different conformations of lysozyme might result in differences in A2C.

3.2 The BSA-SDS SystemFluorescence Measurements: I1/I3 values and the Pyrene Fluorescence

Lifetime to Determine the Onset of Aggregation. The I1/I3 values as afunction of SDS concentration were used to monitor the onset of thecooperative binding of SDS on BSA. A low value of I1/I3 in the absence ofSDS, indicates that pyrene binds in a hydrophobic site on the protein. Theonset of the cooperative binding is recognized by a decrease of the I1/I3 value,and takes place at about the same SDS concentration (about 1 mM, with 1%BSA) independent of pH and ionic strength (figure 5; figures 1 and 2 in paperIII). At pH 5.6 a clear increase of the I1/I3 value before the start of thecooperative binding is seen, which is related to an increase of the polarity inthe immediate vicinity of pyrene and is interpreted as a more open structureof BSA.

Like the I1/I3 values, the fluorescence lifetime of pyrene can be used todetermine the onset of the cooperative binding. In the absence of SDS, thepyrene fluorescence is long-lived, since pyrene bound in hydrophobic sites ofBSA is protected from water and oxygen dissolved in water. A reduction ofthe lifetime with increasing SDS concentration indicates the start of thecooperative binding, with pyrene passing into the micelle-like clusters. The

30

lifetime in the micelle-like clusters is higher than in free micelles. Based on thisobservation, it was concluded that BSA wraps around the micellaraggregates, protecting the pyrene molecule from water and oxygen and theBSA-SDS complex was described by the necklace and bead model. The onsetof aggregation was found to be independent on pH (figure 5a, paper III).

Figure 5 I1/I3 ratio versus SDS concentration in differents buffers with 1% BSA, I=0.2 M.

Aggregation Numbers from Fluorescence Quenching. The aggregationnumbers of the micelle-like structures were determined by steady-statefluorescence quenching (table 1, paper III). Nagg increases with increasing SDSconcentration. The numbers found in different buffers with ionic strengthranging from 0.2 M to 0.6 M and pH from 5.6 to 7.4 are closely similar.Apparently, the absorbed surfactant aggregates are formed in a localenvironment that is electrostatically similar, independent of bulk ionic strengthand protein charge distribution. The number of micelle-like SDS clusters perBSA molecule varies between 3-4. At pH 7.4 and I= 0.6 M, the numbers aresomewhat lower: 2-3.

DLS: Determination of the Hydrodynamic Radius. In figure 6 therelaxation time distributions from a series of measurements at constant BSA

31

concentration and varying SDS concentration (pH 5.6, I= 0.2 M) are shown.At low SDS concentrations the distributions show one peak, which representsthe relaxation of the BSA-SDS complex. At higher SDS concentrations asecond mode at shorter relation time becomes visible, which corresponds tothe relaxation of the free SDS micelles. Figure 7 shows the correspondinghydrodynamic radii Rh of the BSA-SDS complexes plotted as a function ofthe total surfactant concentration.

Figure 6 Relaxtion time distributions for a series of measurements at constant BSAconcentration (10-5 M) and varying SDS concentration (from 0 M in the upper left corner to20 x 10-3 M in the lower right corner, see also figure7); Acetic acid buffer with pH 5.6 andI= 0.2 M

The curve in figure 7 may be compared with the binding isotherm (seesection 1.3.1). Two regions are seen: the cooperative binding region (III) andsaturation plateau (IV). Regions I (specific binding) and II (non-cooperativebinding) will not be visible since the initial binding of SDS to BSA does notresult in a detectable increase in Rh. Saturation is reached at around 7 x 10-3

M total surfactant concentration, and Rh at saturation is ≈ 5.9 nm. Neither thecooperative binding region nor the saturation plateau show pH or ionic

32

strength dependence in the conditions used (i.e., pH 5.67, I= 0.2 M and pH6.85, I= 0.1 M and I= 0.2 M; figure 6, paper IV) Saturation binding foranionic surfactant has already been found to be pH independent.1

Figure 7 Hydrodynamic radii for the BSA-SDS, BSA-DTAB, and BSA-C12E8 complexesas a function of the total surfactant concentration. The inset shows Rh for the BSA- C12E8complex at low surfactant concentration. At high C12E8 concentrations the observed Rhcorresponds to the free C12E8 micelles, since the free micelles dominate the relaxationdistribution.

