dynamics of protein and mixed protein/surfactant adsorption layers at the water/fluid interface

Advances in Colloid and Interface ScienceŽ .86 2000 39]82

Dynamics of protein and mixedproteinrsurfactant adsorption layers at the

waterrfluid interface

R. Miller a,U, V.B. Fainermanb, A.V. Makievskia,b,J. Kragela, D.O. Grigoriev a,c, V.N. Kazakovd,¨

O.V. Sinyachenkod

aMPI fur Kolloid- und Grenzflachenforschung, Max Planck Campus, D-14476 Golm, Germany¨ ¨bInternational Medical Physicochemical Centre and Institute of Technical Ecology,

16 Ilych A¨enue, Donetsk 340003, UkrainecInstitute of Chemistry, St. Petersburg State Uni ersity, Uni ersitetskiy Pr. 2, 1989084

St. Petersburg, RussiadDonetsk Medical Uni ersity, 16 Ilych A¨enue, Donetsk 340003, Ukraine

Abstract

The adsorption behaviour of proteins and systems mixed with surfactants of differentnature is described. In the absence of surfactants the proteins mainly adsorb in a diffusioncontrolled manner. Due to lack of quantitative models the experimental results are dis-cussed partly qualitatively. There are different types of interaction between proteins andsurfactant molecules. These interactions lead to proteinrsurfactant complexes the surfaceactivity and conformation of which are different from those of the pure protein. Complexesformed with ionic surfactants via electrostatic interaction have usually a higher surfaceactivity, which becomes evident from the more than additive surface pressure increase. Thepresence of only small amounts of ionic surfactants can significantly modify the structure ofadsorbed proteins. With increasing amounts of ionic surfactants, however, an opposite effectis reached as due to hydrophobic interaction and the complexes become less surface activeand can be displaced from the interface due to competitive adsorption. In the presence ofnon-ionic surfactants the adsorption layer is mainly formed by competitive adsorptionbetween the compounds and the only interaction is of hydrophobic nature. Such complexesare typically less surface active than the pure protein. From a certain surfactant concentra-

U Corresponding author. Tel.: q49-331-5679252; fax: q49-331-5679202.Ž .E-mail address: miller@mpikg golm.mpg.de R. Miller .}

0001-8686r00r$ - see front matter Q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S 0 0 0 1 - 8 6 8 6 0 0 0 0 0 3 2 - 4

( )R. Miller et al. r Ad¨ances in Colloid and Interface Science 86 2000 39]8240

tion of the interface is covered almost exclusively by the non-ionic surfactant. Mixed layersof proteins and lipids formed by penetration at the waterrair or by competitive adsorptionat the waterrchloroform interface are formed such that at a certain pressure the compo-nents start to separate. Using Brewster angle microscopy in penetration experiments ofproteins into lipid monolayers this interfacial separation can be visualised. A brief compar-ison of the protein adsorption at the waterrair and waterrn-tetradecane shows that theadsorbed amount at the waterroil interface is much stronger and the change in interfacialtension much larger than at the waterrair interface. Also some experimental data on thedilational elasticity of proteins at both interfaces measured by a transient relaxationtechnique are discussed on the basis of the derived thermodynamic model. As a fastdeveloping field of application the use of surface tensiometry and rheometry of mixedproteinrsurfactant mixed layers is demonstrated as a new tool in the diagnostics of variousdiseases and for monitoring the progress of therapies. Q 2000 Elsevier Science B.V. Allrights reserved.

Keywords: Mixed adsorption layers; Liquidrfluid interfaces; Adsorption kinetics; Penetration kinetics;Proteinrsurfactant mixtures; Proteinrlipid mixtures; Dynamic interfacial tensions; Surface viscoelastic-ity; Brewster angle microscopy; Maximum bubble pressure tensiometry; ADSA

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402. State-of-the-art theoretical models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2.1. Thermodynamic models for protein layers . . . . . . . . . . . . . . . . . . . . . . . . 422.2. Thermodynamic model for mixed proteinrsurfactant layers . . . . . . . . . . . . . 452.3. Fundamentals of adsorption kinetics at a liquid interface . . . . . . . . . . . . . . 492.4. Adsorption kinetics modelling for protein systems . . . . . . . . . . . . . . . . . . . 50

3. Adsorption kinetics of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534. Simultaneous adsorption from mixed aqueous solutions . . . . . . . . . . . . . . . . . . . 575. Proteinrlipid adsorption from two separate solutions at the joint interface . . . . . . 616. Penetration of proteins into lipid layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647. Protein adsorption at waterroil interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . 648. Peculiarities in the protein adsorption at drop and bubble surfaces . . . . . . . . . . . 669. Dilational elasticity of a protein adsorption layer . . . . . . . . . . . . . . . . . . . . . . . 69

10. Surface tension and elasticity of blood serum as example of a natural proteinrsurfac-tant mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

11. Summary, conclusions, outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78Notation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

1. Introduction

Proteinrsurfactant mixtures are widely used in many technologies, for examplew xfor the stabilisation of emulsions and foams in the food industry 1 or for coating

( )R. Miller et al. r Ad¨ances in Colloid and Interface Science 86 2000 39]82 41

w xprocesses in the photographic industry 2 . Mixtures of proteins and surfactants arealso common to all biological systems. For example, blood serum is a mixture ofhuman serum albumin with a number of compounds, including low-molecularsurface-active molecules. The dynamic surface tension of such biological fluids isused as a diagnostic and therapeutic tool, for example for renal diseases and other

w xpathologies 3 . This implies the scientific and practical importance of studies onthe adsorption behaviour and its dynamics of mixed proteinrsurfactant systemswhich are intensively studied by many groups using various experimental tech-

w xniques 4]12 .However, only very few systematic studies dealing with this problem have been

published so far. The effect of low molecular non-ionic surfactants, such asalcohols, acids and oxyethylated ethers, on the adsorption dynamics of a proteindepends on their concentration and surface activity and on the solution conditions,such as temperature, ionic strength and pH.

Non-ionic surfactants, for example the highly surface-active oxyethylatedw xTWEEN 20 13 , produce almost no effect on the surface tension of b-lactoglobu-

lin solution at short adsorption times, but leads to a significant decrease of theequilibrium surface tension. One can assume that these types of surfactants adsorbcompetitively with the protein. However, no definite predictions can be madeconcerning the effect of ionic surfactants on the adsorption dynamics because inthis case the inter-ion interaction between surfactants and proteins can result in

w xthe formation of proteinrsurfactant complexes 14 . For very small added amountsŽ .of ionic surfactants 100 times lower than the protein concentration even a small

increase in the surface tension can be observed. For larger amounts of the samesurfactant the surface or interfacial tension generally decreases. In certain concen-tration regions for some proteins an anomalous adsorption behaviour was observedw x15 .

In the present review mainly experimental studies of the adsorption behaviour ofmixtures of various proteins with different ionic and non-ionic surfactant are

Ž .shown. As proteins the most frequently studied human serum albumin HSA ,Ž . Ž . Ž .bovine serum albumin BSA , b-casein b-CS and b-lactoglobulin b-LG are

chosen. The surfactants on which the analysis is focussed here are selected suchthat their surface activity is comparable. On the basis of a qualitative analysis,schematic pictures for the different types of adsorption layers are drawn. First thestate-of-the-art of the theoretical description is reviewed, where mainly models forpure proteins are given. Then the kinetics of adsorption of proteins in the absenceand presence of surfactants are described in Sections 3 and 4. Sections 5 and 6focus on proteinrlipid systems and competitive as well as penetration systems arereviewed. Results for the adsorption of proteins at the waterroil interface andpeculiarities for studies at drops and bubbles are given in Sections 7 and 8, whileSection 9 deals with the dilational rheology of protein layers. Section 10 isdedicated to an interesting new field of research, the cross-linking of surfacescience and medicine, where fundamental knowledge on mixed proteinrsurfactantlayers at a liquid interface is required to use measured interfacial phenomena as a


tool of medical diagnostics. Ideas on the structure of proteinrsurfactant complexesas inspiration for future experimental and theoretical studies to describe suchmixed systems are given in Section 11.

2. State-of-the-art theoretical models

The practical importance of the adsorption of proteins at fluid interfacesstimulated the development of different models under equilibrium and dynamic

w xconditions as demonstrated in recent reviews 16]18 . Besides the statisticalw xtheories and scaling models of de Gennes 19 , for example, there are many

w xthermodynamic models which are based on the Butler equation 20 for thechemical potentials of the components in the bulk phase and at the interface as the

w xstarting point. Examples for such models are those of Joos 21 , Ter-Minassian-w x w xSaraga 22 and Lucassen-Reynders 23 which describe various details of adsorp-

tion layers at interfaces.

2.1. Thermodynamic models for protein layers

The Butler equation is suitable for describing the specific demand, v, ofdifferent adsorbed molecules at an interface. For surfactant solutions this can beexpressed by the adsorption relation of Joos which is the quantitative equivalent

w xfor an interface of the principle of Braun]Le Chatelier 24 . This adsorptionrelation says that when an adsorbed molecule may occupy different parts of theinterface, then at small surface pressures, P, a maximum molar surface area, v, isoccupied, whereas minimum v is achieved at large P. This means that theadsorption layer thickness increases with increasing protein concentration. Anillustration of the idea is given schematically in Fig. 1.

With increasing surface coverage, i.e. stronger competition between the adsor-bed protein molecules, their molar area becomes smaller until finally a minimumarea is reached. A thermodynamic model has been recently derived by Fainerman

w xet al. 25 using the Butler equation as starting point, the results of which can besummarised as follows.

