Hydrophobicity of biosurfaces — Origin, quantitative determination and interaction energies

COLLOIDS SURFACES ELSEVIER

Colloids and Surfaces B: Biointerfaces 5 ( 19951 9l 110

Hydrophobicity of biosurfaces- origin, quantitative determination and interaction energies C.J. v a n O ss* Departments of Microbiology and Chemical Engineering, State University o[ New }brk at Bu(/alo. Bu~itlo, NYI4214-307& USA Received 12 December 1994: accepted 25 April 1995

Abstract

It is shown that the "hydrophobic" attraction energy between two apolar moieties (as well as between one polar and one apolar moiety) immersed in water is the sole consequence of the hydrogen-bonding energy of cohesion of the water molecules surrounding these moieties. It is also shown that "'hydrophobic" surfaces do not repel, but on the contrary attract water. The theory is given of hydrophobic interactions at a macroscopic level, as well as various methods for their quantitative measurement. The properties of hydrophobic, partly hydrophobic and hydrophilic compounds and surfaces are described, including those of amino acids, proteins (incorporating protein solubility), proteins at the air water interface, carbohydrates, phospholipids, phospholipid layers, and nucleic acids. Finally, some effects and applications of hydrophobic interactions are discussed, including protein adsorption, protein precipitation, cell adhesion, cell fusion, and liquid chromatography approaches such as reversed-phase and hydrophobic interaction chromatography. Finally, the influence of hydrophobic forces is treated in antigen-antibody and other ligand receptor interactions. Keywords: Biosurfaces: Hydrophobicity; Interaction energies

1. Introduction

In biological systems, hydrophobic interactions are usually the strongest of all long-range noncovalent interactions. Nonetheless, the mechanism of hydrophobic interactions is still poorly understood by the majority of workers and most remain unaware that it is feasible to arrive at a quantitative determination of their magnitude in S.I. units in most cases. One of the difficulties encountered in conceptualizing hydrophobic interactions is due to the fact that hydrophobic attractions are the net outcome of conflicting interactions, and/or of the * Tel: 1716J829-2900 or 2907: Fax: {7161829-2158. 0927-776595'$09.50 <~: 1995 Elsevier Science B.V. All rights reserved SSDI 0927-7765(95)01217-6

absence of a number of interactions, which are present in other cases. Also, many are misled by the designation "'hydrophobic" [ 1]. In actual fact, "hydrophobic" compounds or surfaces do not repel water: they attract water with rather substantial binding energies, albeit not quite as strongly as very hydrophilic compounds or surfaces. It can be shown that hydrophobic attractions are principally due to the hydrogen-bonding free energy of cohesion of the water molecules of the liquid medium in which hydrophobic molecules, sites or particles are immersed. A succinct but cogent analysis of the hydrophobic effect has been given by Hildebrand [1]. It should be stressed that hydrophobic attractions can prevail between one hydro-

92

CJ. van Oss/Colloids Surfaces B." Biointerfaces 5 (1995) 91 I10

phobic and one hydrophilic site immersed in water, as well as between two hydrophobic sites.

and 7'AB components. For the apolar components, yEW and 3,~w [3,4]: ,LW (YXf~ ~ij =

2. Nature of hydrophobic interactions

X/Y~W)2

(4)

For the polar components, yAS and ?AB [3,5]:

2.1. Theory

;'*" ij

In studying the hydrophobic attraction between solute molecules, immersed in water, it is advantageous to analyze their interaction energy (as a function of distance, if required), in the same manner used to study the stability of solid particles in aqueous suspensions. To that effect, the free energy of interactions between molecules (i), immersed in water (w), can be expressed as dGiwi:

=

2(x/v~ri e + ~/r)*~/F - x/y~v# - ,/ri%'~) (5)

It should be noted that yEW, .,,ie, and yie can be determined by contact angle (0) measurements, with at least three different liquids (of which two must be polar), using Young's equation in the following form [3,5]: , ' ~ / ~LW LW (1 + COS 0))'L = z;(X/}S ~;L ~_ ~

~). @

±. / ~ V , Of .S, ( ~ gL IT ]

(6) zJ Giw i = -- 23'iw

( 1)

where Yiw is the interfacial tension between i and water. The interfacial tension, Yiw, between aliphatic hydrocarbons and water is quite high, usually close to 5 1 m J m -2 [1], whereas it is significantly lower for even slightly polar compounds. For example, for benzene, 7iw = 35 mJ m -2 [2] (where the decrease in 7iw is solely due to the interaction between the u-electrons of benzene and water), while for more polar compounds Y~wis even lower (e.g. for n-butanol, 7iw = 1.8 mJ m 2 [2]). The interfacial tension, 7ij, can in general be determined from the surface tension components, Yi and 7j, of the interacting compounds, i and j. It should be noted that Yi consists of an apolar, or Lifshitz-van der Waals component, yLw (which comprises the dispersion, as well as the induction and orientation contributions to the van der Waals interactions [3,4]) and a polar, or Lewis acid base component, yA~ [3,5], such that "}'i = yLW _1_ ~/iAB

(2)

where 7pu comprises two parameters, y~ (the electron-acceptor parameter of the polar surface tension component) and y~¢ (the electron-donor parameter of the polar surface tension component), according to [3,5]

The manner in which the surface tensions 7i and 7j are combined to yield the interfacial tension between i and j differs drastically between the yLW

where L stands for the contact angle liquid and S for the solid (see Section 3). 2.2. Interaction between identical molecules, immersed in water

Eqs. (4) and (5) combined yield the total interfacial tension (see Eq. 2) " ~ i j = ( ~ N / ~ W - - N / * / L W ) 2-}- 2 ( @ j

+,/ri*rP-

0

(7)

and inserting Eq. (7) into Eq.(1), and equating j with w, one obtains the free energy of hydrophobic interaction between two similar solute molecules (i), immersed in water: a

AGiwi = - 2(YLw - 7J~w)z b

--4(~ d

c

+ y~we~/~ e

- ",/7]'~i Yw - ~ )

(8a)

Thus, the total free energy of hydrophobic interaction comprises the five terms (a-e) on the righthand side of Eq. (8a): a = the apolar adhesive (Lifshitz-van der Waals) interaction energy between molecules, i, and water; b = t h e polar cohesive interaction energy between the electron-acceptors and the electron-donors of the molecules, i;

CJ. van Oss/Colloids SurjLtces B: Biointerfaces 5 (1995) 91 llO

c=the polar cohesive interaction energy between the electron-acceptors and the electron-donors of the water molecules; d=the polar adhesive interaction energy between the electron-acceptors of molecules, i. and the electron-donors of the water molecules: e =the polar adhesive interaction energy between the electron-donors of molecules, i, and the electron-acceptors of the water molecules Taking i, for octane (see Table 1), it is clear that following Eq. (8a): a b ,4Giwi . . . . 0 - 0 - ( 4 =-102mJm

c d ; x 25.5)+0+

2

(8b}

Thus, in the case of a completely apolar compound such as octane, the hydrophobic attraction between octane molecules, immersed in water, is 100% due to Lewis acid-base forces, i.e. to the hydrogenbonding energy of cohesion of the water molecules of the liquid medium [8,9] (see also Tanford [10,11] and Kronberg et al. [12]). In the case of benzene ( ? L W = 2 8 . 8 5 m J m - 2 ; "'i 2.7 mJ m -2 [5,7]), Eq. (8a) becomes , .e =. 0: . -. ie dGiw i =

-0.99-0-(4

=-69.8mJm

× 25.5)+ 0 + ( 4 x 8.3) 2

philic site (usually exemplified by the electrondonor parameter, ),ie [5]) of a solute on the hydrophobic interaction energy. Many hydrophobic compounds (especially lipids} have a ?~W-value close to the ,,{4W-value of water (21.8 mJ m-2), which makes term a of Eq.(8a} close to zero. In biological compounds the highest value encountered for ",,iLw is about 40 mJ m 2 in which case the contribution from term a of Eq. (8aj would be about - 5 . 5 mJ m ~2 or about 5.4% of term c, which thus remains the main driving force of the hydrophobic interaction. To conclude, the hydrophobic interaction between molecules, i, immersed in water, is mainly due to the hydrogen-bonding energy of cohesion of the water molecules, further helped to an extent of 0-5.4% by a van der Waals attraction, and attenuated by the interaction of electron-donor sites, ,ie (which are typical attributes of hydrophilicity), with the electron-acceptor sites of water, ?w e. Completely apolar compounds have no electron-donor (or electron-acceptorl sites, and thus undergo the maximum hydrophobic interaction. By definition, hydrophobic compounds, cells, particles or surfaces are those for which dGiw i < 0, whereas hydrophilic compounds, cells, particles or surfaces, are those for which AGiwi > 0 [13].