According to literature data, BSA is denatured at n=50 (wheren=[SDS]/[BSA], and [SDS] is the total surfactant concentration) asdetermined by calorimetry23 or at n= 80, as determined by NMR.51 Thismeans that the tertiary structure is already unfolded at these low bindingnumbers. Very little change is observed by circular dichroism in thesecondary structure of BSA in the BSA-SDS complex at these low bindingnumbers.14 The corresponding increase in Rh is small, i.e., ≈2% for n=50 and≈10% for n=100, respectively (figure 6, paper IV). Since the change insecondary structure of BSA in the saturated BSA-SDS complex is rathersmall,14 it may be concluded from the light scattering results that the increase

33

in size, about 67% for the saturated BSA-SDS complex, is mainly caused bythe formation of micelle-like clusters on the protein.

SLS: Determination of the Number of BSA Molecules in the BSA-SDSComplex. As described in section 2.1.3, the number of BSA molecules in thecomplex can be determined. The number decreased with dialysis time andapproached unity after several weeks. (In contrast to the lysozyme-SDSsystem (section 3.1, paper I), no additional SDS was added to the innersolution to accelerate the equilibrium process). This means that very longdialysis times (several weeks) are needed to reach equilibrium. Measurementson solutions that had not reached equilibrium resulted in too high values ofthe number of BSA molecules in the complex.

NMR: Determination of the Binding Isotherm. The binding isothermsdetermined in different buffers are shown in figure 8. A rather steep increase(cooperative binding) is followed by a plateau. The saturation binding (about0.98 g of bound SDS/ g of BSA at pH 6.85, I= 0.1 M, leading to a bindingnumber n= 230, and 1.40 g of bound SDS/ g of BSA at pH 5.67, I= 0.2 Mleading to n=320) is reached at a ratio of total SDS to protein (g/g) ofapproximately 3 to 1 in both cases. The difference between the two saturationvalues might be explained by an increase of the size of the micelle-like clustersat higher ionic strength. However, the aggregation numbers of the micelle-likeclusters in different buffers with ionic strength ranging from 0.2 M to 0.6 Mwere found to be closely similar (paper III). Anyhow, a saturation value of0.98 g SDS /g of BSA is low compared with literature, where a value of 1.4 gSDS/ g of protein was found.39

34

Figure 8 Binding isotherms of BSA-SDS systems in different buffers. The fraction ofbound SDS (g) is plotted as a function of total SDS (g), the two values are normalized withrespect to BSA (g). [BSA]= 3 x 10-4 M

3.3 BSA-Surfactant: The Influence of the SurfactantHeadgroup on Complex Formation

DLS. Figure 7 shows the hydrodynamic radii Rh of different BSA-surfactant complexes plotted as a function of the total surfactantconcentration. Under the conditions used (pH 5.6), BSA is net negativelycharged. The surfactants all contain a C12 alkyl chain, but differ in theirheadgroups: negatively charged (SDS), positively charged (DTAB) and non-charged (C12E8). These curves are compared to the binding isotherm, andregions III (cooperative binding) and IV (saturation plateau) are clearly seenfor the SDS-BSA and BSA-DTAB systems. Cooperative binding of DTABoccurs at a higher concentration than for SDS. A reason3 might be becauseof the different contributions of the charged amino acid side chains to thehydrophobic interaction. The negatively charged amino acids, glutamic and