The equation of state of the adsorption layer as dependence of surface pressureŽP s g y g on surface concentration reads g and g are the surface tensions ofo o

.the solvent and solution, respectively

RTŽ . Ž .P s y ln 1 y G v 1S SvS

n

where the total adsorption is defined by G s G , and the average molar area isÝS iis1

given by


Ž .Fig. 1. Idea of the protein adsorption model: a schematic of the conformational changes of adsorbedŽ .protein molecules; b molar area occupied by one adsorbed molecule; the states 1]4 correspond to

increasing surface pressure P or surface coverage G , i.e. decreasing average molar surface area v .S S

nŽaq1. w Ž . Ž .xG v i exp y iPv r RTÝ Ýi i 1

iG1 is1 Ž .v s s v 2nS 1aGÝ i w Ž . Ž .xi exp y iPv r RTÝ 1

iG1 is1

The total adsorption G can be expressed by the adsorption in the state ofS

minimum partial molar surface area G , the adsorption in the ith state G by the1 itotal adsorption GS

n Ž .i y 1 Pv1a Ž .G s G i exp y 3ÝS 1 RTis1

a � wŽ . x w x4i exp y i y 1 Pv r RT1 Ž .G s G 4ni Sa � wŽ . x w x4i exp y i y 1 Pv r RTÝ 1

is1

The adsorption isotherm as dependence of surface pressure P on protein bulkconcentration, c, reads

w Ž . Ž .x1 y exp y Pv r RTS Ž .b c s 5n1a w Ž . Ž .xi exp y iPv r RTÝ 1

is1

Here a is a constant which determines the variation in surface activity of theprotein molecule in the ith state with respect to the state 1 characterised by a


minimum partial molar area v s v , b s b ia. The value i can be either1 min i 1integer or fractional and the increment is defined by D i s Dvrv . For a s 0 one1obtains b s b s const., while for a ) 0 the b increases with increasing v . Thei 1 i i

Ž . Ž .system of Eqs. 1 ] 5 completely describes an ideal adsorption layer of proteins.Ž .For a non-ideal adsorption layer instead of Eq. 1 we obtain the equation of

Žstate containing a term representing the interaction with the parameter a while itis assumed that molecules in all conformations have the same interaction be-

.haviour . Taking into account a non-ideality adsorption layer and the contributionŽ .of the DEL one can transform the equation of state for the surface layer 1 into

w xthe following relationship 26

RT 4RT2 1r2Ž . Ž . Ž . w x Ž .P s y ln 1 y G v q a G v q 2« RTc chw y 1 6S S S S Sv FS

where F is the Faraday constant, z is the number of non-bound unit charges in theprotein molecule, w s zFc0r2 RT , c0 is the electric potential of the surface, « isthe dielectric permittivity of the medium and c is the total concentration of ionsS

within the solution. For protein solutions at high ion concentrations, the DebyeŽ 2 .1r2length l s « RTrF c is small. This means that for protein solutions the DELS

thickness can be smaller than the adsorption layer thickness. Therefore, theŽ .concentration of ions in Eq. 6 is just their concentration within the adsorption

Ž .layer. It follows from Eq. 6 that for large c the approximation w < 1 can beS

used. Thus, one obtains an equation of state for non-ideally charged surface layersw xof a protein 26

RT 2Ž . Ž .Ž . Ž .P s y ln 1 y G v q a y a G v 7S S el S SvS

2 Ž .1r2 Ž .where a s z Frv 8« RTc , and for the adsorption isotherm of Eq. 5 weel S S

now get

� 2 2 w Ž . x Ž .G v exp yaG v i v rv y 1 y i v rv y 2 aG vi S S S 1 S 1 S S S

2Ž . wŽ . xq2 a rz G v q a rz G v 5el S S el S S Ž .b c s 81 iv rvi Sa Ž .i 1 y G vS S

w xThe simplifications discussed by Fainerman and Miller 26 , a 4 a, a rz f ya,el elw Ž .xallow to simplify the equation of state for protein surface layers Eq. 7 and the

w Ž .xadsorption isotherm equation Eq. 8 to

RT2 2Ž . Ž .P s y ln 1 y G v y a G v 9S S el S SvS


G v1 S Ž .b c s 101 v rv1 SŽ .1 y G vS S

The total adsorbed amount in these equations can be expressed via the adsorptionin state 1

n Ž . Ž .v i y 1 i y 1 Pv1 1a Ž .G s G i exp exp y 11ÝS 1 v RTSis1

where the first exponential factor arises due to the non-ideality of entropy ofmixing.

Ž .Knowing the model parameters v s v , Dv, n s 1 q v y v rDv, a ,1 min max minŽ . Ž .a and b the two dependencies P c and P G result, and hence the importantel 1

surface pressurerbulk concentration isotherm can be obtained and compared toexperimental data, as is shown in Fig. 2 for the proteins under discussion here. Theexperimental data are described by the thermodynamic model using the parame-ters given in Table 1.

As one can see, the theory describes the experimental data properly and onlyabove a certain critical concentration is there a deviation which shows the limits of

Ž . Ž .the model. For P G 20]25 mNrm the theoretical model given by Eqs. 9 ] 11predicts an unrealistically sharp increase of surface pressure with a small increaseof protein concentration, and simultaneously a slight increase in adsorption. Thiscontradicts the experimental data which show that, starting from some proteinconcentration, the P value remains almost constant, while the adsorption contin-ues to increase. This leads to an increased coverage up to an almost completesaturation of the adsorption layer at high protein concentration. The differencebetween the model and the experimental data could be attributed to the formationof a second adsorption layer. The fact that the surface pressure of concentratedprotein solutions is independent of the bulk concentration can also be satisfactorilyexplained in the framework of a monolayer model, considering a weakening of the

w xinter-ion interactions in concentrated monolayers 26 or the possibility of two-w xdimensional aggregation of adsorbed protein molecules 27 .

2.2. Thermodynamic model for mixed protein r surfactant layers

There are no thermodynamic models for mixtures of a protein with a surfactantin the literature so far. Let us derive here, however, a simple but quantitativemodel for such systems, using the assumption of an ideal layer with respect to theadsorption enthalpy and the same starting point as above for a protein solution

Ž .without any surfactant. For i different surfactants or proteins which exist in jdifferent states at the interface the following very general equations of state

RTŽ .P s y ln 1 y v G 12ÝS i jvS i , j

()

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Table 1w Ž . Ž . Ž . Ž .xIsotherm parameters of some selected proteins with respect to Eqs. 2 ] 4 , 6 and 7

Protein v v Dv b a Molecularmax min el2 2 2Ž . Ž . Ž . Ž . Ž .nm rmolecule nm rmolecule nm rmolecule lrmol r weight

y8BSA, HSA 80 " 20 40 " 10 40 " 20 2.8 = 10 180 " 40 69 000y8 aŽ .6.5 = 10

y8b-LG 20 " 5 6 " 2 1.0 " 0.5 2.8 = 10 40 " 10 18 400

y6b-CS 100 " 20 5 " 2 2 " 1 6.9 = 10 70 " 20 24 000

y7 aŽ .9.4 = 10

a Values in parentheses are for the waterrn-tetradecane interface.


Ž . Ž . Ž .Fig. 2. Surface pressure isotherms of proteins: BSA B , b-LG l and b-CS I .

and adsorption isotherm

G vi1 S Ž .b c s 13i1 i v rvi1 S

1 y v GÝS i ji , j

w xcan be obtained 28,29 , where the average molar area of all interfacial states andcomponents is defined by

G vÝ i j i ji , j Ž .v s 14S

GÝ i ji , j

The ratio of the adsorption in any arbitrary state j to the adsorption in a certainŽ .state k e.g. the state with minimum area value is expressed by the generalised

w xrelationship 30a i Ž .G v y v v P v y vi j i k i j i j i k i j Ž .s exp exp 15ž / ž /G v v RTik S i k

which can be referred to as the generalised Joos’ formula. For the non-idealbehaviour of the system which contains a single component capable of existing in adifferent state, the rigorous thermodynamic expressions are far more complicated

Ž w x.than those listed above see, e.g. 30 . The relevant mathematical formalismbecomes yet more involved if the ionisation contribution into the surface pressureof the adsorption layer and into the chemical potentials of surface active ions

w xlocated in the diffuse region of the double electric layer is taken into account 30 .Therefore, to describe the adsorption behaviour of the proteinrsurfactant mixture,one should introduce the assumptions which simplify the problem significantly.


Ž .The mixture of protein component 1 existing in the state with minimal surfaceŽ .area i.e. when the surface pressure values are not too low and the surfactant

Ž .existing in the single state component 2 characterised by the ideal behaviour inw xthe monolayer, was considered by Miller et al. 31 . The equation of state for a

mixed monolayer was shown to be

RT2 2Ž . Ž .P s y ln 1 y G v y a G v 16S S el 1 1vS

while the expressions for the adsorption isotherm of protein and surfactants are

G v1 1 Ž .b c s 171 1 v rv1 SŽ .1 y G vS S

G v2 2 Ž .b c s 182 2 v rv2 SŽ .1 y G vS S

where G s G q G . The average molar area of adsorbed components 1 and 2 canS 1 2w xbe expressed by 31

G v q G v1 1 2 2 Ž .v s 19S G q G1 2

The relation between the adsorptions of protein and surfactant can be derivedŽ . Ž .from the adsorption isotherms of Eqs. 17 and 18 :

G v b c1 1 1 1 Ž .v yv rv1 2 SŽ . Ž .s 1 y G v 20S SG v b c2 2 2 2

For v 4 v at a given ratio of the concentrations in the solution bulk, the1 2portion of protein in the surface layer decreases sharply with the increase of the

Ž .total adsorption G . For a s 0, Eq. 16 reduces to the known relationshipS elw xproposed by Joos 24 for the mixture of two surfactants in an ideal monolayer

Ž .G v b c P v y v1 1 1 1 1 2 Ž .s exp y 21G v b c RT2 2 2 2

At low surfactants concentrations and adsorptions, as v rv ( 1 and v rv < 1,1 S 2 S