(8c)

Here, the hydrophobic attraction between benzene molecules, immersed in water, is still 98.6% due to Lewis acid-base forces (i.e. to the hydrogen-bonding energy of cohesion of the water molecules) which is, however, 32.5% attenuated through the (net repulsive) interaction of the re-electrons of benzene with the electron-acceptors of water molecules. A comparison between Eqs. (8b) and (8c) illustrates the counteractive influence of the hydro-

2.3. Interaction between d!/lerent molecules. immersed in water

The interaction between two d(ffbrent species of molecules, 1 and 2, immersed in water obeys the Dupr6 equation [7] (see also Eq. 2~: Z/Glw2 =

,LW ~IA&,' LW j_ ~,AB .,All 132 - - i l w - - ) ' 2 w " , 1 2 --)lw

Octane Water

21.6 21.8

)e 0 25.5

' __ / ' ~LWw /~ LW

A G l w 2 = 2(~")'~ w

" 1.\~

W)w *P~','w - - \ , ' 2

b + 9~./~,e~,e e

),® 0 25.5

.,AB )2w

(9)

which in accordance with Eqs. (4) and (5) becomes 7

TabLe 1 Values of ),Lw, ;,e a n d 7 ®, in mJ m 2 for octane and water, at 20>C [6,7] ?LW

93

c '~,e~ e j

+'~/;2 ~w --2W),~ iw - - \ ' , l h --

N/; 1 t2 )

)

d },e~ •

~2 (10)

CJ. van Oss/Colloids SurIaces B: Biointerjaces 5 (1995) 91 110

94

The apolar term a in Eq. (I0) differs from term a in Eq.(8a) in that it can have a positive or a negative value. A positive value occurs when },]~w> .Lw Lw <.,}w ;w >"~w, or when 7~w < Yw ,. [7,14]. For the polar part of Eq. (10), the most important term is f, which again represents the always-present hydrogen-bonding energy of cohesion of water (see Eq. 8a). Here also, when both the molecular species 1 and 2 are completely hydrophobic, i.e. 7~ = ~'~ = 72e = ~'2e = 0, the hydrophobic interaction energy, A G l wAB2 = - - 102 mJ m 2 at 2 0 C . Van der Waals interactions (term a, Eq. 10) can strengthen the hydrophobic attraction, or attenuate it, as the case may be. (In most cases of adsorption of solutes (i) onto the water-air interface (A), 7~w> 7LwW>?'kw (where ?,kw= 0) so that LW AGwA >0; see Eq.(10) [3,6,7].) Thus. in interactions with the water air interface, a net van der Waals repulsion decreases the overall free energy of hydrophobic attraction [7]; see Section 4.2. Hydrophilic sites on molecules 1 and/or 2, which usually correspond to appreciable values of ?'~ and/or ),~ (71~ and ";2~ are generally zero or close to zero), will attenuate AG~w2 (see terms d and e, Eq. 10) in the same way as term e in Eq. (8a).

It should be noted that hydrophobic attractions between one hydrophobic and one hydrophilic moiety, immersed in water, are not only theoretically possible, but quite common in actual practice. 2.4. Decay of L W and AB interactions as a function of distance Both LW and AB interactions are relatively long-range; their strength decreases as a function of distance (l) in different ways, according to each different geometrical conformation. For flat parallel plates (l ~< 10 nm): :

Lw AÙ(o ( lo/l) 2

(11)

and for spheres, with a radius R (also for I ~< 10 nm):

~G~,'( = -An~121

t12)

where A = - 127zl~AG~Wo), lo ~ 0.157 nm [3,7] and A U(lo) cLw is defined in Eq. (1) or, for other geometrics, is a similar function of 7 Lw. For AB interactions, in the case of fiat parallel

plates:

and for spheres with a radius, R:

AGm=TzR2zlG~exp(U)

(14)

where 2 ~ 0 . 6 - 1 . 0 nm for hydrophilic repulsions [6,7], but may vary between 0.2 and 13 nm or more, depending on whether l is very small (e.g. from / ~ 0 . 1 6 to about 1 nm) or large ( l ~ 1 0 n m or more) [6,7,15]. Contrary to electrostatic interactions (which decay steeply or more gradually as a function of distance, according to whether the ionic strength of the liquid medium is high or low), the rate of decay as a function of distance of LW and AB interactions is not greatly influenced by the ambient ionic strength, at ionic strengths below 1.0.

3. Measurement

3.1. Traditional approaches Traditionally, hydrophobic interaction energies have been typified by the various thermodynamic components (enthalpies, entropies and heat capacities) involved in the actual or imaginary transfer of hydrophobic compounds to water (either directly, or via prior solution in another organic solvent) [10,11,16]. These thermodynamic entities were sometimes based on direct calorimetric measurements [10,11], but they were more usually derived from, for example, solubility data, or vaporization parameters [ 10,11 ]. However, the relation between the free energy of hydrophobic interaction and the energy required to transfer a hydrophobic compound to water, from the liquid state or from another organic solvent, is not the most fruitful approach for elucidating the mechanism of hydrophobic interactions. As mentioned in the preceding section, the free energy of hydrophobic interaction is most conveniently expressed as the free energy of interaction of low-energy molecules (i), immersed in water (w):

C.J. van Oss Colloids

AGiw~. As seen two times the pound (i) and determined: see

SlojacesB: Biointerlaces

in Eq. (1), this is equal to minus interracial tension between comwater, ;'iw, which can easily be below.

3.2. lnterlilcial tensions Interfacial tensions between two immiscible liquids can be measured directly by a variety of methods (e.g. hanging drop, spinning drop and drop weight approaches [7,17]). For most waterimmiscible organic liquids, measured interracial tensions with water have been tabulated [2] and, for a number of compounds, have been determined at different temperatures [18]. Solid water interfacial tensions cannot be measured directly, but can be determined by contact angle measurements with three different liquids, using Eqs. (6) and (7) [3,5,7]. For water-miscible liquids, the 7 Lw, ~'¢j and .,e, values must first be determined by contact angle measurements on different apolar and polar solids, using Eq. (6) [7,19]. Interfacial tensions can then be obtained as usual, via Eq. (7). One of the tenacious axioms of traditional hydrophobic effect theory is the assumption that the interactions are obligatorily entropic [20,21]. This is simply not true. It can be shown that whilst for alkanes the free energy of hydrophobic interactions is indeed mainly entropic [11 ], for other hydrophobic compounds these interactions can be mainly enthalpic (e.g. for CC14, heptanoic acid and dibromoethane) and for some others almost equally enthalpic and entropic (e.g. benzene). These data [7.9] were derived from ;'iw-values measured at different temperatures. One may object that the determination of AG~w~ via measured ;'iw-values ( Eq. 1) is not really a direct measurement of free energies of hydrophobic interaction. There are. however, at least two other approaches for verifying the correlation between AG~w~-values, obtained as above, by more direct physicochemical or purely physical observations. The first approach is based upon the connection between AG~w~-values and the aqueous solubility of hydrophobic compounds (i). The second approach entails the direct physical measurement of the attractive forces between two hydrophobic surfaces (i), immersed in water: see below.