35

aspartic acid, contain two and one side-chain CH2 groups, respectively,whereas the corresponding positively charged amino acids, lysine andarginine, have four and three side-chain CH2 groups, respectively, andtherefore contribute more to the hydrophobic interaction. Saturation isreached at about the same total surfactant concentration, i.e., around 7 10-3

M. However, the hydrodynamic radius of the DTAB-BSA complex atsaturation is smaller: ≈ 4.8 nm compared with ≈ 5.9 nm for the BSA-SDScomplex, which is compatible with the finding that cationic surfactants haverelatively low saturation binding as compared with anionic surfactants.1

Because of the low cmc of C12E8, difficulties exist in the determinationof the hydrodynamic radius of the BSA-C12E8 complex (paper II). However,at low C12E8 concentrations a small increase of Rh is observed (inset in figure7), proving surfactant binding. This inital increase is similar to that observedfor the BSA-SDS system.

Fluorescence Measurements: I1/I3 values, Pyrene FluorescenceLifetime, and Aggregation numbers. SDS and C12E8 were used in thefluorescence study (paper III).

Figure 9 Pyrene fluorescence lifetime versus surfactant concentration for different BSA-surfactant systems.

36

From the dependence of the pyrene fluorescence lifetime on the surfactantconcentration (figure 9), it was deduced that the massive cooperative bindingstarts at approximately the same total surfactant concentration, irrespective ofthe headgroup. It was not possible to determine the critical aggregationconcentration (cac) from the I1/I3 values (figure 4, paper III), since thepolarities of the hydrophobic sites of the protein and the micelle-likestructures on the protein are similar. The increase of Nagg with increasing[surfactant]/[BSA] molar ratio was found to be similar.

3.4 BSA-Surfactant: The Influence of the SurfactantChain Length on Complex Formation

The effect of surfactant chain length on complex formation has beenstudied for both negatively charged surfactants i.e., SOS (C8), SDeS (C10) andSDS (C12) (paper IV) and positively charged surfactants i.e., DTAB (C12) andCTAB (C16) (paper II).

DLS. The hydrodynamic radii, Rh of the complexes formed betweenBSA and the negatively charged surfactants as a function of the totalsurfactant concentration (logarithmic scale) are shown in figure 10. The BSAconcentration is 10-5 M, pH 6.85, and ionic strength I= 0.1 M. The onset ofthe cooperative binding region occurs at a lower surfactant concentrationwith increasing alkyl chain length. This phenomenon is analog to the(cooperative) formation of free micelles, for comparison the cmc’s for SDS,SDeS and SOS in 0.1 M NaCl solution are 1.47, 13.5 mM and 102 mM,respectively.83

The BSA-SDS and BSA-SDeS systems show a saturation region. Rh iscalculated using eq. 7, with D0≈ Dm (i.e., kdc<< 1). The Rh of the saturatedBSA-SDS complex (6.3±0.1 nm) is smaller than that of the saturated BSA-SDeS complex (5.72±0.02 nm). The difference in size is probably caused by adifference in binding number. This may be compared with the aggregationnumber of free micelles which is 93 for SDS and 52 for SDeS, respectively in0.1 M NaCl solution.83 A saturation region is not observed for the BSA-SOSsystem. The concentration of SOS ranges from 20 mM to 160 mM. At thehigher surfactant concentrations, the particles are no longer non-interacting (i.e., kdc>1) and the Rh calculated from Dm is an apparent Rh, and,consequently, a saturation region is not observed.

37

Figure 10 Hydrodynamic radii for the BSA-SOS, BSA-SDeS, and BSA-SDS complexesas a function of the total surfactant concentration at pH 6.85 and I= 0.1 M. [BSA]= 10-5 M.

Just as for DTAB, a cooperative binding region is also observed forCTAB (figure 7, paper II). Since CTAB is more hydrophobic, the complexformation starts at a much lower surfactant concentration. Again, due to therather low cmc of CTAB, difficulties arise when determining Rh of thecomplex (paper II).