Ž . Ž .an approximation follows from Eqs. 17 and 18

Ž . Ž . Ž .G v s b c 1 y b c r 1 q b c and G v s b c 221 1 1 1 2 2 1 1 2 2 2 2

Ž .Using Eq. 16 and the corresponding equation of state for the protein solution,one obtains the expression for the surface pressure jump for the mixture


2RT 1 RTa b cel 1 1 2Ž . Ž .DP s ln y 2b c y b c 2312 2 2 2 2ž / ž /v 1 y b c v 1 q b c1 2 2 1 1 1

where DP is the extra decrease of surface tension for the solution of component121, caused by the addition of component 2. It can be seen that, for certain

Ž .relationships between the parameters entering Eq. 23 , the first term in thisequation can be neglected as compared with the second term. This results in thenegative value of the surface pressure jump of the mixture, that is, the surfacetension of the proteinrsurfactant mixture can exceed that characteristic to thesystem where no surfactant is added. Moreover, calculating the extreme value of

Ž .the second term of Eq. 23 one sees that the surface tension maximum of mixtureis located at b c s 1. In the region where the surfactant concentration is high, we2 2

Ž .calculate from Eq. 20 that almost all protein molecules are expelled from thesurface layer. This constitutes a crucial difference between surfactantrproteinmixtures and mixtures of different surfactant or proteins. For such mixtures, the

Ž .exponential factor can be excluded from Eq. 21 . A simple expression follows forŽDP that is, for the variation of surfactant surface tension caused by the addition21

.of protein in the system studied, the inequalities G < G and v G < v G hold.2 1 2 2 1 1Ž . Ž .Transforming Eqs. 16 ] 18 one obtains

2 v rv1 SRTa RTa 1el el2 2Ž . Ž . Ž .DP s G v ( b c 2421 1 1 1 1 ž /v v 1 q b c2 2 2 2

From a generalised Szyszkowski]Langmuir equation for two component mixturesthe following relationship for DP results12

RT b c2 2 Ž .DP s ln 1 q 2512 ž /v 1 q b c1 1

Ž .As the power index in Eq. 24 is extremely high, the value of DP does not21exceed 1 mNrm. This approximate theoretical model which attempts to explain theanomalous behaviour of the proteinrsurfactant mixture was recently confirmed

w xusing the HSArC DMPO mixture as an example 31 .10

2.3. Fundamentals of adsorption kinetics at a liquid interface

All quantitative descriptions of adsorption kinetics processes are so far based onw xthe model derived by Ward and Tordai 32 . Differences in the models developed

on this basis consist mainly of the boundary and initial conditions as reviewedw xrecently 33 . The diffusion-controlled adsorption model of Ward and Tordai

assumes that the step of transfer from the subsurface to the interface is fastcompared to the transport from the bulk to the subsurface. It is based on thefollowing equation


c 2cŽ .s D at x ) 0, t ) 0. 262t x

As very suitable boundary condition Fick’s first law is used at the surface located atx s 0

G cŽ .s j s D at x s 0, t ) 0. 27

t x

For a mixture of different surface-active molecules a diffusion equation for eachcomponent is needed. To complete the transport problem one additional boundarycondition and an initial condition are necessary. The typical boundary conditions isan infinite bulk phase

Ž . Ž .lim c x ,t s c at t ) 0 28oxª`

and the usual initial condition is a homogenous concentration distribution and afreshly formed interface and zero adsorption

Ž . Ž .c x ,t s c at t s 0 29o

Ž . Ž .G 0 s 0. 30

For surfactant mixtures the conditions are chosen equivalent to those for a singlesurfactant system. The solution of this initial and boundary condition problem is

Ž .2 dG t y t't 'Ž . Ž .c 0,t s c y d t 31Ho ' d tDp 0

or the equivalent relationship

D 't' 'Ž . Ž . Ž .G t s 2 c t y c 0,t y t d t 32( Hož /p 0

Ž . Ž .The use of Eqs. 31 and 32 to describe the adsorption kinetics of a surfactantŽ . w xfrom solution requires an additional relation G c 33 .

2.4. Adsorption kinetics modelling for protein systems

This is true also for proteins. However, proteins can undergo conformationalchanges in the adsorption layer, as is demonstrated in Fig. 3.

Due to increasing adsorption, the degree of unfolding becomes smaller withadvancing time. At low bulk concentrations, however, the area at the interfacecovered by adsorbed molecules is small and the molecules can occupy a maximuminterfacial area. A kinetics model should take all this into consideration. Thereforea comprehensive kinetic adsorption theory for proteins can certainly not be worked


Fig. 3. Changes of protein configuration at the interface during adsorption at different protein bulkw xconcentrations, according to Wustneck et al. 34 .¨

out in the very near future. Here we can give only some general conclusions whichresult from the evolution of protein molecules in different states in an adsorptionlayer.

The very first contact of a protein molecule with the interface is characterised bya surface area v . The adsorbed molecules rearrange then at the interface to1

0 0 Ž .establish the equilibrium value G , which is governed by G , v i.e. P , and thei S S

number of different molar areas. For an ideal equilibrium adsorption layer thew xamount of molecules adsorbed in the ith state is given by 25

cb ia1 iv rv1 S0 Ž . Ž .G s 1 y G v 33i S SvS

Under dynamic conditions G - G0 and protein molecules increase their number ofi iadsorbed segments each occupying an area v , while when G ) G0 earlier adsor-1 i ibed segments rearrange to leave the interface. The transfer between the differentstates may be described by a first-order reaction

kq kqi iq1 Ž .G l G l G 34iy1 i iq1y yk ki iq1

A rate constant with ‘q’ indicates an increase, a constant marked by ‘y’ leads to adecrease of the partial molar surface of the protein by a value Dv s v . From Eq.1Ž .30 follows the equation of adsorption kinetics for a protein in i states

dGi y q q yŽ . Ž .s yG k q k q G k q G k q I 35i i iq1 iy1 i iq1 iq1 id t


with I being the diffusion flux of the ith state from the bulk of the solution to theiŽ .surface. In the present model I s 0 for all i G 2. For i s 1 we get from Eq. 31i

dG c1 y q Ž .s G k y G k q D 362 2 1 2 ž /d t x xs0

Ž .The diffusion flux I can be obtained from Eq. 31 . As a good approximation weiw xcan use 35

1r2c Dw Ž .x Ž .I s D s c y c G 371 o 1ž / ž /x p txs0

Ž .The protein concentration in the sublayer c G can be determined via the1Ž . Ž . Ž .adsorption isotherm Eqs. 5 ] 8 . Eq. 35 is quite complicated for further analysis

so that some simplifications have to be introduced. From experimental experiencew x34,36]40 we know that the proteins follow a diffusion-controlled adsorptionmechanism at the airrwater interfaces, at least in the range of small surface

w xpressure P F 2 mNrm. Particularly from refs 36,39,40 , we can deduce that thetime tU at which the surface pressure P starts to increase, i.e. the time to reach acertain adsorption G , fulfils the relation c2 tU ( const. in the protein concentra-mtion interval c s 0.05]0.001 grl. This confirms a diffusion model in the range of

Ž . Ž .small P for which we get as approximation from Eq. 32 neglecting the integral

Dtw x Ž .G s 2c 38(S oPª0 p

Usually at surface pressure P ) 2 mNrm the kinetics of adsorption is slower thanŽ .predicted by Eq. 38 . Therefore it can be concluded that, at least for low

concentrations, the rearrangement or conformational changes and desorption ofadsorbed segments of the protein molecules are fast enough and do not control theoverall adsorption process, while for higher surface concentrations or surfacepressures, these processes start to be of importance.

Processes of unfolding and rearrangement with increasing saturation of theadsorption layer may be explained by the influence of the protein concentration onthe adjusting time of adsorption equilibrium. These processes may cause somestrong effects on the surface rheology too. Also the effect of compression anddilation of the surface layer on the process of adsorption have to be discussed inthe frame of the model of different molecular states of the protein at the interface.

For mixed proteinrsurfactant systems the adsorption equilibrium is understoodonly qualitatively. About the adsorption kinetics there are no comprehensiveattempts at a theoretical description. For particular cases with respect to composi-tion and time interval, a semi-quantitative interpretation is possible by separating

w xthe effect of the two components 41 . This holds true also for penetrationexperiments where a protein adsorbs at an interface covered partly by an insolublesurfactant layer. In particular, this field of theoretical modelling deserves more


attention in the near future in order to provide a basis for understanding thesebiologically and medically important mixed systems.

3. Adsorption kinetics of proteins

The adsorption kinetics, mainly studied by dynamic surface tension measure-ments, shows many features very much different from that of typical surfactants.The interfacial tension isotherms and dynamic surface pressure curves for thestandard proteins like BSA, HSA, b-casein and b-lactoglobulin were measured bymany authors using various techniques, however, mainly for the solutionrair

w xinterface only 41]49 . First the beginning of the adsorption process showspronounced periods of time where adsorption proceeds, but the interfacial tensiondoes not change. This period is called induction time.

Once the interfacial tension starts to decrease due to adsorption it often exhibitsextremely steep dependencies, caused by the shape of the adsorption isotherm,which is also very steep in a relatively narrow concentration interval. The model

w Ž . Ž .xequations Eqs. 9 ] 11 give a qualitative explanation of the nature of theinduction time. In agreement with the dependencies P on G ? v as given byS S

w xFainerman and Miller 26 , the surface tension begins to decrease only from aŽ .certain monolayer coverage G ? v . This minimum coverage is determined byS S min

the adsorption model parameters. For standard proteins like BSA, HSA, b-caseinŽ .and b-lactoglobulin G ? v is in the range 0.1]0.2. The time necessary toS S min

reach this minimum coverage is called induction time. The reason for its existenceis the rather small contribution of the surface layer entropy to the surface pressure.