5 (1905) 91 110

95

3.3. Solubility The aqueous solubility (s) of slightly, soluble hydrophobic compounds may be expressed as [6,7] zJGiw i S c =

kTln ~

( 15t

where Sc is the contactable surface area between two interacting hydrophobic molecules, k is Boltzmann's constant, T is the absolute temperature and s is expressed in units of moles of (i} dissolved per mole of H20. In other words, the (short-range) free energy of interaction between two hydrophobic molecules immersed in water, expressed in k T units, equals the natural logarithm of their solubility in water, expressed in mole fractious, Equations for expressing the aqueous solubility or organic compound, of the type of Eq. (15), have been proposed previously, [22-26]. However. in all these cases a constant, /7, was employed instead of AGiwi/kT, and instead of using the contactable surface area, Sc, between two hydrophobic molecules, the total surface area of the cavity filled by one hydrophobic molecule (extended by the radius of the water molecules surrounding the cavity) was employed. Nonetheless, for a given class of compounds, e.g. alkanes, fl was found to have a constant vahie [22,23,25]. This is not surprising as, for all alkanes, AG~wi varies little from the value of - 102 mJ m e (see Eq. 8bL Thus, for purely apolar compounds such as octane or cyclohexane, the solubility, s )see Eq. 15) at 20 C may be expressed by. the general approximation s ~ e x p ( - 25 Sc)

( 16t

where s is expressed in mole fractions 1 tool 1 ~ = 0.018 mole fraction) and Sc in nm e. For instance, using Eq.(16), for octane ( S c = 0 . 5 1 6 n m -~. see Table2) s z 2 . 5 × 10 -~i mole fractions, which is reasonably close to the experimental value of 2.2 x 10 ~ mole fractions (Table2), considering that Eq. (16) is an exponential equation. From the AGi,,,~ data given above, it is possible to derive the Sc-values for e.g. octane and benzene, from their solubilities; see Table 2. It is clear from Table 2 that the contactable surface areas, Sc, found for octane and benzene,

C.J. van Oss/Colloids Surfaces B: BiointerJaces 5 (1995) 91-110

96

Table 2 z/Giwi values in k T (obtained from aqueous solubilities, using Eq. 15) compared with AGiw~ values in mJ m 2 (obtained from interfacial tensions, Eq. 1), to obtain the Sc-values for octane and benzene

Octane Benzene

AGiwi (mJ m 2)

Solubility (s)a (mole fractions)

-102 -69.8

2.21 x 10 -6 5.185 x l0 4

AGiwi

(kT) 13.023 -7.565

Sc (nm 2) 0.516 0.438

a From Ref. [27].

correlate well with the surface areas of one side of these molecules, measured at their van der Waals boundary, known from their molecular dimensions [28]. Conversely, when Sc is obtained from the molecular dimensions of an apolar organic molecule, its hydrophobic interaction energy, zlG~wi, can be found directly from its aqueous solubility, s, using Eq. (15). For partly apolar and partly polar molecules (e.g. alcohols) the z/Giw i values (in kT) for the apolar and the polar parts have to be determined separately (and then added together), to obtain reliable correlations between the AGiwi and Sc-values. In applying this principle to non-ionic [29] as well as to anionic [30] surfactants, their critical micelle concentrations (which may be equated with s [ 17]) can be predicted fairly accurately from interracial tension data.

3.4. Direct measurement With force balances of the type developed by Israelachvili [20], the attractive forces between two crossed half-cylinders, coated with smooth mica, which in turn had been coated with hexadecyl [20,31] or octadecyl groups [32], immersed in water, could be measured directly. Considering that the hydrophobization of these mica surfaces (which was done with quarternary ammonium bases) was always less than complete (as shown by contact angle measurements), the correlation between calculated (Eq. 8a) and measured AGiwivalues is quite close [7,8].

3.5. Influence of temperature The influence of temperature on hydrophobic interactions between completely apolar moieties, immersed in water, is rather slight. The decrease in hydrophobic attraction energy between e.g. alkanes, immersed in water, upon heating from 20 to 40°C is about 5% [7]. For partly polar compounds, however, temperature effects can be much more significant. For instance, upon heating polymethylmethacrylate particles, immersed in water, from 20 to 40°C, AGiwi changes from - 3 0 . 3 to - 17.0 mJ m z, signifying a 44% decrease in attractive energy (data from Ref. [7], p. 176). Nylon-6,6 particles, immersed in water, actually show a change in sign upon heating from 20 to 40"~'C: at 20°C AGiwi= - 5 . 4 m J m 2, while at 40°C AGiwi becomes + 11.6 mJ m - 2. The reasons for these two different phenomena are as follows. The hydrophobic interaction energy between completely apolar moieties, immersed in water, is proportional to ~/AB (for water), which decreases only from 51 to about 49 mJ m 2 when heating from 20 to 40°C. However, partly polar compounds, immersed in water, are also influenced by the separate values of ~w ~ and yw e (for water), which change from 25.5 mJ m -2 for both (at 20cC), to },w ~ = 32.4 and ,,e~w= 18.5 mJ m -2 at 38:C: water becomes a much stronger Lewis acid at higher temperatures [-6,7]. Using Eq. (Sa) it will become clear that for electron-donor monopolar compounds such as polymethylmethacrylate and nylon, the strong increase of Yw* with temperature will markedly increase the (absolute) value of term e of Eq. (8a), which results in a decrease in the negative value of AGiwi upon heating (polymethylmethacrylate) or even in a change in AGiw~from negative to positive (nylon-6,6).

4. Properties of hydrophobic and partly hydrophobic surfaces and compounds

4.1. General properties of hydrophobic surfaces Usually, hydrophobic surfaces (i) are surfaces with a low ~,~, as well as a low y~ parameter. Most biological surfaces and macromolecules have

CJ. van Oss Colloids Surlaces B. Biointe~jitces 5 (1995) 9l 110

a , ,e value which is exceedingly low (i.e. of the order of 0.1 mJ m 2 or less) and, when hydrophilic, a high ,,e (more than 28.5 mJ m 2) and thus m a y be designated as essentially " m o n o p o l a r " [5,7]. The best definition of a h y d r o p h o b i c surface (i) is a surface for which z i a i w i < 0 [13]. For most lipids, ,,I.W ,i ~ 22 mJ m 2 and ;i~ 0 , so that AGiwi ~< 0 when ~,ie ~: 25.5 mJ m -i, at 2 0 - C (see Eq. 8a). For biological macromolecules and other non-lipidic surfaces, it is more realistic to assume "'FlW, ~ 4 0 mJ m 2 in which case AGiwi < 0 when .,e < 28.35 mJ m 2 ,t

4.2. Completely hydrophobic compounds and st,~liwes In this category are all completely saturated hydrocarbons, e.g. alkanes, cycloalkanes, decah y d r o n a p h t h a l e n e (decalin), and in general all c o m p o u n d s or surfaces with an interfacial tension with water. ~'iw, of a b o u t 4 5 m J m -2 or more [2,7]. Even c o m p o u n d s with a 7~w-value of almost 45 mJ m 2 (e.g. l - b r o m o n a p h t h a l e n e , with /iw ~42.1 [2]) are often considered to be completely hydrophobic, for all practical purposes [7]. More generally, all c o m p o u n d s or surfaces with zero values for their ;'* and ~,o parameters are completely hydrophobic, e.g. Teflon F E P , polytetrafluorethylene, polyisobutylene, polypropylene, polyethylene [7]. Thus, c o m p o u n d s or surfaces with A G i w i ~ - 8 4 m J m 2 m a y be classified as completely hydrophobic. The air water interface is one of the most hydrophobic surfaces known. However, its h y d r o p h o b i c attraction energy to biological solutes, c o m p o u n d s or cells that have ;,~w > 22 m J m 2 (which applies to most biopolymers and other biological surfaces), is mitigated by the net repulsive van der Waals interaction between these biopolymers or other biological surfaces ( 1 ) and the a i r - w a t e r interface, where air is designated as 2 (i.e. AGlw2 Lw > 0). For instance, in the case of most proteins (for which, in the dry state, 7]~w ~ 4 0 m J m 2 [6]), AGlw 2Lw (2 again signifying the air phase) is a b o u t + 15 mJ m 2. Thus, whilst the attractive energy of e.g. fibrinogen for teflon has a AG~w2 value of about --26 mJ m 2 the AG~w2value for fibrinogen

9 "7

with respect to the air--water interface is only approximately - 11 mJ m -' [7].

4.3. Partly hydrophobic compounds amt smilbces C o m p o u n d s or surfaces with AGiwi-values between - 8 4 and 0 mJ m e m a y be considered as partly hydrophobic. This category comprises most of the c o m p o u n d s or surfaces normally considered as hydrophobic, with e.g. benzene on the more h y d r o p h o b i c side of the scale (zJGiwi~-..-70 mJ m - - ' for benzene; see above). More moderately h y d r o p h o b i c surfaces are those of: polymethylmethacrylate (AG~wi= - 3 7 . 8 mJ m -'/ [7]: talc (AGiw i = - 37.4 mJ m 2): ZrO2 (~]Gi~ i = - 52.7 mJm-:t: SnO2 ( A G i w i = - 3 0 . 2 m J m :) [33]; cellulose acetate film (JGiwi = - 2 0 . 7 m J m 2). Of these, ZrOz is clearly the most and cellulose acetate the least hydrophobic.