3.5 Reduced Protein-SDS SystemsFirst, solutions of reduced proteins in the absence of SDS are

considered. DLS on solutions of reduced lysozyme revealed Rh to be slightlyless than that of the native protein (figure 1b, paper I), which indicates thatreduced lysozyme is compact and probably has secondary structure.Reducing lysozyme causes aggregation. This has also been found for BSA(figure 8, paper II), which aggregated to such an extent that the monomericform of BSA was not observed.

For both lysozyme (figure 4) and BSA (figure 9, paper II), it has beenfound that the complexes formed between the reduced proteins and SDS aresmaller compared with the corresponding complexes formed with the native

38

proteins. It was suggested that the different values of A2C for the reducedlysozyme-SDS complex and the native lysozyme-SDS complex, might becaused by different conformations of lysozyme (paper I). Fluorescencemeasurements on the BSA-SDS system showed that the number of boundSDS molecules does not depend on the presence of the disulfide bridges(paper III). The results indicate that the reduced proteins wrap more efficientlyaround the micelle(s), which results in the somewhat smaller complexes. Thisis in agreement with results from fluorescence anisotropy decaymeasurements,84 where reduced BSA-SDS complexes were found to haveshorter rotational correlation times compared with native BSA-SDScomplexes (BSA was labeled with dansyl). Shorter correlation timescorrespond to smaller volumes, i.e., the reduced BSA-SDS complex is smallerthan the native BSA-SDS complex.

3.6 Ternary phase diagram: BSA-SDS-3.1 mM NaN3

Determination of the gel region. The phase equilibria of the BSA-SDS-3.1 mM NaN3 system at room temperature are represented as atriangular phase diagram in figure 11. The samples have equilibrated for atime period of about one month. A gel phase is surrounded by a solutionphase and a region where gel and solid (i.e., undissolved material) coexist. Thesolution phase has also been observed by Morén et al.85 Solutions close to theborder of the gel phase are highly viscous. The time required to form the gelis composition-dependent, it may take hours (high BSA concentration) orweeks (relatively low BSA concentration). Solutions containing large amountsof BSA are yellowish. The gel phase is transparent and also yellowish inappearance.

Concentrated BSA solutions without SDS in the presence of 3.1 mMNaN3 do not gel at room temperature within a time period of months.Aqueous solutions of BSA have pH values above the isoelectric point, andBSA carries a net negative charge. Apparently, electrostatic repulsionsprevent BSA from extensive aggregation and gel formation. However, whena certain amount of SDS is added, gel formation may occur. The binding ofSDS to BSA changes the tertiary11 and, to some extent, the secondary14

structure of BSA. Internal hydrophobic groups might become exposed and

39

LG

10 20 30 40 50

BSA

SDS

60

70

80

90

100

10

20

30

40

50

3.1

mM

NaN

3

Figure 11 Phase diagram for the BSA-SDS-3.1 mM NaN3 system at room temperature:open circles, solution; solid circles, gel; and crosses, gel and solid

aggregation occurs.13 Also, the possibility of the formation of intermoleculardisulfide bridges increases when internal groups are exposed (it was shownthat intermolecular disulfide bridges are involved in gel formation, paper V).The gel phase is transparent, which indicates a network consisting of fine-stranded structures.13,66 Generally, the formation of fine-stranded structuresis found in systems where the degree of screening of the electrostaticrepulsions is low (in contrast to random clumping at high screening). Bindingof negatively charged SDS increases the surface charge density of BSA. Gelsare formed using as much as 26 wt% SDS (≈0.9 M), which introduces asubstantial amount of counterions. Nonetheless, clear gels are formedindicating filamentous structures. Gels formed at low BSA and high SDSconcentration (e.g., 8 wt% BSA, 27 wt% SDS, [SDS]/[BSA]= 775) probablycontain free micelles, i.e., micelles that are not bound to BSA.