Ž .One can see from Eq. 9 that the contribution of entropy of mixing to the surfacepressure, determined by the factor RTrv is two orders of magnitude lower thanS

the factor RTrv for surfactants. This essentially distinguishes the adsorptionbehaviour of proteins and surfactants: for the latter no induction time exists.

In this section first a discussion of the problems related to low concentrationsused in experiments with proteins is given. Then an example of extremely longinduction times is discussed observed for very low protein concentration. For thestandard proteins the typical dynamic surface tensions are shown then, however, aquantitative interpretation fails so far due to lack of theoretical models.

In several experimental techniques the adsorption at the interface, or sometimeseven at the surface of the container, tubes and connectors of dosing systems, canlead to a depletion of protein in the bulk. Estimations have shown that the proteinmass in the bulk of a drop and in the adsorption layer, are comparable for drops of

w xa radius 1.5 mm and a bulk concentration of c - 20 mgrl 50 . The use of thependant drop method, however, may be considerably extended to small surface

w xpressures, usually P - 2 mNrm 36,38]40 and hence to low bulk concentrationswhen taking into account the protein mass balance.

On the other hand, it becomes possible to determine the protein adsorption forw xsmall P directly from drop experiments, as was shown by Miller et al. 50 , which

makes this technique complementary to experiments such as radiotracer tech-


Ž .Fig. 4. Dynamic surface tensions g log t for b-CS at different low bulk concentrations exhibiting they1 0 Ž . y1 1 Ž . y1 1 Ž . 3phenomenon of an induction time, c s 10 l , 2 = 10 I , 10 ^ molrcm , accordingb -CS

w xto Miller et al. 50 .

w xniques or ellipsometry 51,52 . On the basis of the above-mentioned thermody-namic model, the results from drop measurements have been compared to datafrom other experiments for b-CS and b-LG and very good agreement was foundw x53 .

The typical feature of protein adsorption kinetics, the existence of an inductionw xperiod, was analysed semi-quantitatively by Miller et al. 50 using b-CS as model

protein. The general physical picture of the induction period is that there isminimum adsorption of a protein necessary at the interface until the surfacepressure starts increasing. The flexible b-CS molecule is known to unfold and

w xrearrange at low adsorption layer coverage 51,52 .To ensure that no artefacts simulate the induction time, different complemen-

tary methods have been used, such as maximum bubble pressure method, dropvolume technique, and pendant drop technique, which differ in the time scalesfrom 0.001 s up to 105 s. Dynamic surface tensions of b-CS solutions at concentra-tions from 10y11 to 10y7 molrcm3 were measured. For the lowest concentrationsthe results are shown in Fig. 4.

A decrease of surface tension of approximately 2 mNrm is found at c s 10y11

molrcm3 after approximately 5000 s, and for c s 2 = 10y11 molrcm3 this surfacepressure is reached after approximately 700 s. According to the theoretical model

w xderived by Miller et al. 50 , a diffusion coefficient for b-CS of approximatelyD s 1.5 = 10y6 cm2rs and an adsorption of G s 0.2 mgrm2 at P s 2 mNrm isobtained. Similar results have been obtained from radiotracer and ellipsometry

w xmeasurements 38,51,52,54 . The minimum adsorption layer thickness d of b-CSminis calculated to be approximately 0.5 nm. Such a thickness corresponds to a flexibleunfolded protein chain at the interface.

y6 2 Ž .Using a diffusion coefficient of 10 cm rs for HSA, the time induction timeand the minimum adsorption G can be estimated at which the surface tensionmin


Ž .Fig. 5. Dynamic surface tension g t of b-LG at the waterrair interface, phosphate buffer at pH 5,y1 0 Ž . y9 Ž . y9 Ž . y8 Ž . 3228C: c s 10 l , 10 B , 5 = 10 v , 2 = 10 ' molrcm , according to Wustneck et¨b -LG

w xal. 34 .

Ž .Fig. 6. Dynamic surface tension g t of b-CS at the waterrair interface, phosphate buffer at pH 7,y1 1 Ž . y1 1 Ž . y1 0 Ž . y9 Ž . y8 Ž . y8 Ž . 3228C: c s 10 l , 5 = 10 B , 10 ' , 10 e , 10 I and 2 = 10 ^ molrcm ,b -CS

w xaccording to Wustneck et al. 34 .¨


starts to decrease. Assuming that G does not depend on the protein bulkminconcentration for HSA of 7.25 = 10y10 and 1.45 = 10y9 molrcm3, the correspond-ing induction times are approximately 20 s and 5 s, respectively. These results agree

w xvery well with those reported by Miller et al. 55 , i.e. the process of unfoldingseems to be very quick. A different situation was found for b-CS where a

w xrelaxation time of unfolding of 500 s was estimated 50 .The adsorption kinetics of proteins have been first systematically investigated by

w x w xGraham and Phillips 38 and later by other authors, as reviewed in 34 . Thetypical course of the dynamic surface tension for some b-LG and b-CS solutionsare shown in Figs. 5 and 6.

Different models based on effective diffusion coefficients D have been appliedefffor the interpretation of these data, however, the results show D far fromeffphysically reasonable values. In particular with decreasing concentration, values ofD are found several orders of magnitude higher than expected from the size andeffshape of the molecules. The reason for these discrepancies is surely that themodels had been based on a Langmuir isotherm as no other quantitative modelsexist. Moreover, no assumption of any changes of the conformation of adsorbedmolecules at the interface has been made. Future theoretical work has to befocussed on a generalisation of the adsorption kinetics models using the newthermodynamic models and the kinetics of protein unfoldingrrefolding in the

Ž . Ž .interfacial layers, as discussed by Eqs. 34 ] 37 .For HSA solutions the dynamics of surface tension decrease is rather different

from that of surfactants. One can see from Fig. 7 that for HSA concentrationsc F 10y10 molrcm3, almost no surface tension decrease was observed during the

w xfirst 200 s and it took more than 10 h for equilibrium to be attained 49 . For higherconcentrations the induction time as discussed above decreases quickly and thedynamic surface tension decreases in a way observed for usual surfactants. The

Fig. 7. Dynamic surface tension of HSA at the waterrair interface, phosphate buffer solution at pH 5,y1 1 Ž . y1 1 Ž . y1 1 Ž . y1 1 Ž . y1 0 Ž .temperature 228C: c s 2 = 10 v , 3 = 10 ` , 5 = 10 l , 7 = 10 e , 10 ' ,HSA

y1 0 Ž . y9 Ž . y8 Ž . 3 w x5 = 10 ^ , 10 B , 10 I molrcm , according to Miller et al. 31 .


comparison of the data with existing theoretical models, however, does not yieldreasonable results so that also for HSA better kinetic models have to be derivedtaking into consideration the change in the molar area with increasing surface

w xcoverage 31 .

4. Simultaneous adsorption from mixed aqueous solutions

While studies on mixed proteinrsurfactant systems have been qualitative over along period of time, in recent years more systematic investigations on specific

w xfeatures have been performed: food proteins mixed with surfactants 14,31,34,56 orw xwith lipids 57]59 .

For the investigation of mixed systems, various surfactants have been chosen inthe literature. The adsorption isotherms of the following surfactants are displayed

Ž .in Fig. 8: the cationic hexadecyl trimethyl ammonium bromide CTAB ; the anionicŽ .sodium dodecyl and tetradecyl sulphate SDS, STS ; and the non-ionic surfactants

Ž .decyl dimethyl phosphine oxide C DMPO and polyoxyethylene 20 sorbitan10Ž .monolaurate Tween 20 . SDS and Tween 20 are the most frequently used

compounds in this area of research. As one can see, their surface activity is quitedifferent and thus a reasonable comparison of their effect on mixed

w xproteinrsurfactant adsorption layers is difficult 60 . On the other hand, thesurface activities of CTAB, STS and C DMPO are comparable, which was the10

w xreason for the extensive studies started recently 61 .The dynamic surface tension of b-LGrCTAB mixtures at the waterrair inter-

Fig. 8. Surface pressure isotherms at the waterrair interface of the surfactants discussed in this study:Ž . Ž . Ž . Ž . Ž .C DMPO ` , CTAB I , STS ^ , Tween 20 B and SDS v .10


Fig. 9. Dynamic surface tension of b-LGrCTAB mixtures at the waterrair interface at 258C,y9 3 y10 Ž . y9 Ž . y8 Ž . y6phosphate buffer solution: c s 10 molrcm ; c s 10 I , 5 = 10 ` , 10 ^ , 10b -LG CTAB

3 Ž . w xmolrcm e , according to Miller et al. 61 .

Fig. 10. Schematic of the proteinrsurfactant interaction changing with the ionic surfactant concentra-Ž . Ž . Ž .tion: a free protein molecule; b free surfactant molecules. Steps of interaction: i first interaction via

Ž . Ž .ionic interaction; ii increasing ionic interaction until all charges are saturated; iii starting hy-Ž .drophobic interaction; and iv increased hydrophobic interaction.


face is shown in Fig. 9 for a constant b-LG concentration and increasing amountsŽ .of surfactant. While the dynamic curves g t for the two lowest CTAB concentra-

tions are more or less identical to the data for pure b-LG, the highest studiedconcentration yields dynamic surface tensions completely controlled by the surfac-tant. In the intermediate range, the contribution of the proteinrsurfactant complex

w xformed in such mixed systems dominates 14,60,61 . The adsorption kinetics for thismixing ratio is thus controlled by the adsorbing complex having a surface activitydifferent from that of the protein molecules.

The principle of interaction between protein molecules and ionic surfactants isshown schematically in Fig. 10. At low surfactant concentration a first interaction

Ž .starts due to electrostatic interaction state I , which proceeds until the availableŽ .charges are saturated by the surfactant ions state II . In this state the complexes

are much less soluble than the protein and also have the highest surface activity. Itw xcan happen that in this state a part of the complexes precipitate 2 .

