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Proteins at liquid interfaces
Proteins at Liquid Interfaces Ill. Molecular Structures of Adsorbed Films D. E. G R A H A M 1 AND M. C. P H I L L I P S z Unilever Research Laboratory Colworth/Welwyn, The Frythe, Welwyn, Herts, U. K. Received September 9, 1978; accepted November 9, 1978 The adsorption data presented in the preceding paper (Part II of this series) have been used to deduce the molecular structures of/3-casein, bovine serum albumin (BSA), and lysozyme films at the air-water and oil-water interfaces. The hydrophobic, disordered/3-casein molecules are more surface-active than the globular BSA and lysozyme molecules./3-Casein is in an all-train configuration at low surface pressures (zr < 8 mN m -1) at the air-water interface and can be described by Singer's equation of state for linear polymers. At higher 7r values, loop formation ensues, and the loops increase in density at the expense of the trains until in the close-packed, condensed state the ratio of amino acid residues in loop and train configurations is about 2:1. This conformational change does not occur at the oil-water interface because loops already form at low ~r; enhanced loop formation in the nonaqueous phase is favored because oil molecules solvate the hydrophobic residues. Multilayer formation occurs at high substrate protein concentrations (> 10-2 wt%) giving films thicker than 100 ,~. Although the primary layer is irreversibly adsorbed, molecules in subsequent layers are reversibly adsorbed. Lysozyme molecules are denatured at low lr values Or < 8 mN m-~), and at higher values essentially native molecules (which are reversibly adsorbed) coexist in the surface film. Much residual native structure remains even in the denatured film because lysozyme is very resistant to denaturation. At the oil-water interface, the adsorbed lysozyme molecules are denatured to a greater extent than those at the air-water interface; native molecules are not stable at this interface. The adsorbed BSA molecules contain residual native structure, but there is no abrupt conformational change in the film at a particular packing density. The structure at the air-water interface is intermediate to those of lysozyme and 13-casein. BSA has the same 7r-A curve as lysozyme at the oil-water interface. INTRODUCTION
Most o f the earlier work on proteins at
fluid interfaces has been involved with spread films so that while there is much information available on spread protein films at the airwater interface (for reviews see Refs. (1-4)) the literature on adsorbed protein films is very limited (see Refs. (5-11) for some isolated papers and (12-14) for reviews). The molecular details inferred from surface pres1 Present address: New Technology Division, BP Research Centre, Sunbury on Thames, Middlesex, TW16 7LN, U. K. 2 Present address: Department of Physiology and Biochemistry, The Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, Pennsylvania 19129, U.S.A.
sure (Tr)-molecular area (A) curves obtained in these earlier studies have led to the following two concepts of protein films. (i) In dilute films, the protein molecules are completely unfolded and spread out at the surface so that no protein tertiary or secondary structure remains. (ii) In concentrated films, native and completely unfolded molecules coexist at the surface. Four detailed models have been derived (for a review see (14)). Two of these models depict the surface as being covered with a primary layer of completely unfolded protein molecules and an additional layer of protein molecules packed either immediately above, or below the primary layer. The other two models depict the protein films as containing native molecules
427
Journal of Colloidand Interface Science, Vol.70, No. 3, July 1979
0021-9797/79/090427-13502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.