4.4. Surtaces and compounds that can chanee/)'om hydrophilic to hydrophobic Whilst m a n y biopolymers can be strong m o n o polar electron-donors, and thus are very hydrophilic without having any electrostatic potential (e.g. various polysaccharides [5,71t, the reverse is not true. There are virtually no strongly (e.g. negatively) charged biosurfaces that are not also strong electron-donors l i.e. they have a high ~'e-valuel [7]. However, when such strongly charged surfaces are neutralized, their ,,e p a r a m e t e r diminishes severely, which then causes these surfaces to become hydrophobic. This can happen in the two following ways. (1) Amphoteric surfaces can be made h y d r o p h o b i c by bringing the pH of the ambient aqueous medium to the isoelectric point [7,34 36]. (2t Electrostatically charged surfaces can be partly or wholly neutralized by the admixture of plurivalent counterions (i.e. plurivalent cations in the case of negatively charged surfacesl. In the process, they become hydrophobic [7,37 40]. The activity of cations in hydrophobizing negatively charged molecules or surfaces follows the S c h u l t z e - H a r d y rule [41], i.e. the cation concentrations required for h y d r o p h o b i z a t i o n are as follows: N a - > Ca 2- > La 3" > Th 4-. For Na concentrations larger than 0.1 M are typically

98

C.J. van Oss/Colloids Surfaces B: Biointerfaces 5 (1995) 91 110

needed [42], for Ca 2 + the required concentrations are larger than 2 0 r a M [25], for La a+ they are only a few tenths of 1 mM [38] and for Th 4+ even smaller concentrations suffice [40].

4.5. General properties of hydrophilic surfaces In contrast to hydrophobic compounds and surfaces, hydrophilic compounds and surfaces have: AGiwi~>0. Thus, for hydrophilic biopolymers, cells and other non-lipidic surfaces (with 7~w ~ 40 mJ m -2 and -/ff ~ 0), y e > 28.35 mJ m -2 (see above) [5,7]. Even very hydrophilic biopolymers can adsorb onto hydrophobic surfaces. For instance, dextran (1) (Mw ~ 150000), with y~w = 42 mJ m 2, 7~ = 0 and y~ = 55 mJ m-2, will adsorb onto clean polystyrene (2), with v ~ w = 4 2 m J m -2, ~/2"= 0 and y2e = 1.1 mJ m -z [17], from an aqueous solution (~,LW 21.8 mJ m - z ),w ~ = yw ° = 25.5 mJ m - 2), because (see Eq. 10), AGlwz = -23.1 mJ m -z. The ability of hydrophilic biopolymers and biooligomers (e.g. proteins and peptides) to adsorb onto hydrophobic surfaces from aqueous solutions is the basis of the adsorption step in reversed-phase liquid chromotography (see below).

4.6. Surface properties of amino acids A fair number of different classifications of the more prevalent amino acids according to the relative hydrophobicity or hydrophilicity of their side chains can be found in the literature [43-47]. Some are based on the energy involved in the transfer of various side chains from a given solvent to water [44,45], or on their partition coefficient in e.g. an aqueous Ficoll/Dextran system [43]. The amino acid orders found by these approaches are rather similar. Kyte and Doolittle have produced a list of amino acids ordered according to their putative decreasing hydrophobicity, obtained by means of a computer program that takes a variety of properties of amino acids into account [46]; the amino acid order in this list differs significantly from that in all the others, inter alia featuring tryptophane as hydrophilic. It is probably prudent not to attach to much weight to this list. Finally, Parker et al. [47] give a classification

based on the retention times of synthetic peptides in reversed-phase liquid chromotography. The synthetic peptides are the same, except for the insertion of two (identical) amino acids two places from the carboxy terminal. The order, again, deviates little from that found by most of the other approaches. In general, the order of amino acid side cains, beginning with the most hydrophobic, is probably best summarized as follows: Trp, Phe, Leu, Ile, Met, Val, Tyr, Cys, Ala, Pro, His, Arg, Thr, Lys, Gly, Glu, Set, Asx, Glu, Asp (according to Parker et al. [47], where the amino acids to the right of Thr are the more hydrophilic ones). It should be stressed that an amino acid with a larger hydrophobic side chain is more hydrophobic (in terms of kT) than an amino acid with a small hydrophobic side chain. Thus, Trp is more hydrophobic, in practice, than e.g. Val, even though the (small) side chain of Valine is actually more apolar than the (large) side chain of Tryptophan. The above list may allow one to arrive at a rough estimate of the likely degree of hydrophobicity (or hydrophilicity) of a given site of a peptide or protein, once its amino acid composition is known. There is, however, another possible pitfall in estimating the approximate hydrophobicity of a given site from its amino acid composition, i.e. otherwise hydrophilic acidic and basic amino acids, when present within reasonable proximity of one another in the same site, will not only approximately neutralize each other elecrostatically, but will also render the site much more hydrophobic [7,36,48]. The pH of the medium thus clearly plays a crucial role in the relative hydrophobicity of amino acids, as does the presence of plurivalent counterions; see Section 4.4.

4.7. Surface properties of proteins 4.7.1. Protein at the air water interface Water-soluble proteins may be regarded as monomolecular micelles. Multimolecular micelles are soluble aggregates of amphipathic molecules, such as surfactants, with the hydrophobic sites at the micelle interior and the hydrophilic sites at the water interface. Similarly, water-soluble proteins have the majority of their hydrophilic amino acids at the water interface (or else they would not be

C.J. van Oss/'Colloid~" SurJaces B." Biointer/iwes' 5 (1995) ~1 110

soluble in water), while the more hydrophobic amino acids tend to be buried inside the threedimensional framework of the macromolecule, in its native, dissolved state [7]. The surface properties of proteins are relatively easily determined, e.g. by contact angle measurement on a layer of the protein air-dried from a solution deposited on a glass plate. It should be realized that many (but not all) proteins become reversible denatured upon air-drying. This is because in the process of airdrying, proteins dry first at the solution air interface. Now, proteins adsorb strongly at the a i > water interface, which is quite hydrophobic (see Section 4.2}, and upon adsorption onto hydrophobic surfaces proteins tend to orient their most hydrophobic sites to the hydrophobic surface [7,49]. This is manifested by the fact that many proteins, for instance human as well as bovine serum albumin, quickly lower the surface tension of the water in which they are dissolved (at fairly low concentrations) by about 20 mJ m-2, or more [50]. Once a protein has been air-dried, its more hydrophobic moieties are exposed to the surface at which contact angle measurements would be performed. One thus finds that many (but not all) proteins that were air-dried have become fairly hydrophobic [7,51,52]. Examples of proteins in that category are serum albumin, immunoglobulin-G, egg lysozyme and tobacco mosaic virus. However, other proteins do not appear to become hydrophobic upon air-drying, e.g. fibrinogen and fibroncctin, or become only slightly less hydrophilic upon air-drying, e.g. myelin basic protein [7,52].

4.7.2. Solubility ot'proteins Proteins that fall in the first category are not soluble in water from the air-dried state. This is readily noticed when attempting to dissolve such air-dried proteins in water. At first, the dry granules just float around or sink; they are then seen to swell l i.e. they become rehydrated) and finally observed suddenly to dissolve completely. A tentative reason for the difference between proteins that reversibly become more hydrophobic upon airdrying and those that remain hydrophilic is that those becoming more hydrophobic all tend to be rather globular, whilst the unaffected proteins consist of unfolded chains that are much longer and

99

thinner (e.g. fibrinogen and fibronectin), so that they have little scope for internalizing hydrophobic amino acids and thus depend on the total mean hydrophilicity of their primary amino acid composition for aqueous solubility. Contact angles on hydrated proteins are measured on thick layers of extremely concentrated protein in aqueous solution. These are prepared by further concentrating already concentrated solutions of protein in a pressure ultrafilter [7,36,52]. By this approach, the surface properties of proteins can be determined as a function of their degree of hydration. For instance, for human serum albumin the surface properties could be determined with one, as well as with two, molecular layerlsl of water of hydration, showing an orientation of the water molecules of hydration (with the oxygen atom outward) of about 74% in the first molecular layer of water of hydration, and of 31% in the second layer [7,36]. For the solubility of proteins, it is essential that virtually all of the amino acids at the protein outer interface with the aqueous medium be hydrophilic. One or two hydrophobic amino acids somewhere at a protein's outer surface may be tolerated, but a hydrophobic patch on the surface of, say, about 1 nm 2 surface area would certainly cause aggregation, i.e. insolubility. Immunoglobulin G (IgG) may be taken as an example. Its i-potential is less than 1 - 1 0 mVl [53], so that the net electrostatic (EL} repulsive interaction energy between lgG molecules under physical conditions, at close range, is extremely small (AGi~(i~ + 0 . 2 m J m 2), and thus may be neglected. This leaves van der Waals (LW) and hydrophobic (AB) interactions. Now, IgG molecules tend to have hydrophobic patches, e.g. in the paratope region of the combined VL and Vu chains [54,55], but these hydrophobic paratopic patches occur in concavities, or clefts [48,56], so that a collisional encounter between two or more IgG molecules will not result in aggregation. It is easy to see what would happen if a hydrophobic patch of, say, 1 nme in surface area were to exist in an accessible site of the lgG surface. For the properties of such a hydrophobic site, it is plausible to assume that they are similar to the surface properties of dried IgG, i.e. ,,!,vv,,=42, 3'i~ = 0.3 and )'ie = 8.7 mJ m 2. Using Eq. t 8a), one