With further increase of the surfactant bulk concentration, increasing amountsof surfactant molecules interact with the complexes via hydrophobic interactions,making the complex step-by-step more hydrophilic and hence more soluble inwater and less surface active. This hydrophobic interaction proceeds until thesurface activity of the complexes reaches a comparatively low value. Due tocompetition in the adsorption layer, more and more complexes are replaced by

Žsurfactant molecules so that finally, typically at the CMC of the surfactant in term.of free surfactant , the adsorption layer is mainly formed by the surfactant. This

picture is confirmed by studies of various other parameters of the mixed adsorptionŽ w x.layers e.g. 60 , where data on surface shear viscosity, adsorption layer thickness

Fig. 11. Dynamic surface pressure of HSA at a concentration of c s 10y1 0 molrcm3 mixed withHSAthe non-ionic surfactant C DMPO at various concentrations; phosphate buffer solution at pH 7,10

y9 Ž . y8 Ž . y8 Ž . y8 Ž . y7 Ž .temperature 228C: c s 1 = 10 v , 1 = 10 ` , 4 = 10 ' , 7 = 10 ^ , 10 B ,C DMPO10y7 Ž . y7 Ž . y7 Ž . y6 3 Ž . w x2 = 10 I , 4 = 10 l , 7 = 10 e , 10 molrcm w , according to Miller et al. 31 .


and surface mobility have been determined independently. These data agreeexactly with the findings for the equilibrium and dynamic surface tensions.

The mixed proteinrnon-ionic surfactant systems behave significantly differently.For example, the dynamic surface tensions for mixed HSArC DMDO solutions at10228C are shown in Fig. 11. From the first addition of surfactant it becomes clearthat there is a competitive adsorption and with increasing surfactant concentrationthe kinetics become more and more surfactant-like.

w xWhen one looks into the equilibrium data as given by Miller et al. 31 one cansee that for c ) 10y7 molrcm3, the isotherms of the pure surfactant andC DMPO10

Ž y10 3.the mixed system constant protein concentration of 10 molrcm are almostidentical and the adsorption of HSA can be assumed to be negligible.

Ž y9 y7. 3In the concentration range c s 10 ]10 molrcm , the surface ten-C DMPO10

sion of the mixtures exceed the values for the pure HSA solution, although fromŽ .Eq. 25 we get a substantial decrease of the surface tension of the solution due to

the addition of the second component, except when b c < b c . For 4 = 10y81 1 2 2

molrcm3 - c - 10y7 molrcm3 this surface tension excess amounts only toC DMPO10

approximately 1 mNrm, however, for c F 4 = 10y8 molrcm3, the g exceeds thatŽ .of the pure HSA solution by 3]4 mNrm. The theoretical model of Eq. 25 cannot

explain such increase of surface tension, i.e. negative value of DP . However, this12Ž .phenomenon is explained quite well by Eq. 23 . The location of the maximum at

Ž .the curve corresponds approximately to the value estimated from Eq. 23 , that is,b c f 1. For low values of a , the mixing of components 1 and 2 cannot lead to2 2 el

Fig. 12. Schematic of the proteinrsurfactant interaction changing with the non-ionic surfactantŽ . Ž .concentration. Steps of interaction: i first interaction via hydrophobic interaction; ii increasing

Ž .hydrophobic interaction; and iii increased hydrophobic interaction.


an increase in surface tension. Therefore, an anomalous behaviour of surfacetension for HSArC DMPO mixtures results from the large free charge of protein10

w xmolecules 25,62 .This phenomenon can also be explained rather simply from a physical point of

view. The interaction with the non-ionic surfactant leads to less surface-activecomplexes, which then cause the increase in surface tension. The effect of increas-ing amounts of non-ionic surfactants in a protein solution is shown schematically inFig. 12. The simultaneous adsorption is almost a pure competitive adsorption,enhanced even by the stepwise hydrophilisation of the protein due to hydrophobicinteraction with the surfactant.

w xSurface shear viscosity studies support these findings 31 . For concentrationsc F 2 = 10y8 molrcm3, the surface layer possesses a rather high viscosity,C DMPO10

characteristic for pure HSA solutions while at a concentration of 7 = 10y8

molrcm3, the shear viscosity decreases sharply to almost zero, characteristic forpure surfactant solutions. Therefore, both tensiometric and rheologic studiesindicate that the compatibility of HSA and C DMDO in the mixed monolayer is10

w xvery poor, in contrast to mixtures of surface active homologues 63]65 , where anyaddition of a second component results always in an extra surface tension decrease

Ž .for the mixture, as given by Eq. 25 .To summarise, it can be seen from the above qualitative discussion of the

adsorption behaviour of proteinrsurfactant mixture that these systems can exhibitrather unusual features. In general, for the arbitrary concentrations of the compo-nents, surfactant ionisation in the monolayer and the intermolecular interaction ofthe components in the bulk and in the monolayer, one can expect that equilibriumand dynamic mixed proteinrsurfactant monolayers could display various and evenunpredictable features.

5. Protein rrrrr lipid adsorption from two separate solutions at the joint interface

Investigations on the formation of mixed lipidrprotein layers at interfaces arevery helpful in understanding the nature of interactions in such interfacial layersw x66]70 . Such interactions can change the hydrophobicity or hydrophilicity and

w xconformation of proteins significantly 66,69,70 . The interaction between proteinand phospholipids can be studied by interfacial tension measurements at thewaterrchloroform interface via the adsorption of the single components fromseparate bulk phases. This means that a lipid, such as DPPC, adsorbs from thechloroform phase while the protein, such as b-LG, adsorbs from the aqueous phasew x71 . Compared to conventional studies where surfactants were mixed with theprotein in an aqueous solution, the present type of investigation provides twoadvantages. It avoids the formation of complexes in the bulk phase due toelectrostatic binding or hydrophobic interaction and subsequent adsorption ofthese complexes, and it excludes the influence of lipidrprotein interaction insolution on the adsorption mechanisms.

The interfacial tensions of such systems, for example a-dipalmitoyl phosphatidyl


Ž .Fig. 13. Dynamic surface pressure P t for a mixed adsorption of b-LG from an aqueous 0.1 mgrlsolution outside the chloroform drop containing different DPPC concentrations: c s 5.4 = 10y1 2

b -LG3 y10 Ž . y1 0 Ž . y9 Ž . y9 Ž . y8 Ž . y8 Ž .molrcm ; c s 10 B , 5 = 10 I , 10 v , 5 = 10 ^ , 10 ' , 5 = 10 `DPPC3 w xmolrcm , according to Li et al. 71 .

Ž .choline DPPC mixed with b-LG, was measured by the pendent drop and dropw xvolume techniques 71 . Figs. 13 and 14 show selected results obtained at two b-LG

concentrations in a broad DPPC concentration interval.In a wide concentration interval the adsorption kinetics of the mixed system is

influenced by both components. However, at low protein concentration, c sb -LG5.4 = 10y12 molrcm3, the adsorption rate of the mixed interfacial layer is mainlycontrolled by the DPPC. When c is increased up to c s 4.3 = 10y11

b -LG b -LG

Ž .Fig. 14. Dynamic surface pressure P t for a mixed adsorption of b-LG from an aqueous solutionoutside the chloroform drop containing DPPC at different concentrations: c s 4.3 = 10y1 1

b -LG3 y10 Ž . y9 Ž . y9 Ž . y8 Ž . y8 Ž . 3molrcm ; c s 10 ` , 10 I , 5 = 10 ^ , 10 ' , 5 = 10 l molrcm , accordingDPPC

w xto Li et al. 71 .


y1 1'Fig. 15. Surface pressure P as a function of t for a mixed adsorption of HSA from a 5.0 = 10molrcm3 aqueous solution outside a chloroform drop containing DPPC different concentrations:

y8 Ž . y8 Ž . y8 Ž . y8 Ž . y8 Ž . y8 Ž . y81 = 10 B , 2 = 10 I , 3 = 10 v , 4 = 10 ` , 5 = 10 ' , 6 = 10 ^ , 8 = 10Ž . 3 w xl molrcm ; according to Wu et al. 73 .

molrcm3 the interfacial activity of the protein abruptly increases, and at low lipidconcentrations, 10y11 molrcm3 - c - 10y10 molrcm3, the DPPC has veryDPPClittle effect on the whole adsorption process, i.e. the adsorption rate is dominatedby the protein adsorption. This behaviour is also observed at higher DPPCconcentrations when the protein concentration is further increased to 4.3 = 10y11

3 Ž . y10 3 w xmolrcm Fig. 14 or even at c s 1.9 = 10 molrcm 71 . When the lipidb -LGconcentration c exceeds 3 = 10y8 molrcm3, the adsorption behaviour is veryDPPC

w xsimilar to that of the pure DPPC independent of the protein concentration 72 .ŽA complementary and very extensive study of four phospholipids DPPE, DPPC,

. ŽDMPE, DMPC mixed with one of three proteins b-LG, b-CS, and HSA, respec-. w xtively was performed by Wu et al. 73 in order to generalise the conclusions about

the role of the phospholipid and protein structure on the equilibrium as well asdynamic interfacial tension behaviour. For this aim the lipid concentration wasincreased such that an almost saturated adsorption layer was reached. It was shownthat the head group has a much stronger effect on the equilibrium adsorption statethan the chain length.

For the dynamic interfacial tensions the lipid structure plays a minor role. Anexample for simultaneous adsorption of DPPC from the chloroform and theprotein HSA from the aqueous phase is shown in Fig. 15.

A quantitative analysis of the dynamic interfacial tensions is possible only at thehigher lipid concentrations where the protein adsorption plays a minor role.Independent of the lipid and protein structure, the adsorption kinetics of the wholemixed system appears to follow a diffusion-controlled mechanism. This might bedifferent for the waterrair interface where, in contrast to the waterrchloroforminterface, interfacial phase separation was observed in penetration experiments.