428
GRAHAM AND PHILLIPS
together with either completely unfolded molecules or molecules having different degrees of unfolding. Because most of the studies mentioned above were performed before the three-dimensional structures of proteins were known, the above models for protein films are necessarily vague about the molecular details. Here we attempt to improve the situation by using the surface pressure and adsorption isotherms for fl-casein, BSA, and lysozyme presented in Part II to construct rr-A curves for films of these proteins adsorbed at the air-water and oil-water interfaces. This work extends our earlier study of the adsorption of caseins at the air- water interface (15). The molecular area and film thickness data (Part II) are used to deduce the steady-state conformations of the adsorbed protein molecules. In addition, the isotherms are compared with those of linear flexible polymers which have been calculated from surface equations of state based upon statistical theories of polymers. In this way, the conformations of the different protein films at both the air-water and oil-water interfaces can be evaluated in terms of protein segments lying in the surface (i.e., in trains) or being solvated by either bulk phase (i.e., loops or tails), and in terms of residual native protein structure. EXPERIMENTAL Materials
Most of the materials have been described in Parts I and II. A crude a-lactalbumin (B variant) was prepared from the milk of a single cow by the method of McKenzie (16). Following large scale (5 g) fractionation on Sephadex G-100 in water at pH 7 the protein was further purified by applying 200- to 500-mg portions to a 2.5 x 20-cm column of DEAE-Bio-Gel A (Bio-Rad, Bromley, U. K.) equilibrated with 5 mM Tris buffer at pH 8.1. A linear gradient of NaC1 (0-0.1 M) eluted the proJournal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
tein; the material obtained was free ofglycoa-lactalbumin and the F fractions described by Hopper and McKenzie (17). N-Ethylmaleimide a-lactalbumin (NEM-a-lactalbumin) was prepared from reduced a-lactalbumin following methods indicated by Riorden and Vallee (18). Methods
In order to monitor the adsorption of lysozyme at 50°C, the procedure described in Part II for the experiments at 22°C was modified as follows. The sealed cabinet which housed the microbalance, gas flow counter, and Teflon trough was heated to 50 _ 2°C by circulating hot water through coils mounted inside the back of the cabinet. In addition, submerged glass capillary heating coils maintained the 100-ml aqueous phase in the trough at 50 __+0.5°C. The atmosphere in the cabinet was saturated with water vapor from constantly moistened fibers, thus minimizing evaporation from the trough subphase. The aqueous phase (0.1 M HC1) and glass Wilhelmy plate were equilibrated at 50°C before the interface was cleaned, the gas flow counter was positioned over the trough, and the protein solution, which had been equilibrated similarly at 50°C for 20 minutes, was injected into the subphase. The subphase was stirred continuously during protein adsorption. The effects of pH on protein adsorption were measured using the methods of Part II but with substrates containing appropriate levels of NaCI, NaOH, and HC1. The equilibrium spreading pressures (We) of the proteins were determined using a procedure described previously (19). Briefly, finely powdered, solid protein was introduced on to the aqueous substrate and left until no further increase in rr was noted; further solid was then added. This procedure was repeated until no increase in pressure was noted on the addition of further solid. This final pressure was taken as 7re.
PROTEINS
AT LIQUID
THEORY
Several theories relating the surface concentration (F) and the conformation of the adsorbed molecules to the surface pressure 7r have been developed. Singer (20) derived an expression (Eq. [1]) for rr for flexible macromolecules adsorbed flat to a surface; he utilized a statistical-thermodynamic treatment of these surface polymers which is analogous to that of Flory and Huggins for polymers in solution. x - 1 z' 7F=
7'/"0
x
2
~ro = kT/ao, k is the Boltzmann constant, T is the temperature, ao is the limiting, condensed area per statistical unit of the linear polymer, z' is a number closely related to the lattice coordination number, 0 is the degree of surface coverage given by the ratio ao/a where a is the observed area per statistical unit of the polymer (0 is also defined asAo/A where A0 is the condensed area per linear polymer molecule and A is the observed area per molecule), and x is the number of statistical units per polymer molecule. In the case of the proteins discussed here, x is large and (x - l)/x ~ 1. The coordination number of the surface lattice has been designated (20) to be a value between 4 (when the polymer is completely flexible and can sample any adjoining latttice site on the surface) and 2 (when the polymer is rigid and has a very limited choice of lattice site). For proteins where all segments are in trains in the surface, Eq. [1] can be written 7r= rr0 ~ - l n
1-
-In(l-
0)
[21