100

CJ. van OssT'Colloids Surjaces B." Biointerjaces 5 (1995) 91 110

then obtains dGiwi = - 4 4 . 4 m J m -2, which is about 15% due to van der Waals and 85% due to hydrophobic (AB) interactions. For a hydrophobic site with a contactable surface area, Sc ~ 1 nm 2, this amounts to z~Giwi = l l . l k T (or - 6 . 4 kcal mol 1), which strongly favors attraction, i.e. aggregation. The contactable surface areas of paratopic sites of IgG are usually larger than 1 nm 2 (they vary roughly between 0.4 and 10 nm 2 [48]), but may not necessarily be entirely hydrophobic. Nonetheless, the size of a hydrophobic patch within a paratopic site would quite reasonably be about 1 nm 2. It is, therefore, necessary for the free solubility of I g G molecules that such hydrophobic patches remain excluded from accidental contact by sequestration within a cleft. One may thus estimate in general that when hydrophobic amino acids are present at the accessible surface of water-soluble proteins, they must occur in patches of only a few tenths of a nm 2 in surface area, giving rise to a mutual attachment energy of not more than about -2kT. On the outer surface of water-soluble proteins, one may therefore find an occasional single hydrophobic amino acid and very rarely two neighboring hydrophobic amino acids (e.g. of the group of alanine or isoleucine). Amino acids with rather large hydrophobic chains (e.g. tryptophan or phenylalanine) are seldom found singly at a prominent site on the outer surface of water-soluble proteins [57], and even more rarely in pairs. There are, of course, proteins which are insoluble in water. For instance, the corn protein, zein, is insoluble in cold as well as in hot water, but soluble in dimethylsulfoxide, formamide and ethylene glycol, and partly soluble in a 70:30 (v/v) acetone water mixture. Its surface properties = 41.1, 71e = 0.04 and 3,ie = 18.4 mJ m - 2 a r e : ,,Lw ,i [7,36,58]. Gelatin (with ,,~w = 37.6, ~ = 0 , ?,~ = 18.5 mJ m 2 [7]) is also insoluble in (cold) water; it becomes soluble in water upon boiling, but forms a gel at temperatures below about 40°C. Both proteins are clearly too hydrophobic to be soluble in water; in both cases, AGiwi~-19.5 mJ m 2. As such proteins clearly attract each other when immersed in water, their contactable surface area is of the order of ~> 10 nm 2. Thus, they attract each other with an energy of at

least about -48kT, which gives rise to complete insolubility in water (see Eq. 15). However, when thread-shaped gelatin molecules have been dissolved in boiling water (their aqueous solubility at high temperatures, is, at least in part, a consequence of the high ,,e,w and the low ~,e~wvalues at higher temperatures [7]), its long peptide chains repel each other largely at right angles. Thus, at higher temperatures the Sc-values for gelatin are rather low, i.e. probably of the order of only 0 . 5 n m 2. Upon cooling, the points of close encounter, which undergo a change from repulsion to attraction, still only attract each other at sites of about the same small surface area of, say, about 0.5 nm 2. This gives rise to the formation of a peptide-chain network held together with pointwelds each with d G i w i ~ - 2 . 4 k T , which fits well with the properties of gelatin gels. Gelatin consists mainly of glycine, proline and hydroxyproline [59].

4.8. SurJace properties of sugars and polysaccharides In contrast with proteins, which can comprise hydrophilic and/or hydrophobic amino acids in any proportion, polysaccharides consist mainly of multiples of monomeric sugars which are all rather hydrophilic and quite soluble in water (with solubilities at 20cC varying from 11% for lactose, to 48% for glucose and 67% for sucrose). Such high to medium aqueous solubilities indicate a low degree of hydrophobicity. These small sugars are not completely hydrophilic (i.e. their AGiwi value is still negative [7]), but their molecular weights, and thus their Sc values, are relatively small, so that a high aqueous solubility still prevails. Nonetheless, in the form of polymers these simple sugars may either become more hydrophilic or more hydrophobic, dependent only on the structure of the polymer molecule and not at all on the monomeric building block. For instance, the cyclic heptamer of glucose, cyclomaltoheptaose, or fi-cyclodextrin (fi-CD)is, on a weight percent basis, 26 times less soluble in water than glucose and about 162 times less soluble on a molar basis. The inner surface of the fi-CD ring is very hydrophobic [60] and serves to bind many organic inclusion

c.J. van Oss/Colloids Sutjaces B: Biointer/aces 5 ~1995; 91 110

compounds, for purposes of separation [60,61]. However, the decreased aqueous solubility of fl-CD (compared to its monomer) cannot only be due to the hydrophobicity of its inner cavity [61]. The outer surface of the cyclic structure is also significantly more hydrophobic than its monomer, glucose [7]. Curiously, both the smaller cyclic glucose polymer, cyclomaltohexaose, or ~-cyclodextrin, and the larger cyclic polymer, cyclomaltooctaose, or 7-cyclodextrin, are much more soluble in water than fi-CD [60]. Thus, the subtle orientation of the monomeric glucose moieties that constitute these cyclic polymers plays a crucial role in the surface hydrophobicity of the polymers. Linear polymers of glucose, such as dextran, are more hydrophilic than their monomeric subunit [7], and at least as soluble in water. Other polymers of glucose are about as (mildly) hydrophobic as glucose, but usually insoluble in water owing to (a) their larger size, and (b) their crosslinking. Examples of this are cellulose and amylopectin [7]. The linear analog of amylopectin, amylose, is more hydrophilic than amylopectin, and soluble in water [7] (amylopectin is the major component of cornstarch: amylose is an important component of potato starch). Some cellulose esters are similar in hydrophobicity to cellulose (e.g. cellulose acetate [7]~, others are more hydrophobic (e.g. cellulose nitrate [7,62]). The hydrophilicity or hydrophobicity of aqueous gels of polysaccharides can strongly depend upon which surface of such a gel is used for measuring the degree of hydrophilicity or hydrophobicity via contact angle determinations. For instance, aqueous agarose gels are slightly hydrophobic when measured at the air interface where the gel solidified, and totally hydrophilic (almost like water) at surfaces that have been freshly cut open [7,63]. (Agarose is a polygalactose, made from the seaweed polysaccharide agar agar, from which the sulfate groups have been removed by hydrolysis). When hot agarose solutions are poured into fiat-bottomed petri dishes made of clean hydrophilic glass, the gel, after setting, is also quite hydrophilic at the surface where it touched the glass. If, however, hydrophobic petri dishes made of polystyrene are used, the gel's former interface with the plastic is more hydrophobic.

101

In conclusion, surfaces of potysaccharides made of the same sugar (e.g. glucose) can vary from exceedingly hydrophobic (e.g. the toroidal inner surface of the /#cyclodextrin ring) to extremely hydrophilic (e.g. the surface of the largely linear polymer, dextran), which seems to indicate that a polysaccharide's hydrophilicity is closely linked to the maximum extent of freedom of movement of its constituent monomer moieties. When a polysaccharide has been rigidified in a certain conformation, e.g. by gelling, surfaces that were originally oriented at a hydrophobic interface (e.g. air or polystyrene) will become at least somewhat hydrophobic, and surfaces that had been oriented towards a hydrophilic surface (e.g. the bulk waterphase at the interior of the gel, which is about 99% water, or a hydrophilic glass surface) remain very hydrophilic. This also indicates that the monomeric sugar moieties of polysaccharides can be hydrophobic or hydrophilic, dependent solely on their three-dimensional conformation. 4.9. SutJ~lce properties of phospholipids 4.9.1. Formation of phospholipid hilavers

Phospholipids are essentially surfactants, which are either anionic (e.g. phosphatidic acid (PA) and phosphatidyl serine (PS)), or non-ionic (e.g. phosphatidyl choline (PC) and phosphatidyl ethanolamine (PEA)), with (usually fairly long) hydrophobic tails which attract each other in water, and hydrophilic heads which repel each other in water. Like other surfactants, they tend to form organized micelles in water at concentrations that are higher than the aqueous solubility' of single phospbolipid molecules. However, the aqueous solubility of phospholipids is so low that dried phospholipid powders do not spontaneously disperse in water to form micelles or vesicles. To form phospholipid vesicles with unilamellar bileaflet membranes, phospholipids (e.g. PA, PS or PC)need to be dried from a solution m an organic solvent, and then suspended in water, vortexed, and finally sonicated [64,65]. 111 this manner, "'naked" phospholipid vesicles are readily obtained. However. naturally occurring vesicles or cells usually also carrying glycocalyx just outside the cell membrane. Thus, the physicochemical properties (e.g. surface tension

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CJ. van Oss/Colloids Surjaces B. Biointerfaces 5 (1995) 91 110

properties and (-potential) of phospholipid membranes cannot readily be measured on unaltered native material directly obtained from in vivo preparations. In practice, one must therefore resort to measurements on artificially prepared phospholipid vesicles.

unsaturated and polyunsaturated lipid chains all will tend to segregate into discrete regions that together constitute the cell or vesicle membrane. Thus, a mosaic of separate patches will be formed of saturated, unsaturated and polyunsaturated lipid chains.