3.7 Heat-Set BSA-SDS GelsHeat-set gels were studied in the concentration range of 9-14 wt%

BSA and 0.4-18 wt% SDS, i.e., [SDS]/[BSA]= 10-425 in 3.1 mM NaN3

40

solution. The tertiary structure of a protein is destroyed by heat, as well as bythe binding of SDS.11 When about 50 SDS molecules are bound to BSA, thetertiary structure that could be destroyed by heat is already destroyed bySDS. A smaller number of bound SDS molecules results in a partiallyunfolded tertiary structure only, and heat will destroy the tertiary structurefurther.11 Heating of BSA-SDS solutions with molar ratio ≥ 50 causes anacceleration of aggregation and gel formation. Without heating, gel formationis much slower, e.g., a solution containing 14 wt% BSA, [SDS]/[BSA]= 100gelled in about 7 weeks (see also section 3.6). Before describing the structureof the heat-set gels (3.7.2), heat-induced aggregation of BSA-SDS complexesat low concentration is discussed.

3.7.1 Heat-Induced Aggregation of BSA-SDS ComplexesHeat-induced aggregation of BSA and BSA-SDS complexes was

studied with DLS and the hydrodynamic radii of the aggregates versustemperature are shown in figure 8, paper IV. The BSA concentration was 10-5

M, and the molar ratio [BSA]/[SDS] varied from 10 to 50 at pH 6.85, ionicstrength 0.1 M. Rh of BSA is constant in the temperature range 25-55 0C. At60 0C a small increase of Rh, corresponding to the denaturation86 of theprotein is seen. At higher temperatures a large increase of Rh is observed,together with a large increase (tenfold) of the intensity of the scattered light,which indicates aggregation.

At low [BSA]/[SDS] ratios (i.e., 10 and 20), BSA resists thermaldenaturation.11 Rh starts to increase at temperatures of ~ 75-80 0C. The initialincrease is due to denaturation of BSA. Since the tertiary structure of BSA isnot totally unfolded at n= 10 or 20, heat will destroy the tertiary structurefurther.11 Aggregation of the BSA-SDS complexes causes a further increasein Rh at higher temperatures. The slower increase of Rh (i.e., sloweraggregation) of the [BSA]/[SDS]= 20 system can be explained by the highernet-negative charge on the BSA-SDS complex. For the [BSA]/[SDS]= 50system a small increase in Rh is observed at higher temperatures. However,the tertiary structure of BSA is already destroyed by binding of 50 SDSmolecules, and the increase is thus due to aggregation. Since the complexesare negatively charged the aggregation is relatively slow.

41

3.7.2 Structure of Heat-Set BSA-SDS GelsThe gels obtained by heating concentrated BSA solutions are opaque,

which indicates that the aggregation under the conditions used can bedescribed by random clumping.13 Heating solutions containing both BSA(10-14 wt%) and SDS (0.4-18 wt%) however, resulted in the formation oftransparent gels. Solutions containing large amounts of BSA are yellowish,and, also, the transparent gels are yellowish in appearance. The transparencyof the gels indicates a network of fine-stranded structures.13,66 It was shownthat intermolecular disulfide bridges are involved in gel formation. Bothsolution and the corresponding gel were studied.

Aggregation Numbers from Fluorescence Quenching. A family ofdecay curves of the fluorescence intensity F(t) recorded with differentquencher concentrations in the sample, gives directly the important qualitativeinformation that the solution (or gel) contains finite size aggregates (figure 3,paper V). The quantitative estimate of the aggregate size within theInfelta73,74-Tachiya75-77 model is determined, using eqs. 13 and 14. Theaggregation numbers obtained for the solutions are similar to those for thecorresponding heat-set gels (table 1, paper V). When the quenching in themicelles is fast (kqτ0 >>1),87 static quenching measurement according to Turroand Yekta70 can be used. An increase of Nagg with increasing molar ratio isobserved. The aggregation numbers are smaller compared with the Nagg

values found for 1% BSA with similar molar ratio, which is reasonable sincein the latter system a high ionic strength (I= 0.2 M) was used (paper III).