Equation [21 predicts that the surface pressure ~r increases with 0 and becomes infinite when 0--~ 1; it is therefore clear that Eq. [2] can only explain the low-surface-pressure region of the 7r-A curve. It must be borne in mind that proteins are more corn-
429
INTERFACES--III
plex than the macromolecules treated in the above theory because they are not homopolymers, and they contain a large variety of different amino acid residues, have more internal structure, and are also charged. The Singer model of a polymer on a surface lattice neglects both segment-segment interactions and loop formation, and so it is not surprising that the theoretical and experimental zr-A curves can deviate for proteins. The model by Singer has been extended by Motomura and Matuura (21) to account for segment-segment and segment-solvent interactions, and by Frisch and Simha (22, 23), Silberberg (24, 25), and Hoeve (26) to account for loop formation when some of the polymer segments are not in direct contact with the surface. Frisch and Simha's (22) equation for zr (see Eq. [3]) when only part of the polymer is in contact with the surface is similar to that of Singer. Assuming that the amino acid residues can be equated to the polymer segments discussed by Frisch and Simha it follows that (S v-1 In(1 rr = 7ro 2 v(~ 1/t) X (1-~)pO)-ln(l-pO)
2 S 1 ,
[3]
where zr0 = kT/ao and a0 is the limiting area per amino acid residue and also the actual area of a statistical site on the surface, 0 = ao/a where a is the observed area per amino acid residue, t is the number of amino acid residues per protein molecule of which v are in direct contact with the surface and t - v are in looped configurations, p is the ratio v/t, and S (~z') is a number close to the two-dimensional surface lattice coordination number. For the case of proteins, t -> 1, Eq. [3] reduces to Sv-1 ,r = fro 2 v xln(1
- - ~2- p 0 ) -
In
(1--pO)]
[4]
Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
430
GRAHAM AND PHILLIPS 3t3
25 'E 2 0
:
i
g, 5--
0
L
L
I
I
I
I
J
I
I
0-5
I
I
I'0
1"5 Areo / m 2 mg-I
I
I~
I
I
t
I
~
~
2"0
*
I
I
25
FIG. 1. Surface pressure-molecular area isotherms for adsorbed films of//-casein at the air-water (phosphate buffer, pH 7) interface (O, steady-state values; A, values during the adsorption process) and oil-water interface at 22°C (©, steady-state values): . . . . , theoretical isotherm calculated from Singer's Eq. [2] with z' = 4 and ao = 15 A2 residue-k F o r the specific case when all the amino acid residues are in trains, then v = t,p = 1, and Eq. [4] b e c o m e s identical to the Singer Eq. [2]. Equation [4] can effectively describe a linear p o l y m e r which adopts both loop and train conformations. Consequently, agreem e n t b e t w e e n theory and e x p e r i m e n t can be induced by arbitrarily choosing values of e i t h e r p or a~ (where a~ is the effective condensed area per amino acid residue and e q u a l s p "a0 such t h a t p ~< 1), and by assuming that the protein has a fixed flexibility so that the lattice coordination number, z', can be taken as a constant. RESULTS
7r-A Curves Derived from Adsorption Isotherms of B-Casein at Air-Water and Oil- Water Interfaces The steady state adsorption ( F - C p ) and surface pressure (¢r-Co) isotherms h a v e been given in Part II for B-casein at the a i r w a t e r (Fig. 1, Part II) and o i l - w a t e r (Fig. 5, Part II) interfaces. By combining the respective isotherms, z r - A (whereA = F -~) curves can be deduced for steady-state a d s o r b e d B-casein films at the two interfaces (see Fig. 1). Journal of Colloid and Interface Science,
Vol.
70,
No.
3, July
1979
It is clear from Fig. I that the B-casein film is more condensed at the o i l - w a t e r than at the a i r - w a t e r interface. Another significant difference is that the z r - A curve at the a i r w a t e r interface has an inflection at 7r - 8 m N m -a which is not a p p a r e n t in the m o r e condensed o i l - w a t e r curve. H o w e v e r , the two z r - A curves tend to collapse at similar ~- (23.5 ___ 0.5 m N m -a) and A (0.35 ___ 0.03 m 2 mg -1 or 6.6 ___ 0.5/~2 residue-i). When the monolayer of protein becomes saturated, zr tends to reach a saturation value; in the case of B-casein this occurs at both interfaces when Cp = 1 × 10 -4 wt% (see Figs. 4 and 5 in Part II), and both zr and F remain constant o v e r the Cp range 10 -4 to 10-2 wt%. At Cp > 1 × 10 -2 wt%, F continues to increase while zr remains constant, and this is depicted on the ¢r-A curve as the " c o l l a p s e . " The collapse pressures shown ~n Fig. I are close to the equilibrium spreading pressure of 22 m N m -~ obtained by placing solid B-casein on the a i r - w a t e r interface ( s e e Table I). In the case o f B-casein, a flexible protein which a p p r o x i m a t e s to a r a n d o m coil, the molecule can be considered to be a linear sequence o f amino acid residues, and z' for the lattice model can be taken to be 4. Theoretical ~--A curves have b e e n calculated
PROTEINS AT LIQUID INTERFACES--III TABLE I Equilibrium Spreading Pressure rre for Proteins on W a t e r (pH 5.5) at 21°C Protein
"a'e(mN m i)
fl-Casein BSA Lysozyme fl-Lactoglobulin Ovalbumin Conalbumin
22 17 15a 19 20 17
a This value m a y be low b e c a u s e spreading occurred very slowly and equilibrium m a y not h a v e b e e n attained.