4.9.2. Properties of lipid moieties of phospholipid layers

4.9.3. Insertion of proteins into phospholipid layers

Saturated alkyl chains of phospholipids attract each other very strongly, in water, with a hydrophobic energy of attraction, AGlwl ~- - 102 mJ m -2 in all cases [7]. For a contactable surface area for e.g. hexadecyl chains, Sc ~ 1.0 nm 2, this amounts to AGlw I ~ - 2 5 . 4 k T (dGlwa AB = -- 25.2kT and AGlwl L W = -0.16kT). In the absence of water, the free energy of cohesion between two hexadecyl chains, AGtl = - 5 5 mJ m -2, or -13.6kT. It should be noted that in the absence of water, AG11 = AG~ w in this case (and in almost all other cases of lipid-lipid interaction). The hydration energy of hexadecyl chains at first sight is not negligible: AGlw = - 4 9 mJ m 2. However, because of the smallness of the contactable surface area of the individual water molecules (Sc ~ 0.15 nm2), this only amounts to AGlw = -1.8kT. Thus, in any case where IAGlwlIAB< [AG~[, the water of hydration can be expelled. However, in the case of saturated alkyl chains (here, hexadecyl), ~" > IAGLWl, so that the expulsion of water [AG~wll of hydration is not favoured and the hydrophobic (AB) attraction does not change into a cohesive (LW) bond; see Fig. 1. With unsaturated lipid chains, AGaw~ A B ~ A G l lL W , and with polyunsaturated lipid chains IAGlwl[ AB < [AGLllW[(see Fig. 1), so that here expulsion of water may, in theory, be expected. In practice, however, this is somewhat more difficult to predict, as with unsaturated and especially with polyunsaturated lipid chains the "fit" between two adjoining chains is often less perfect than among saturated unbranched alkyl chains. This tends to give rise to the continuing presence of residual interstitial water (and concomitantly to a weaker bond), for steric reasons. Given the much stronger hydrophobic attraction between saturated lipid chains than between unsaturated or polyunsaturated lipid chains (see Fig. 1), it can be predicted that saturated lipid chains and

Using Eq.(10), it is easily shown that hydrophobic (e.g. Y~ot~in~ 1 0 - 1 6 m J m -2) as well as hydrophilic (e.g. 7~otein ~ 36 mJ m -2) protein moieties are capable of binding saturated as well as unsaturated and polyunsaturated lipid chains (Fig. 2). However, hydrophobic protein moieties bind any lipid chain more strongly than hydrophilic protein moieties; either one binds saturated lipids more strongly than unsaturated lipids, and these more strongly than polyunsaturated lipids (Fig. 2). Thus, with proteins that have a hydrophilic and a hydrophobic moiety, the hydrophobic moiety is the most likely to be inserted between lipid chains. Also, when such proteins are present, their insertion (via their hydrophobic moiety) is most strongly favored to occur between saturated lipid chains (Fig. 2). It is, therefore, likely that the proteins that are inserted in phospholipid cell membranes are preferably situated in the saturated lipid patches, described above.

4.9.4. Properties of hydrophilic moieties of phospholipid layers Owing to the relative ease of obtaining phospholipid vesicles in aqueous suspensions, most macroscopic surface data obtained experimentally on phospholipds have provided information on their hydrophilic moieties, which are found at the outside of the vesciles, i.e. at the vesicle-water interface. Direct contact angle measurements have been done on hydrated layers of PC vesicles deposited on glass, yielding: 7~w=29.1, 7ie=2.65 and ?,ie = 60.0 mJ in 2, from 0 D i i o d o m e t h a n e = 50.25'; 0~_ B ...... phthalene = 51.5 ~; 0Hexad. . . . . = 29.9 °; 0Water= 7°; 0Olyc~roI = 32" [66]. Thus, the stability of PC vesicles in aqueous suspension [64,65,67] (which cannot be due to their (-potential, as that is close to zero [64,67], is entirely a consequence of hydrophilic (AB) repulsion. From the data given above,

CJ. van Oss Colloids Sut~laces B." Biointer/aces 5 (1995) 91 110

103

"25kT t I

-lOkT

-5kT

AGIwI AG11 AGIw

AGIwl AG11 AGIw

Saturated

Unsaturated

AGIwl AG11 AGIw Polyunsaturated

Fig. 1. Free energies (in kT) of (from left to right) saturated (hexadecyl), unsaturated and polyunsaturated hydrophobic chains (1) of the same size (Sc = 1.0 nmZ}, immersed in water (JGlwl) and in the absence of water (AGII), as well as the free energies of hydration (AG~). In all cases, ,IGlw~ consists mainly of hydrophobic (AB), and AGH and AG~,~ mainly of LW interactions.

it can be shown that for vesicles with a diameter of 50 nm, using Eqs. (8a) and (14), A Giw i = + 140k T at close range; this value is more than 97% due to repulsive AB interactions, opposed by a small LW attraction (representing less than 3% of the total interaction energy). With the (negatively) charged phospholipids (PA and PS), vesicle stability is also for an important part due to the electrostatic repulsion component, EL AGlwl. However, this value is only about 40% of the total repulsive energy. Whilst for a PC vesicle of 50 nm diameter, zIGiAw]= + 140kT (see above), for a PA or PS vesicle of the same size, with a surface potential, ~o, of about - 6 0 m V , zlGi~w~= + 100kT, yielding a total repulsion energy, GTOT ~ + 240kT (with a small opposing LW attracIWI tion of about -4kT). An important difference between hydrophilic particles without an electric charge (e.g. PC) and hydrophilic particles with a significant surface

charge (e.g. PA, PS), is that the hydrophilicity of the latter two is, to an important degree, coupled to the surface potential. When the surface potential of charged particles is decreased, for instance by the admixture of plurivalent counterions (e.g. C a 2+ ), they become concomitantly much more hydrophobic [-7,37 39]; see also Section 4.4. The hydrophobizing action of C a 2 + ions on phospholipid membranes is of particular importance in cell or vesicle-fusion (see Section 5.4).

4.10. Sulfate properties of nucleic acids As seems reasonable from their chemical composition, DNA is somewhat more hydrophobic than RNA [7]. Thus, DNA adsorbs more strongly onto cellulose ester membranes than RNA. This forms the basis of the Southern Blotting technique I-7,62]. Strands of DNA may be considered to consist

C.,L van Oss/Colloids Surjaces B." Biointer/aces 5 (1995) 91 110

104 c

Q. O

o

.~

?,

,,,

~

P-

-15kT c-

=:

~-

o

~

O Q.