We will only briefly touch on this type of experiment here in Section 6, as it will bew xdiscussed in greater detail in another paper of this issue 74 .

6. Penetration of proteins into lipid layers

Since the phenomenon of monolayer penetration was first reported by Schulmanw xand Hughes 75,76 , kinetics and equilibrium properties of many penetration

w xsystems have been intensively investigated 77]86 . Penetration systems consistingof proteins and lipids, because of their particular importance in nature, are ofparticular interest most of all in biophysics, biochemistry and medicine. Because ofits ideal amphiphilic properties, phospholipids form stable monolayers at theair]water interface and can therefore be used as the simplest model for studyingprotein penetration. In recent years, new methods have extended the knowledge ofvarious two-dimensional monolayer phases. With the recently developed Brewster

Ž .angle microscopy BAM , which allows direct visualisation of the long-rangeorientation order, owing to the optical anisotropy induced by the tilted aliphatic

w xchains 87]89 , it was possible to observe that b-LG penetration can induce afirst-order phase transition in a fluid-like Langmuir monolayer of dipalmitoyl

Ž .phosphatidyl choline DPPC . A corresponding theoretical model was derived tow xdescribe this interesting phenomenon 90 . Interesting results on b-LG penetration

w xinto a DPPC monolayer have been obtained recently 91 . A mechanism wasdeveloped based on the strong interaction between the lipid molecules that leadsto a separation of the compounds at the interface. Using a typical surfactant, aC DMPO, instead of the protein, the same features where observed for the DPPC10

w xmonolayers 92 . Thus, the phase transition of the lipid monolayer is not the resultof a specific effect of the penetrating protein molecules, but a general property ofpenetration layers. The conditions, however, under which a penetration systemundergoes phase transition are not yet clear. In a further contribution to this issue,the penetration process of proteins into spread lipid monolayers is describedthermodynamically in detail and experimental results are given supporting the

w xtheoretical models 74 .

7. Protein adsorption at water rrrrr oil interfaces

The adsorption behaviour of proteins at waterroil interfaces should follow thesame general physical picture, however, specific differences have to be expectedcaused by the protein structure and chemistry. At the waterrair interface theprotein molecules try to expose the hydrophobic parts upon the air phase whichleads to an unfolding of the molecules. This process can proceed as long and as faras there is time and space at the interface available. Thus, the process of unfoldingis restricted by a competitive adsorption of other molecules, proteins or surfac-

Ž .tants. Once a molecule is spread over a free interfacial area unfolded it is unclear


Fig. 16. Dynamic surface pressure of HSA solutions at pH 7 for two concentrations: 2 = 10y1 1

3 Ž . y1 0 3 Ž .molrcm B,I , 2 = 10 molrcm ',^ . Closed symbols, waterrair interface; open symbols,waterrtetradecane interface.

to what extent these molecules can refold, and what will be the conformation ofthe molecules after refolding.

For waterroil interfaces the situation is different, as the adsorbing proteinmolecules can penetrate into the hydrophobic oil phase with the hydrophobic partsof the molecule. This means that this type of unfolding can proceed even at acomparatively strong competition at the interface due to adsorption of othermolecules, as the unfolding does not happen at the interface but within the oil bulkphase. Again, the question of refolding due to increased competition or compres-sion of the interfacial layer is difficult to answer. This question has been addressedby many authors and is discussed in terms of interfacial protein denaturation. This

w xquestion was summarised recently by MacRitchie 93 .w xComparative studies have been performed by Graham and Phillips 38,51 and

w xmore recently by Makievski et al. 53 for HSA, b-casein and b-lactoglobulin at thesolutionrair and solutionroil interfaces. The dynamic interfacial pressure P as afunction of time for HSA, b-casein and b-lactoglobulin at the solutionrair andsolutionroil interfaces for various protein concentrations are discussed by

w xMakievski et al. 53 . The data indicate that the nature of the interface significantlyaffects the dynamics of the adsorption process and the equilibrium adsorptioncharacteristics. In particular, for HSA and b-casein, both the rate of interfacialtension increase as well as the equilibrium values of P at the solutionroilinterface are higher than the corresponding values at the solutionrair interface.For b-lactoglobulin, however, comparable values are observed at high bulk concen-trations, while at low bulk concentrations the observed increase in P was morepronounced for the solutionrair interface.

Ž .In Fig. 16 an example of the dynamic surface pressure as a function of time P tis given for two HSA concentrations, measured by the pendent drop technique at


Ž . Ž .Fig. 17. P]c isotherm for HSA at the waterrair ' and waterrtetradecane interface ^ at pH 7.

the waterrair and waterrtetradecane interfaces. The differences in the kineticsand in the absolute values are easily recognised.

To estimate the equilibrium surface pressures in order to construct the isothermsŽ .the curves P s P t were extrapolated to t ª `. The values obtained from two

'Ž . Ž .different extrapolation procedures, lim P 1r t and lim P 1rt , show differ-tª` tª`w xences smaller than "0.5 mNrm 94 . In Fig. 17 the extrapolated equilibrium

surface pressure isotherms for HSA at pH 7 at the solutionrair and solutionroilinterfaces are plotted as a function of the initial protein concentration in thesolution.

One can see that the surface pressure isotherms measured for the waterroilinterface have significantly higher values as discussed above. This may be explainedby a larger adsorption layer thickness at the waterroil interface as compared to thewaterrair interface due to penetration of protein loops or tails into the oil phase.

w xThe data of Makievski et al. 53 agree with those in the literature with respect tothe location of the isotherm, however, they are incompatible with the data

w xpublished by Graham and Phillips 51 in respect to the maximum surface pressureŽ .at the solutionroil interface approx. 20 mNrm . In particular, at high concentra-

tion the surface pressure for HSA reaches values of approximately 28 mNrm. Forb-casein, values of even approximately 32 mNrm have been reported by Makievski

w xet al. 53 while the data reported by Graham and Phillips were only 24 mNrm.

8. Peculiarities in the protein adsorption at drop and bubble surfaces

It was already discussed above in Section 3 that the use of small solutionvolumes can lead to a significant depletion of the bulk phase due to proteinadsorption, as the absolute concentrations common for such systems are extremelylow. It was also mentioned that this disadvantage inherent to single drop studiescould be turned into an advantage as the loss of protein mass can be correlated tothe adsorbed amount at the interface. In order to evaluate the correctness of this


idea and to estimate the accuracy of such a procedure, experiments with drops andw xbubbles have been recently performed 95 . The main difference between these

differences is the limited and more or less unlimited bulk volume, respectively.The adsorption of a surfactant or protein from inside a drop or from a large

external solution bulk to a bubblerdrop surface, is characterised by some additio-nal features, which can result in a different adsorption behaviour in particular forproteins. Firstly, the conditions for the transport by diffusion to a spherical surfaceare not only different due to the available reservoir but also due to a depletion ofmolecules from the bulk leading to a decrease in the bulk concentration. Thiseffect can be significant particularly for proteins where the concentrations ofinterest are in the range of 10y12 to 10y8 molrcm3. In such cases the mass of theadsorbed protein or surfactant can be of the same order as the mass of proteininside the drop. Secondly, there are differences due to the curvature of thedroprbubble surface: in a drop, the radial field diverges decelerating the diffusionwhile transport from outside a drop or bubble is enhanced due to a convergingradial field. The third difference to be mentioned is that the waterrair interfacerepresents an environment for adsorbing proteins on a drop surface different fromthat of a bubble surface, which can have an impact on the protein conformation.

The analysis of the differences in the diffusion process have been performed byw x ŽMakievski et al. 95 . For the diffusion from an infinite bulk to a spherical drop or

.bubble surface the following approximate solution for the dynamic adsorption G isw xobtained 96]98

cDt DtŽ .G s q 2c 39(r p

where r is the drop radius.The adsorption from inside a spherical drop at its surface of area A can be

Ž .expressed by an average protein concentration c as a function of time in the dropw xvolume V 50

V rŽ . Ž . Ž .G s c y c s c y c 40

A 3

w xAn approximate solution for the adsorption from inside a drop was given in 50

cDt DtŽ .G s y q 2c 41(r p

Ž . Ž .As compared to a flat surface, where the second term in Eqs. 39 and 41 is a firstapproximation for the adsorption process, the diffusion to a bubble surface from an

winfinite solution leads to an increased adsorption rate positive sign of the curva-Ž .xture term in Eq. 39 and hence to a larger adsorption, in contrast to the diffusion

from inside a drop where a deceleration of the adsorption rate is obtainedw Ž .xnegative sign of the curvature term in Eq. 41 . For a diffusion coefficient of


Fig. 18. Dynamic surface tension g of b-casein solutions as a function of time: 5 = 10y12 molrcm3

Ž . y1 1 3 Ž . y9 3 Ž .',^ , 5 = 10 molrcm v,` , 10 molrcm B,I . Closed symbols, drop experiments; openw xsymbols, bubble experiment, according to Miller et al. 50 .

y6 2 Ž . Ž .D s 10 cm rs, a typical value for b-casein, the first terms in Eqs. 39 and 41exceed 10% when t ) 1000 s. For larger diffusion coefficients the differencebetween the two cases becomes significant at shorter times. In Fig. 18 an impres-sive example is given for b-casein solutions at three different concentrations.

The differences are significant and even at comparatively high bulk concentra-tions the reasons mentioned above lead to a distinct change in the adsorptionkinetics. It can be suspected that the minimum surface tension at a drop surfacereached at sufficiently large bulk concentration will be remarkably higher than thatat a bubble surface. This will be mainly caused by the difference in the adsorptionrate which restricts unfolding at the bubble surface and enhances it at a dropsurface.