from Eq. [2] with z' = 4 for values of a0 in the range 5-30/~2 residue-1 (Fig. 2); in addition, two curves for z' = 2.1 are presented. The 7r-A curves become more condensed as the values of a0 or z' are decreased.
~r-A Curves for BSA and Lysozyme Adsorbed at the A i r - W a t e r and Oil-Water Interfaces The 7r-A curves for native BSA and lysozyme adsorbed at the air-water and oil-
30
(a)
(f) (b)
431
water interfaces are presented in Figs. 3 and 4. The adsorption and surface pressure isotherms of lysozyme denatured in 0.1 M HCI at 50°C are given in Fig. 5, and the resulting ~r-A curve is compared with that of the native form in Fig. 4. The denaturation temperature of lysozyme in 0.1 M HC1 (pH 1) is 45°C (27) so that with the subphase at 50°C the lysozyme has undergone an unfolding transition which is 80% reversible on cooling (28). It is clear that the 7r-A curves of the native globular proteins at the air-water interface are more condensed than that for flcasein at the air-water interface with lysozyme being more condensed than BSA (cf. Figs. 1, 3, and 4), In contrast to fl-casein, lysozyme and BSA form relatively expanded films at the oil-water interface. Prior denaturation of the lysozyme molecule by heating also leads to expansion (Fig. 4). It is interesting that lysozyme at the air-water interface exhibits an inflection in the 7r-A curve at ~- = 8 mN m -1 whereas BSA does not. Lysozyme, in particular, has been studied by many workers using both spread (29, 30, 31) and adsorbed (5, 29, 32) films, but
(9) (e)
(d)
(e)
2~
-rE
20
eJ 15
o
I0
0"5
I'0
15
2'0
Area / m2 n~-I
FI~ 2. Surface p r e s s u r e - m o l e c u l a r area i s o t h e r m s calculated from Singer's Eq. [2]. (a) a0 = 5 A2/ residue, z' = 4; (b) ao = 10 A2/residue, z' = 4; (c) a0 = 15 AS/residue, z' = 4; (d) a0 = 20 A2/residue, z' = 4; (e) ao = 30 A~/residue, z' = 4; (f) ae = 10 A2/residue, z' = 2.1; (g) ao = 15 A2/residue, z' = 2.1. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
432
GRAHAM AND PHILLIPS 3o
I /
25
~
--
zo
\
-
5
0"5
I-0
1"5
2"0
I
2"5
Area /m2mg "1
FIG. 3. Surface pressure-molecular area isotherms for adsorbed films of bovine serum albumin (BSA) at the air-water (V) and o i l - w a t e r interface (V) at 22°C. The theoretical curve is the same as in
Fig. l. the inflection has h i t h e r t o b e e n u n o b s e r v e d a l t h o u g h there is s o m e e v i d e n c e for it in a partial i s o t h e r m (5). A similar inflection at 7r = 12 m N m -1 has b e e n o b s e r v e d b y Bull (33) for a d s o r b e d o v a l b u m i n films, and we h a v e f o u n d that a - l a c t a l b u m i n , w h i c h is h o m o l o g o u s to l y s o z y m e , exhibits the s a m e effect at rr = 12 m N m -1 w h e n a d s o r b e d at the a i r - w a t e r interface a l t h o u g h r e d u c e d N E M - a t - l a c t a l b u m i n exhibits no such inflection (Fig. 6). It is a p p a r e n t f r o m Fig. 4 that heating o r replacing the air p h a s e with tolu-
30t "rE
z5
ene r e m o v e s the inflection in the 7r-A c u r v e of lysozyme. T h e effects o f c h a n g i n g p H on the a d s o r p tion b e h a v i o r o f fl-casein, B S A , and lysoz y m e are s u m m a r i z e d in Fig. 7. DISCUSSION
Air- Water Interface fl-Casein. In o r d e r to i n d u c e Eq. [2] to fit the e x p e r i m e n t a l z r - A c u r v e s , the value o f either a0 or z' can be c h o s e n arbitrarily.