Pyrene Fluoresence Lifetime and I1/I3 values. The variation of thefluorescence lifetime of pyrene as function of the [SDS]/[BSA] molar ratio inaerated samples is shown in figure 12. Dilute, concentrated and gel samplesare compared. An increase of the molar ratio results in a reduction of thelifetime for solution and gel, and at high molar ratio the value in free SDSmicelles is approached. In the absence of SDS, the pyrene fluorescence islong-lived, since pyrene bound in hydrophobic sites of BSA is well shieldedfrom water and dissolved oxygen. From the extent of the τ0 values and thesame dependence of its values on [SDS]/[BSA] molar ratio, it can beconcluded that the structure of the clusters formed in solution and gel phaseare similar, i.e., micelle-like. The I1/I3 values support this conclusion (table 1,paper V). The values before and after gelation are similar. When pyrene issolubilized in micelle-like clusters, the I1/I3 values are somewhat lower, both in

42

solution and gel, than the value obtained for free micelles. This means that thepolarity sensed by pyrene in micelle-like clusters is lower than in micelles.

Figure 12 Fluorescence lifetime dependence on [SDS]/[BSA] molar ratio.

DLS: Determination of the correlation length ξ. The correlationfunctions of the transparent BSA-SDS gels are all characterized by an initialfast decay. This decay is not seen for the opaque BSA gel (figure 5, paper V).The angular dependence of the relaxation time of the fast mode shows thatthis mode is diffusive. The cooperative diffusion coefficients Dcoop of the BSA-SDS gels were calculated from Dcoop= 1/(τ q2). The cooperative diffusion, orgel-mode diffusion, is related to the limited Brownian motion of the colloidalparticles. The correlation length ξ (eq. 11) defines a mean distance betweentwo points of entanglements.65 This relation has been used frequently inpolymer theory for polymer gels. Here, the transparent gel can be describedas a network of fine-stranded structures formed by the colloidal particles.From fluorescence measurements it was obtained that SDS forms micelle-likeclusters on BSA in the gel. Thus, the colloidal particles forming the networkare BSA-SDS complexes.

43

For the 14 wt% BSA gels, a decrease in ξ is observed with increasing[SDS]/[BSA] molar ratio [i.e., ξ decreased from 0.9 nm to 0.8 nm, when themolar ratio increased from 10 to 200 (figure 6, paper V)]. At higher molarratios more SDS is bound to BSA, resulting in a larger complex and adecrease of the mean distance between two points of entanglements, ξ. Also,the micelle-like structures might act as junction zones between different BSAmolecules; this cross-linking would give rise to smaller ξ values. This kind ofcross-link was suggested for a non-ionic polymer/ cationic surfactantsystem.88

SLS: Qualitative Estimation of Inhomogeneities. Clear differencesbetween BSA-SDS gels and BSA gels concerning the presence of large-scaleinhomogeneities are observed using SLS. First, the normalized scatteredintensity I(t)q=cst / Itol of the opaque BSA gel showed strong fluctuations, whenthe sample was rotated during the measurement. This indicates the presenceof inhomogeneities. For BSA-SDS gels no strong fluctuations were observed(figure 7, paper V). Second, the decrease of ⟨I(q)⟩E / Itol with increasing[SDS]/[BSA] molar ratio at constant [BSA], implies a decrease in thenumber and size of inhomogeneities. Furthermore, with increasing[SDS]/[BSA] molar ratio, a decrease in the angular dependence of ⟨I(q)⟩E / Itol