As mentioned above, the equilibrium surface tensions of the b-casein solutionsobtained by extrapolation of the dynamic tensions to t ª ` from bubble experi-

w x Ž .ments are in good agreement with the data of Graham and Phillips 51 cf. Fig. 2 .At the same time, the data obtained from the drop method and plotted as a

w x Ž .Fig. 19. Surface pressure isotherm of b-casein: data of Graham and Phillips 51 B , obtained fromŽ . Ž .pendent bubble ` and pendent drop v . Theoretical line corresponds to present bubble experiments

and data of Graham and Phillips.


function of the initial protein concentration, give much lower values for the surfacepressure P. For b-CS this is shown in Fig. 19.

For c - 10y7 molrl this effect can be ascribed to the redistribution of theprotein between the bulk and the drop surface. The values of b-casein adsorption

w Ž .xcalculated from the mass balance condition Eq. 36 are shown to be in a perfectw xagreement with the results directly measured by Miller et al. 50 .

When the initial concentration of the protein within the drop exceeds 5 = 10y7

molrl, the loss of mass caused by adsorption is rather small and it is impossible toexplain the higher P-values by this argument. Here it is certainly the difference inadsorption rate that is responsible for diverging results: when the b-casein adsorp-tion in the drop is very slow the molecules have sufficient time to becomecompletely unfolded and spread over the solutionrair interface.

From Miller et al. Fig. 18, one can see that at any concentration the adsorptionprocess at the drop surface proceeds much more slowly than for the bubblemethod. Thus, at low P values the probability for a complete unfolding of theb-casein molecule is higher as compared to the bubble surface. However, formedium and high P values, one can expect that a refolding of molecules, whichwere previously unfolded, could happen. This segment-by-segment refolding processis certainly quite slow, and can be described in future by a theory given in principle

Ž . Ž .by Eqs. 34 ] 36 .

9. Dilational elasticity of a protein adsorption layer

Rheological studies are an additional source of information about the structureof adsorption layers, in particular of proteins at liquid interfaces. While shearrheology yields only qualitative structure information, dilational rheology is basedon the thermodynamics and kinetics of the respective adsorption layers and ishence a complementary method to describe a protein adsorption layer quantita-tively.

The surface dilational modulus « is defined as the increase in surface tension fora small increase in surface area A by

dgŽ .« s 42

dln A

Ž .The surface dilatational modulus or viscoelasticity modulus is a complex numberand incorporates a real and imaginary part, which correspond to the elasticity and

w x Žviscosity, respectively 99 . In the simplest case the modulus is purely elastic the.Gibbs elasticity modulus with a limiting value

dgŽ .« s y 43

dlnG

This limiting value is reached if no exchange of protein with the bulk solutionŽ .G s const. exists and the surface tension is in equilibrium. Deviation from this


Fig. 20. Dynamic surface pressure P and drop area A as a function of time for a 5 = 10y10 molrcm3

HSA solution at the waterrtetradecane interface at three subsequent trapezoidal deformations of thedrop surface.

simple limit occurs when relaxation processes at the surface or near the surface setin. The viscoelasticity is much more sensitive to small changes in the adsorptionlayer coverage than any dynamic or equilibrium interfacial tension, as it is propor-tional to the slope of the adsorption isotherm dgrdG.

Dilational rheological experiments of liquid interfaces can be performed byw xvarious methods 99 . Transient relaxation methods are possibly most suitable for

w xprotein layers due to the extremely low bulk concentrations 100,101 . Suchrelaxation experiments can be performed with pendent drops or bubbles as

w xdescribed by Makievski et al. 53 . After 100]200 min pre-adsorption time rapidŽ .during 3]5 s compression or expansion of the droprbubble surface by 5]10%have been produced by respective drop volume changes. After a 10]30 minrelaxation the surface of the drop was restored to its initial size. The wholeoperation performed for the solutionrair and solutionroil interfaces was repeatedseveral times. An example of such experiments is illustrated in Fig. 20 for a HSAsolution at the waterrtetradecane interface. Besides the change in interfacialtension also the change in the drop surface area is shown.

As one can see slow relaxation processes set in after a compression or expansionof the interfacial layer, certainly due to conformational changes, as the time fordiffusional exchange would be much slower at the present bulk concentration of5 = 10y10 molrcm3 HSA.

To estimate easily the dilational elasticity it is convenient to represent « in a


dimensionless form

« dlnPU Ž .« s s y 44

P dln A

Ž .Neglecting any diffusional exchange from Eq. 39 , it follows that

dlnPU Ž .« s 45

dlnGS

Ž .For some particular cases, Eq. 45 yields rather simple expressions. For example,Ž . Ufor an ideal gaseous monolayer P s RT G , Eq. 45 yields « s 1. Another muchS

more realistic approximation is obtained when the first term on the right-hand sideŽ .of Eq. 6 is small as compared to the second term and can be neglected. This leads

to the approximate expression

RT2 2 Ž .P s a G v 46el S SvS

Ž . Ž . UIntroducing Eq. 46 into Eq. 45 , one obtains « s 2. If the contribution of theŽ .first term in Eq. 2 remains significant, which is the case for low and medium

surface pressures, then we get «U ) 2. On the contrary, transitional relaxationsbetween the adsorption states would lead to a decrease in DP and hence in adecrease of «U.

To calculate the dilational elasticity from experiments the differential quotientŽ .in Eq. 44 is replaced by a finite difference which contains experimentally

available values only

DP AU Ž .« s y 47

P D A

The dependence of «U on P obtained for HSA at c - 10y9 molrcm3 at both0interfaces, waterrair and waterrtetradecane are presented in Fig. 21. One can seethat for P ) 5 mNrm, the experimental «U values only slightly depend on thesurface pressure and lay between 1 and 3, mainly between 1.5 and 2.5. One can

Ž .thus conclude that Eq. 2 agrees satisfactorily with the data obtained in thesetransient relaxation experiments. The values of «U for HSA at the solutionrairinterface exceed those at the solutionroil interface. The value of «U averaged overall measured data at the solutionrair interface was 2.9, and at the solutionroilinterface «U s 1.8. The increase in HSA concentration at c ) 10y9 molrcm3

0resulted in a slight decrease of the dilational elasticity at both interfaces.

w xBenjamins et al. 102 calculated the elasticity for BSA interfacial layers at thesolutionrair and solutionrvegetable oil interfaces from results of Langmuir troughexperiments with a movable barrier and from interfacial tension changes of slowly


Fig. 21. Dependence of the dimensionless visco-elastic modulus «U on the surface pressure for HSAsolutions. Open symbols, waterrair interface; closed symbols, waterrtetradecane interface.

Žoscillating drops oscillation frequency was 0.1 Hz in both experiments, amplitude.of surface area variations F 20% . For the waterroil interface and P - 10 mNrm

the obtained values for «U are in the range 2.5]2.8 and independent of surfacepressure, and then decrease down to 1 when P increases up to 17 mNrm. Theseresults are in good agreement with the approximated value of «U s 2, and the

w xexperimental data for HSA. The elasticity values by Benjamins et al. 102 for thesolutionrair interface were also somewhat higher, quite similar to the presentedHSA data.

Fig. 22. Dependence of dynamic surface tension for two blood serum samples taken from patientsŽ .suffering from different pathologies B,I and ^,', respectively plotted on the logarithm of the

surface lifetime. Closed symbols, MBPM; open symbols, ADSA data.


Fig. 23. Dependence of surface tension for blood serum sample on ty1 r2. Open symbols, ADSA data;closed symbols, MBPM data; solid line, extrapolation of the MBPM results.

10. Surface tension and elasticity of blood serum as example of a natural protein rrrrrsurfactant mixtures

All biological liquids of the human organism contain surface-active compounds,such as proteins, lipids, and molecules of other natures. The main surface-active

Ž .compound of serum is albumin HSA with a concentration in blood of 35]50 grl.The results of the measurements performed with two samples of blood serum,

taken from two patients suffering from different pathologies, are presented in Fig.w x22 103 .

The experimental results from the two methods are combined here in order tocover a broader time interval: values from the maximum bubble pressure methodŽ .MBPM were measured in the effective time range from 0.01 to 50 s, while the

Ž .values from the drop shape method ADSA cover the range from 10 to 1000 s.The mutual consistency of the methods is demonstrated by the fact that in theoverlapping interval from 10 to 50 s, the results are in an agreement to withinexperimental error. It is also seen that more than half of the total surface tensiondecrease falls into the MBPM interval. On the other hand, the ADSA dataessentially complement the MBPM results in the long lifetime range. It can be alsoshown that at times longer than 1000 s, almost no change of the serum surfacetension can be observed.

Ž .To calculate the equilibrium surface tension from the MBPM results g , the`

dependence of the surface tension vs. ty1r2 was extrapolated to the infinite time,w xaccording to the procedure described in Refs. 3,32 . For example, the lower

w xtensiogram of Fig. 22 is plotted in these co-ordinates in Fig. 23 103 .ŽFor this case, the value of g estimated from the MBPM data the point of`

.intersection of the extrapolation straight line with the ordinate is 57.7 mNrm,


while the slope of this line is l s dgrd ty1r2 s 19.8 mNrm ? sy1r2. It is seen thatthe slope of the extrapolation line coincides with the slope of the linear part of theADSA curve in the range above 0.2 sy1r2. However, at lower values of ty1r2, theADSA data are lower than those obtained by the MBPM. A similar extrapolation

Ž y1r2 y1r2 .to infinite time, performed for the ADSA data in the range t - 0.06 sleads to the equilibrium value gU s 47.8 mNrm and the slope of the linear part to`

lU s 140 mNrm ? sy1r2.The shape of the experimental dependence as presented in Fig. 23, with a

narrow interval with an inflection point and two linear parts of different slopes, istypical for mixtures of surfactants characterised by different activities and concen-

w xtrations 104,105 . Curves quite similar to that presented in Fig. 23 were obtainedŽ .both experimentally and theoretically for mixtures of two surfactants, when theconcentration of one of the two was 10]100 times higher than the concentration ofthe second surfactant, while the adsorption activity of the first surfactant was to the

w xsame degree lower that that of the second component of the mixture 104,105 .y1r2 y1r2 ŽTherefore it can be concluded, that for t ) 0.13 s i.e. for time values

.below 60 s the surface-active compound with the main mass portion in the bloodŽ .serum the blood serum albumin is adsorbed. However, at lifetimes above 100 s

blood serum components of higher surface activity adsorb, having concentrations10 or 100 times lower than the albumin concentration. It follows from the theoryw x104 that the slope l is proportional to the square of the adsorption, and inverseproportional to the surfactant concentration. Comparing the two values of l

Ž y1r2 y1r2 .obtained above 19.8 mNrm ? s and 140 mNrm ? s one can conclude thatthe concentration of highly surface active impurities in the sample of blood serumstudied is extremely low.