I /
-
-
"C
~, 10-
0"5
1.0
{'5
2.0
2.5
Area / m2mQ-I
FIG. 4. Surface pressure-molecular area isotherms for adsorbed films formed at the air-water interface from native (I-/, phosphate buffer (pH 7) substrate at 20°C) and denatured (©, 0.1 M HCI (pH 1) substrate at 50°C) lysozyme. II, Native lysozyme at the oil-water interface. The theoretical curve is the same as in Fig. 1. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
433
PROTEINS AT LIQUID I N T E R F A C E S - - I I I 6
~4 Q
Q
0 10-6
10-5
10-4 i0-~, 10-2 Initial Substrate Protein Concentration /wt %
10-1
FIG. 5. Adsorption and surface pressure isotherms for a heat-denatured form of [l-l~C]acetyl lysozyme adsorbed at the Mr-water interface. The substrate was 0.1 M HCI (pH 1,I = 0.1) at 50 +- 0,50C. e , surface concentration; ©, surface pressure; A, surface pressure obtained with unlabeled lysozyme.
A reasonable value f o r a 0 seems to be 15 A 2 because it has been shown that the mean interfacial area of an amino acid residue of a protein molecule is 15 A s when in either a close-packed a-helical or B-sheet conformation (34), and also when in an all-train configuration with the amino acid side chains oriented more-or-less perpendicular to the plane of the surface (48). With this physical significance in mind and since the data in Fig. 2 suggest that the calculated value of 7r is more sensitive to ao than z', we use a0 = 15/~2 residue-~ and z' = 4 for the following tests of Eq. [2]. The curves in Fig. 1
show that on this basis Eq. [2] describes the experimental curve for /3-casein when ~r < 8 mN m-'. This fit implies that under these conditions, the monolayer consists o f /3casein molecules flattened into the surface in an all-train configuration (see Fig. 8a); i.e., most residues are located in the plane of the surface in the classical structure o f a dilute film. It is apparent from Fig. 1 that neither Eq. [2] nor Eq. [4] withp = 1 fit the experimental /3-casein 7r-A curve when 7r > 8 m N m -1 (F-' = A < 1 m 2 mg-a). Since a0 = 15 /~2 residue-' (or A = 0.8 m 2 mg-') corre-
25
2O
5
o'
io-5
~o-4
io-3
Lnilio[ Substrate Protein Concentration/wt %
FIG. 6. The variation of surface pressure at the air-water (phosphate buffer, pH 7) interface with initial substrate concentration of e , bovine c~-lactalbumin; II, NEM-reduced form of bovine ct-lactalbumin; and A, lysozyme at 22°C. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
434
GRAHAM AND PHILLIPS
20
(o)
o
o
R
"rE 16
~6
8
4 l
3-0
I
1
I
I
1
I~
12
(b)
% ~' 2-0
) p.c
I
2
I
4
i
6 ~ Substrate pH
I
14
FIG. 7. The pH dependence of the (a) surface pressures and (b) surface concentrations of the proteins (Cp = 1 x 10-4 wt%) adsorbed at the air-water interface at 22°C. The substrate pH was obtained with HCI or NaOH, and the ionic strength was adjusted to 0.1 with NaCI. (a) [],/3-casein; O, lysozyme, and A, BSA. (b) l , fl-casein; O, lysozyme; and A, BSA.