-lOkT

O ¢.. O.

o 10

-5kT

I

' 1:

i' i

t'1

1:

AGlw 2 AGlw 2 AG12

AGlw 2 AGlw 2 AG12

Saturated

Unsaturated

I

3: |

I

I|

AGlw2 AGIw 2 AG12 Polyunsaturated

Fig. 2. Free energies, AGlw2 (in kT) of hydrophobic (7~ = 16 mJ m -2) and hydrophilic (5,~ = 36 mJ m 2) protein chains, interacting with (from left to right) saturated, unsaturated and polyunsaturated lipid chains (as in Fig. l). In all examples shown, the free energies, AGI=, of interaction between proteins and lipid chains in the absence of water have a value of 17kT, which is more negative than each of the six AGlwz values, so that expulsion of water is favored in all cases. Here also AGIw= consists mainly of hydrophobic (AB) interactions, while ZJGl2 consists mainly of LW interactions. The AG~2 bars are repeated three times, for comparison.

of ribbons with one hydrophilic edge (the deoxyribose side) and one somewhat hydrophobic edge (the nucleoside side). The hydrophobicity of the nucleoside edge is a consequence of the fact that they comprise alternating electron-acceptor and electron-donor sites. Thus, the interaction between two complementary DNA strands is a mildly attractive one between the hydrophobic (nucleoside) edges and a repulsive one between the more distal hydrophilic (deoxyribose) edges of the ribbons. The outer edges then tend to form an angle of about 90 ° with one another (due to the mutual repulsion) [68], which is also the angle typically found in the double helix of double-stranded DNA [-7]. The inner edges, which macroscopically attract each other through a relatively weak hydrophobic interaction, only form strongly attractive direct H-bonds in the cases of adenine-thymine or guanine cytosine encounters, with (microscopic)

attractive energies of about - 8 k T respectively [7].

and - 1 2 k T

5. Some effects and applications of hydrophobic interactions 5.1. Protein adsorption

The degree of adsorption of hydrophilic proteins (i.e. soluble proteins, at pH values significantly below or above their isoelectric points), at low concentrations, onto hydrophobic surfaces is roughly a decimal order of magnitude greater than their adsorption onto hydrophilic surfaces [69a]. Thus, the isotherms for protein adsorption onto hydrophobic surfaces are higher and rise more steeply than those for protein adsorption onto hydrophilic surfaces [69a]. This causes log-log

CJ. van O,vs/('olloidsSut;liwesB."Biointer/aces5 (19952 91 110 plots of isotherms of hydrophobic adsorption to be fairly well represented by straight lines (thus resembling Freundlich isotherms), whereas hydrophilic I"ionic') adsorption isotherms do not form as straight a line in a log-log plot, but are shaped like typical Langmuir isotherms in a non-log-log configuration [69b,c]. Apart from the obvious quantitative differences between protein adsorption modes onto hydrophobic and hydrophilic surfaces, outlined above, it is probably not advisable to attach much significance to the superficial resemblances to Freundlich or Langmuir isotherms [69b,c] shown by these two types of adsorption. It should also be noted that many plotted curves (including the shallower Langmuir isotherms) easily transform into straight lines when converted to log log plots. The adsorption of such proteins onto hydrophobic surfaces is strongly subject to hysteresis, which is not the case when they are adsorbed onto hydrophilic surfaces [69a]. Hysteresis here implies that the slope of the adsorption isotherm is steeper than the slope of the desorption isotherm, which is more nearly horizontal. Thus, protein adsorption onto hydrophilic surfaces is readily reversible upon redilution, while protein adsorption onto hydrophobic surfaces is much more irreversible: complete desorption with the original aqueous solvent is usually impossible. As a first approximation, this is due to the fact that whilst upon adsorption from aqueous solution onto hydrophilic surfaces the tertiary configuration of protein molecules remains largely unaffected, upon adsorption from aqueous solution onto hydrophobic surfaces many proteins undergo a significant change in the tertiary configuration. This is driven by the orientation of the hydrophobic moieties from the adsorbed proteins' interior, to the interface with the hydrophobic surface of the solid. Thus, in the case of hydrophobic adsorption, the energy of the primary adsorption step is smaller than the energy involved in the final, secondary adsorption of the confirmationally altered protein. Complete desorption of hydrophobically adsorbed proteins adhering to low-energy surfaces is nonetheless possible by decreasing the hydrogenbonding energy of cohesion of the liquid medium lwhich is the driving force of hydrophobic attrac-

105

tion). The hydrogen-bonding free energy of cohesion of water at 20 C, AG ~ B = - - 1 0 2 m J m -~ The hydrogen-bonding energy of cohesion of aqueous media can be lowered through dilution with organic solvents which have nmch smaller hydrogen-bonding energies of cohesion than water. Examples of such liquids (Lt are: glycerol (AG~LUk= --60 mJ m 2), formamide (//Gi~j~= - 3 8 mJ m 2 ), ethylene glycol (AGIA~ = - 3 8 mJ m _,), acetonitrile tAGA~ of acetonitrile is not known with precision, but should be of the order of - 1 0 m J m 2). Acetonitrile is widely used as the eluting agent of proteins and peptides in reversedphase liquid chromatography (see below). ww

5.2. Protein precipitation Among the forces that give rise to the precipitation of proteins from aqueous solution, hydrophobic attraction (i.e. AGiAw~{< 0} practically always plays a major role. Without including protein precipitation by gross denaturation, e.g. through heating, there are four major mechanisms of protein precipitation. 5.2.1. Precipitation through dehydration By dehydrating protein molecules, their surface at the water interface becomes more hydrophobic, thus turning the normally prevailing net mutual repulsion between protein molecules, which allows their aqueous solubility, into a net attraction, which lowers their solubility and thus causes precipitation. Precipitation methods that operate mainly through dehydration include: "'salting-out", which is usually done by the admixture of multimolar amounts of (NH4)2SO4 [7()]; cold alcohol precipitation [7,71 73]; precipitation through the admixture of polyethylene oxide [ 72,74]. 5.2.2. Precipitation through comple.\ation with inot\~anic or organic counterions Whilst a certain amount of crossbinding may take place when e.g. negatively charged protein molecules combine with pluriwflent inorganic or organic cations, the main cause of the insolubility resulting from that interaction is the hydrophobization of the proteins" surfaces resulting from their charge neutralization (see Section 4.4l. Cations

106

C J. van Oss/ Colloids SurJitces B: Biointerfaces 5 (1995) 91-110

that can promote the precipitation of some proteins are: Zn 1+, Hg z+, A13+, as well as e.g. Rivanol (6,9-diamino-2-ethoxyacridine lactate monohydrate), protamine, and quaternary ammonium bases [72].

5.2.3. Precipitation of euglobulins This is the precipitation of certain globulins, at a pH that is close to their isoelectric point, at low ionic strength. Euglobulins are somewhat more hydrophobic than "pseudoglobulins" (i.e. globulins which do not precipitate at low ionic strength, at isoelectric pH). However, at pH-values removed from the isoelectric point, euglobulins are still soluble in water, and euglobulins are also soluble in water at physiological ionic strength (# = 0.15), even at their isoelectric point. It is only at their isoelectric point and at low ionic strength that euglobulins precipitate. Under these conditions, the electrostatic attraction between an excess positive site on one protein molecule and an excess negative site on another protein molecule is at a maximum and is just strong enough to prevail over the residual hydrophilic repulsion, which is at a minimum. Upon the addition of moderate amounts of NaC1 ("salting-in'), precipitated euglobulins redissolve. This is because at physiological ionic strength, the i-potentials of positive and negative sites on the protein molecules are significantly smaller than at very low ionic strength [7], so that the electrostatic attraction between these proteins is at a minimum at higher (e.g. physiological) ionic strength.

5.2.4. Specific precipitation The precipitation of dissolved antigens by their specific antibodies is, as a first approximation, caused by the formation of much larger complexes of the same protein. This is especially true with complex formation of immunoglobulin-G (IgG) with anti-IgG antibodies, because in this case one simply starts out with single IgG molecules (which are soluble in water) and ends up with large IgG complexes (which are not soluble in water). There are two reasons for the insolubility of the larger IgG complexes: the first is related to the larger size, and the second to a net increase in hydrophobicity.

(A) A larger sized protein complex is less soluble in water than its smaller monomer, because there are always some slightly more hydrophobic patches on the exterior surface of a protein, which do not give rise to precipitation as long as their contactable surface area is small enough so as not to give rise to a mutual attraction that is sufficiently strong to cause complex formation and thus to result in insolubility. For example, if the free energy of attraction between opposing slightly hydrophobic patches on two protein molecules is AGiwi= - 1 . 0 mJ m -2, and if each of these patches has a contactable surface area, Sc = 1 nm 2, then, in units of kT, AGiwi = - 0 . 2 5 k T , which still denotes solubility. However, when complexes are formed that can interact to give rise to e.g. 20 such patches, then a total contactable surface Sc = 20 nm 2 is reached, resulting in AGiw~=-5kT, indicating incipient insolubility. Thus, soluble proteins with a given surface energy can form larger complexes which still have the same surface energy per unit surface area, but which have a larger surface area [75]. Above a given size, such complexes can become insoluble without the need for a change in surface properties. The critical radius for insoluble immune complexes is of the order of 100 nm [76]. (B) In addition, however, upon formation of antigen antibody complexes some increase in overall hydrophobicity also occurs. This is because upon formation of antigen-antibody complexes (even with IgG as the antigen as well as the antibody), the antigen-active site, or epitope, which tends to be especially hydrophilic, specifically binds to the antibody-active site, or paratope. The hydrophilic epitopal site becomes masked in the process, which renders the entire complex more hydrophobic. There is, therefore, a general tendency of the surfaces of antigen-antibody complexes to be somewhat more hydrophobic than their single constituent antigen and antibody molecules [75].