The viscoelasticity modulus obtained in stress experiments of the surface layerŽ .can be calculated from Eq. 42 . In these experiments the expansion of the drop is

followed by the relaxation of the surface tension to its initial value. The change ofsurface tension which follows the stress deformation can be described by anexponential dependence

Ž . Ž .Dg s Dgexp D trt 48t

where Dg is the initial jump of the surface tension, D t is the time elapsed after thedeformation, t is the relaxation time. The relaxation time characterises the abilityof the monolayer to restore its initial state, that is, this value reflects the kinetics ofadsorption from the solution and the processes involved in the rearrangement ofthe state of adsorbed molecules.

The results obtained from stress experiments with the samples of blood serumw xreferred to in the discussion of Fig. 22 are presented in Fig. 24 103 .

ŽThe measurement time in the dynamic regime was 1200 s adsorption after the.formation of a fresh drop surface . At this time moment, an expansion of the drop

surface by 7]8% was made. The smaller the deformation is, the more rigorous isŽ .the correspondence of the data with Eq. 42 , while finite differences are applied

instead of differentials. On the other hand, if the deformation is too small, then the


Fig. 24. Dynamic surface tension of blood serum measured using ADSA for the same samples as inFig. 22; the stress deformation caused by an expansion of the drop was imposed 1200 s after the start ofthe experiment with a fresh drop.

errors in the surface tension measurements become comparatively large. Thus,deformations of 5]10% are optimal. It is seen from Fig. 24 that the surface

Ž .pressure jumps and the viscoelasticity modulus are different for the blood serumsamples studied. For this fluid, the range of the viscoelasticity modulus is quitebroad: 10]80 mNrm, while the usual values of the relaxation time are between 50and 300 s. Thus, the application of the axisymmetric drop shape analysis comple-mentary to the maximum bubble pressure method allows not only to understandmore clearly the results obtained by the two methods, but provides also a deeperinsight into the tensiometric and rheological characteristics of the interfacial layersformed by human biologic fluids.

Finally, the characteristics of blood serum tensiograms and rheological parame-Ž .ters for the samples taken from three females are compared in Table 2: 1 a

Ž . Ž .healthy person; 2 a patient suffering from kidney disease; and 3 a patientsuffering from neoplastic disease of reproductive organs.

It is seen that the combined use of the two methods enables one to differentiate

Table 2Tensiometric and rheometry parameters of some selected samples of blood serum

U Ua aPatient g l g l « t` `y1r2 y1r2Ž . Ž . Ž . Ž . Ž . Ž .no. mNrm mNrm s mNrm mNrm s mNrm s

1 61.4 12.3 53.7 80.3 27.9 64.92 54.9 24.2 49.5 89.1 28.6 95.73 57.3 22.1 42.2 278.4 50.0 73.3

a From ADSA experiments at longer adsorption times.


more precisely between the pathologies. For both pathologies, the g value is`

lower, and the l value is two times higher than those characteristic for the healthyperson. At the same time, for patient 3, a significant decrease of gU is observed`

Ž Uthe difference between g and g is 15.1 mNrm, as compared with the value of` `

.7.7 mNrm for the healthy person , and the dilational viscosity modulus « is almosttwice the increase as compared with the data obtained for patients 1 and 2. On the

U Ž .contrary, for the patient 2 the difference between g and g is small 5.4 mNrm ,` `U Žwhile the l value is essentially higher three times higher as compared with

.patients 1 and 3, respectively , while the « value is high. One of the possibleexplanations for the change in the parameters gU and lU for patient 3 may be an`

increased concentration of sialic acid in the blood common to the neoplasticw xdisease of reproductive organs 106]108 .

11. Summary, conclusions, outlook

The present overview discusses experimental results obtained for selected pro-teins, namely HSA, BSA, b-casein and b-lactoglobulin, and mixed systems withsurfactants and lipids. The surfactants on which we report here have been selectedsuch that their surface activity is comparable, i.e. the cationic CTAB, the anionicSTS, and the non-ionic surfactant C DMPO have been mainly referred to in this10comparative study.

The equilibrium and dynamic adsorption behaviour of pure protein solutions canbe described very well by a recently developed thermodynamic model and therespective diffusion controlled adsorption theory based on the correspondingequation of state.

For the mixed systems, quantitative theoretical models do not exist, and thus theadsorption kinetics data cannot discussed quantitatively, however, the equationsderived in Section 2.2 allow a semi-quantitative understanding of mixed adsorptionlayers. The assumption that different types of interaction between protein andsurfactant molecules, and a specific surface activity and conformation of theproteinrsurfactant complexes exist, is in good agreement with the adsorption dataobtained. The complexes formed by proteins and the ionic surfactants can behigher surface active than the protein alone due to a modification of the structureof adsorbed proteins. Thus even b-casein becomes rather compact at the interfaceat a certain STS or CTAB concentration. In the presence of non-ionics theadsorption layer is mainly formed by competitive adsorption between the com-pounds. Hydrophobic interaction can also support the formation of complexes.However, such complexes are typically less surface-active than the pure protein sothat they do not participate in the competitive adsorption. This type of complexsupports the depletion of proteins from the interface at increasing amounts ofsurfactants, which is observed for non-ionic as well as ionic surfactants.

In addition to adsorption dynamics studies also penetration kinetics experimentsare of interest for the better understanding of protein layers mixed with surfactants


Fig. 25. Relaxation fluxes in a mixed proteinrsurfactant layer under harmonic area changes, relax-Ž .ation between the two states A and B due to diffusion flux a and due to bindingrrelease of surfactants

Ž .from the formed complexes b , respectively.

at liquid interfaces. In particular the penetration of proteins into lipid layers are ofmuch practical relevance for biological and medical studies.

The strength of interaction between protein and the different surfactants can bestudied by relaxation experiments. Using harmonic perturbations in a broadfrequency interval it should be possible to separate relaxation due to transportŽ . Ž .Fig. 25a from relaxation due to bound surfactants Fig. 25b . Fig. 25 showsschematically the two different relaxation fluxes for a mixed proteinrsurfactantadsorption layer.

There are a number of open questions the answer of which requires moresystematic studies. For example, in the studies of the liquidrliquid interface thesolvent for the lipids should be varied as the chosen chloroform can certainlydestruct proteins. Using a more gentle solvent it can be better cleared up whetherlipid and protein also separate at the waterroil interface. For adsorbed mixedproteinrsurfactant layers studied so far it has not yet been observed that the


components separate at the interface. Techniques for the investigation of thisquestion exist, however, experiments are not known so far.

It will also be interesting to learn under which conditions the modification of aprotein by specific interaction with ionic surfactants can make it possible that theresulting complex transfers from the aqueous into the adjacent oil phase. Thisphenomenon seems possible as the ionic interaction leads to a strong hydrophobi-sation of the protein molecule so that the changed conformation would make itsoluble in oil.

These are only a few of the questions resulting immediately from the presentedstudies. All the given results are important to improve the scientific basis on mixedproteinrsurfactants systems for many practical purposes. One of them, as brieflydiscussed in Section 10, is the use of tensiometry and rheology as tools in medicaldiagnostics and therapy control. This overlap of two scientific areas seems to beextremely interesting and fruitful and there is great hope that it will developfurther as successfully as it did in the short period of the last 5 years. The book by

w xKazakov et al. 3 is an impressive token of this fortunate scientific cross-linking.Another contribution in the present special issue of the journal is dedicated to this

w xnew type of interfacial studies of high medical relevance 109 .

Acknowledgements

Ž .The work was financially supported by the Max-Planck-Gesellschaft MIL1 , byŽ .projects of the Deutsche Forschungsgemeinschaft Mi 418r9-1, Mi 418r7-1 and

Ž .the European Union INCO Copernicus .

Notation

A drop surface areaADSA axisymmetric drop shape analysisa interfacial interaction parameterelBAM Brewster angle microscopyBSA bovine serum albuminb adsorption constant1c bulk concentration of surfactant or proteinc average protein concentrationCTAB hexadecyl trimethyl ammonium bromideC DMPO dodecyl dimethyl phosphine oxide10D diffusion coefficientD effective diffusion coefficientseffDMPC dimyristoyl phosphatidyl cholineDMPE dimyristoyl phosphatidyl ethanolamineDPPC dipalmitoyl phosphatidyl cholineDPPE dipalmitoyl phosphatidyl ethanolamine


HSA human serum albuminj mass fluxk rate constantiMBPM maximum bubble pressure methodn number of adsorption statesR gas law constantr drop radiusSDS sodium dodecyl sulphateSTS sodium tetradecyl sulphateT absolute temperaturet timeTween 20 polyoxyethylene 20 sorbitan monolaurateV drop volumex spatial coordinate normal to the interfacea model parameterb-LG b-lactoglobulinb-CS b-caseinDv molar area difference between two statesG average surface concentrationS

G surface concentration in state iiG minimum adsorptionming surface tensiong equilibrium surface tension`

« dilational elasticity«U dimensionless dilational elasticity

y1r2 Ž y1r2 .l s dgrd t slope of the dependence g tP surface pressurev molar surface areav average molar surface areaS

v minimum molar surface area1t relaxation time

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