is seen. At high [SDS]/[BSA] ratios ⟨I(q)⟩E / Itol depends only slightly on q,but in the absence of SDS the q dependence is pronounced. This indicates thepresence of inhomogeneities with a spatial extent comparable to thewavelength of light, i.e., several hundred nanometers (figure 8, paper V).Finally, the values of ⟨I(q)⟩E / Itol of the solutions and corresponding gels maybe compared. The intensity of the light scattered by a BSA gel is much higher(about 10 times at θ= 900) compared with the corresponding solution, whichindicates the presence of inhomogeneities in the gel. The difference betweenthe scattered intensity of a gel and that of the corresponding solutiondecreases with increasing [SDS]/[BSA] molar ratio. At higher molar ratiosonly small differences are seen.

44

4 Concluding Remarks and Future Outlook

The main part of this thesis concerns the investigation of severalprotein-surfactant systems in dilute solutions. Binding isotherms,hydrodynamic radii of the complexes and the size of the micelle-likesurfactant aggregates formed on the protein were determined. A study ofmore concentrated BSA-SDS solutions was initiated (paper V). The gel regionin the ternary phase diagram was determined, and concentrated solutions aswell as the corresponding heat-set gels were investigated using DLS andfluorescence probe methods. Correlation lengths in the gel were found to bein the order of 1 nm, and it was concluded that SDS, as in dilute solution,forms micelle-like aggregates on BSA in concentrated solution and gel phase.

Transparent heat-set BSA gels (no SDS) are found to consist of anetwork of linear polymers.89,90 In this thesis it was proposed, based on thetransparent appearance of the heat-set BSA-SDS gels, that also these gels canbe described by a network of fine-stranded structures. The DLS results wereexplained using this interpretation. It is clear that a further investigation of thegel structure is needed. Similar to studies concerning protein gels, microscopictechniques might give structural information.89,91 Furthermore, by examiningthe heat-induced aggregation of BSA-SDS complexes one could obtaininformation on the structure of the aggregates formed (before gelation).Various scattering techniques, i.e., light, neutron and X-ray scattering wouldbe suitable for these investigations. Concentrated BSA-SDS solutions and thecorresponding heat-set gels using NMR methods are currently underinvestigation. Rheology has frequently been used in studies concerningprotein gels, but seems not to be used for protein-surfactant systems.Knowledge of the rheological properties of protein-surfactant systems as afunction of temperature would increase the understanding of these systems.

45

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50

Acknowledgements

I feel privileged to have been able to complete my Ph.D. studies with MatsAlmgren as supervisor; his support, interest, and criticism and have been veryvaluable.

Wyn Brown is acknowledged for providing the opportunity to start my Ph.D.studies.

I owe special gratitude to my co-authors: Marilena Vasilescu, for kindlyinvolving me in her research, and Cécile Vigouroux, for making a real effortwhen time was running short.

I would also like to thank: Ignasi Velázquez for initiating the MD project andGit Roxström for her help continuing it; Tomas Edvinsson for his enthusiasticinterest, always taking time discussing light scattering matters; ChristerElvingson for help with statistical thermodynamic problems; Göran Svenskfor performing sedimentation measurements and good help in various ways;Göran Karlsson for performing cryo-TEM measurements; Bob Johnsen forlight scattering and computer advise; Margit Ejerblad, Laila Bodbacka-Fältman, Sven Centring, Gösta Person, and Kjell Barmark for their kind helpin various ways.

Maxim Kuil, my supervisor at Leiden University when I was anundergraduate student, and Frans Varkevisser are much appreciated for theirencouragement especially at the beginning of my Ph.D. studies. It was verynice receiving telephone calls from Leiden!

I would like to thank my relatives and friends back home in Holland, for notforgetting me and sending letters.

I am much grateful to my mother for her encouragement and belief in me.

Finally, I wish to thank Lars, my best friend, for always being there for me.

Uppsala, August 2000

Ank Valstar