5.3. Cell adhesion The adhesion of cells to hydrophobic surfaces would in principle obey the same rules as the adsorption of proteins, if it were not for the fact that cell adhesion practically always is preceded

C.,L van Oss/Colloid~ Sur/'aces B: Biointetjitces 5 ~ 1995) 91 110

by protein adsorption. Cells will, therefore, not adhere much to hydrophobic surfaces, because omnipresent proteins adsorb strongly onto these surfaces before the cells can adhere to them. The adsorbed protein layer then presents a hydrophilic surface to the cells, thus attenuating their adhesion [77]. In vivo, however, cell adhesion to biological surfaces and/or to other cells usually occurs via specific attachment to proteins such as fibronectin, "adhesins" and other cell adhesion molecules (CAMs) [78 80]. Specific attachment is usually, at least in part, due to hydrophobic interactions; see Section 5.6.

107

(see Fig. 1). Thus fusion is most likely to occur when unsaturated lipid patches are brought close together. In addition, as shown earlier, the unsaturated lipid patches are those least likely to have (glyco-)proteins inserted in them and are thus also least likely to be hampered by tufts of glycocalyx material. The local absence of such tufts further facilitates a close approach between membrane patches that comprise unsaturated lipids. Roos and Choppin [83] observed experimentally that cells with more saturated phospholipids in their cell membranes were the least prone to fuse.

5.5. Chromatographic applications 5.4. Cell litsion For two cells or vesicles to fuse with each other, it is necessary as a first step to have them surmount, at least locally, the prevailing macroscopic EL and AB repulsion. This is best done with (negatively charged) phospholipids (i.e. PA and PS), by hydrophobization through the admixture ofdi- (or pluri-) valent cations such as Ca -,+ or Mg 2+ [37,64,81]; see Section 4.4. Bringing cells and vesicles more closely together by admixture of polyethylene oxide further aids fusion [81,82]. It is harder to induce fusion with low ;'-potential cells or vesicles which consist mainly of PC and/or PEA, because Ca 2+ or Mg -'~ do not bind significantly to them. Their mutual "'hydrophilic" repulsion caused purely by hydration pressure (AGi~w]>O) is much more difficult to suppress in these cases, as these repulsions depend solely on high 3,~-values that are not linked to the (-potential. There is one further condition which favors cell fusion: I~/~i > [/[iwi

(17)

where W = -AG [7]. This condition states that the work of adhesion between lipid chains (i) in the absence of water (W~i) is greater than the work of adhesion in the presence of water (i.e. the work of adhesion, lklwi, due to hydrophobic interactions). This occurs when :'~i > ;'i,~

(18)

In practice, W1~> Wiw~ only prevails with unsaturated and especially with polyunsaturated lipids

There are two fairly closely related liquid chromatographic methods that are based on the net hydrophobic attraction between hydrophilic compounds (e.g. proteins, peptides) and hydrophobic surfaces (e.g. octadecyl group-bearing carrier beads), in aqueous media. These are: reversedphase liquid chromatography and hydrophobic interaction chromatography.

ReL~ersed-phase liquid chromatography ( RPLC) consists of adsorbing hydrophilic proteins, peptides, etc., onto hydrophobic beads, in an aqueous medium. As the driving force of the hydrophobic attraction which binds the protein or peptide to the bead is the hydrogen-bonding energy of cohesion of the water of the medium, reversal of binding in RPLC can be effected by gradually adding a low-hydrogen-bonding water-miscible organic solvent to the medium (e.g. ethylene glycol, formamide, acetonitrile) [84]; see Section 5.l. There are. however, proteins that are too hydrophilic (or, probably more precisely, too hydrated) to attach hydrophobically to these low-energy beads. In such cases, one can resort to hydrophobic interaction chromatography. Hydrophohic interaction chromatography (HIC) consists of binding very hydrophilic (or very hydrated) proteins to hydrophobic (e.g. phenylsubstituted) beads under relatively high salt conditions (typically, at ~-1 M {NH4)2S04). At these high ionic strengths, partial dehydration of the protein molecules occurs through a "~salting-out" effect (see Section 5.2.1), and their exceptionally strong hydrophilicity is sufficiently reduced to

108

CJ. van Oss/Colloids Surlaces B: Biointer/aces 5 (1995) 91 110

allow their adsorption onto low-energy surfaces. After adsorption to hydrophobic carriers under high-salt conditions, the proteins can be desorbed with buffers with a decreasing salt content, which restore the original surface hydrophilicity of these proteins and thus favor their detachment. A very hydrophilic plasma protein which can be purified by HIC, but not by RPLC, is human immunoglobulin A (IgA) [7,84]. Thus, the difference between RPLC and HIC is that in RPLC proteins or peptides adsorb spontaneously onto the hydrophobic carrier in ordinary aqueous media, whereas H1C is used in cases where very hydrophilic proteins or peptides only adsorb onto a hydrophobic carrier upon high-salt dehydration (and thus hydrophobization). The other difference between RPLC and HIC is that elution in RPLC occurs through the admixture of organic solvents, while elution in HIC is effected by the simple expedient of lowering the salt content [7]. 5.6. Spec!fic interactions

Specific interactions are those that operate between antigens and antibodies, carbohydrates and lectins, substrates and enzymes, and, in general, ligands and receptors. With the exception of substrate enzyme reactions, which can also involve disulfide bonds, all these specific interactions are non-covalent in nature. The bonds involved are Lifshitz van der Waals (always), electrostatic (in about half the cases) and hydrophobic (in most cases). These are the same kinds of interaction which normally keep biopolymers and/or cells a certain distance apart, by macroscopic, long-range EL and AB repulsions. Thus, the microscopic-scale interactions between specific complementaritydetermining regions (CDRs), which are attractive, must locally be able to overcome the macroscopicscale repulsions which tend to keep the biopolymer or cellular carriers of the CDR apart [48]. Among the long-range attractive forces between specific sites, LW forces usually are negligibly small, and only attractive EL and/or AB (hydrophobic) forces play a role in the primary interaction [48]. In antigen-antibody interactions, the real specific interaction is localized between the epitope (on the antigen) and the paratope (on the antibody).

Normally, all immunodominant epitopic sites are hydrophilic. They may have a net electrical charge in some cases, or be electrically neutral in others. When a net epitopal charge exists, the corresponding paratopes have an electrical charge of the opposite sign. However, when there are alternating positively and negatively charged amino acids in one site and a complementary array in the other side (e.g. + - + on the epitope and - + - + on the paratope), the overall charge on either CDR is very low and no net long range EL attraction will occur. However, such CDR, which locally are close to their isoelectric point, are, for that reason, very hydrophobic [36] and thus will instead engage in a long-range hydrophobic (AB) attraction [48]. In the subsequent short-range interaction, however, an EL attraction between, for instance, + - + - and - + - + sites, as used in the example given above, can play a role in strengthening the bond. In addition, more often than not, paratopes tend to be hydrophobic, which makes it a necessity for paratopes to be situated inside a concave "'cleft" of the antibody molecule [48]; see also Section 4.7.2. Complementary epitopes and paratopes need to fit together sterically as precisely as possible, so as to be able to interact with each other with the largest possible surface area, over the smallest possible distance, 1, where ideally 1--+1o, at which point the highest binding energy is attained (see e.g. Eq. 13). The surface areas of epitopes and paratopes are rather small, i.e. of the order of 0.4-10 nm 2. After initial contact is made between epitope and paratope (the primary interaction), secondary reactions set in. First, interstitial water is expelled, which can change the hydrophobic interaction, expressed by ~'-,lw2,aca~into a direct contact interaction, L1G12 , which consists mainly of AG}~ v and has a sizeable energy (AG}2' may vary from - 3 0 to - 8 0 mJ m 2) [48]. Then, peptides on antigen and antibody molecules in the vicinity of the epitope and paratope are brought closer together through the neighboring primary epitope p a r a t o p e reaction, and then often engage in a secondary hydrophobic attraction, which further strengthens the overall antigen antibody bond [48]. Specific interactions between carbohydrates and

C J. van Oss/Colh~ids Sur/aces B." Biota teUaces 5 ~ 1995) 91 110

lectins, substrates and enzymes, and other ligand and receptor systems operate in a similar manner.

[28] [29]

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