contain more loops and tails because there is insufficient time for molecular rearrangement to occur before compression. This is consistent with the kinetic data in Part I since, although it was shown that the molecular processes involved in adsorption of/3casein molecules to steady-state ~- conditions are fast (T on the order of 1 hr) compared to the overall rate of adsorption, they are slow compared to the overall rate of spreading (usually minutes). As 7r is increased progressively above 8 mN m -1 more loops and tails form, and in order to fit Eq. [4] to the experimental 7r-A curve in Fig. 1, the proportion of residues in trains (p) has to be decreased. For instance, at 7r = 12 mN m -1, the required value ofp = z/3(a~ = I0/~e residue -~) so that about 140 residues are in loops and tails, and only about 70 residues are in trains or direct contact with the air-water interface. Consequently at F s a t , the film is - 5 0 ,~ thick and consists of amino acid residues in Surface Pressure and Concentration
Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
Oil-water Interface
]]'¢8mN m-I
b)
sponds to the minimum possible area for the close-packed all-train structure, loop and tail formation must occur at lower areas (see Figs. 8b, c). It has been suggested (10, 11, 15) that the inflection in the air-water •~r-A curve (Fig. 1), which is also observed in the ~--A curve of the spread monolayer (29) and during continuous compression of an adsorbed film with a barrier, marks the point at which loop and tail formation becomes dominant. The 7r-A curve for an adsorbed B-casein film is more expanded at most rr than that for a spread monolayer (see data in Fig. 1 and Fig. 3 of (29)) although the convergence at higher rr indicates that this is not due to protein loss from the surface in the spreading experiment. The spread film may
Air-water Interface
~:lmg
m-2
]J. =SmNrn-
-,-.,_,.~--~...r~,..~.'x~,-,--.
c] Fsat>r> I mg rn-2 ~
.
.
.
.
.
['r,sat>~>8 mNm-I
17=/-[sot
e)
>rsat 11= IIsat
F
~
~
~
FIG. 8. Schematic representation of the structures of adsorbed films of fl-casein at the air-water and oilwater interfaces. See text for an explanation.
PROTEINS AT LIQUID I N T E R F A C E S - - I l l
loops and trains in the ratio 2:1 (see schematic structure in Fig. 8c). The limiting area is - 0 . 4 m 2 mg-1 (-7/~2 residue-l) at which point about half the residues are in loops and tails (Fig. 8d) and 7r = 24 mN m -1 (Fig. 1). This value of ,r is similar to the eqUilibrium spreading pressure (Tre)of solid/3-casein (Table I) suggesting that the collapse phase is in the same state as freeze-dried protein and that overcompression of the film has not occurred. It is necessary to attain a pressure of 27 mN m -~ in order to induce coagulation of the strongly coagulating ovalbumin molecule (35), and this 1r is significantly above the 7re of 20 mN m -1 (Table I). Since/3-casein does not adsorb to zr > 7re this protein does not coagulate strongly on shaking (36), and gross overcompression with a barrier is required to create coagulum. When the substrate concentration of/3casein is raised sufficiently, 7r does not increase further, but F increases again (C, > 10-2 wt%) and the film thickness becomes > I00 A (Part II). This effect appears to be independent of the state of aggregation of the protein molecules in the substrate because the same result is obtained at 4 and 22°C and is presumably due to fl-casein molecules stacking below the primary layer to form a multilayer (see Fig. 8e). These molecules are reversibly adsorbed and can exchange with /3-casein molecules in the substrate. Both ~r and F for/3-casein are independent of substrate pH in the isoelectric region (pH - 5 ) indicating that at neutral pH the hydrophobic contribution to the adsorption free energy is dominant. The surface concentration of/3-casein is reduced only at pH values sufficiently high (Fig. 7b) to induce a large net negative charge on the protein molecules so that mutual electrostatic repulsion inhibits adsorption; the decreases in 7r and F at high pH are reasonably consistent with the 7r-A curve for B-casein (Fig. I). Lysozyme. The 7r-A curve for native lysozyme is the most condensed of the iso-
435
therms studied, and the deviation from the curve expected from Eq. [2] for an all-train conformation is very large, even at low 7r (Fig. 4). This indicates that the lysozyme molecules are not fully unfolded (see Fig. 9a); the elements of native structure remaining are likely to include disulphide bridges and a-helices (cf. 34, 37). It seems that more unfolding occurs when lysozyme is spread (all the molecules are delivered to the surface when 7r = 0) on 3.5 M KCI because their 7r-A curve is more expanded (cf. 9, 31), The degree of unfolding is likely to be a function of the 7r against which the lysozyme molecules have to unfold rather than the ionic strength of the substrate because a partial 7r-A curve for adsorbed lysozyme on I M KC1 (5) is very similar to that in Fig. 4 for phosphate buffer (I = 0.1). T h e fact that conformational changes in the adsorbed protein molecules are sensitive to 7r probably explains the inflection in the lysozyme Surface Pressure and Concentration a) F ~ 2rag m-2 < 8raN m-I
rI~SmN m-I /
l~ > 8mN m - '
d) r=rsa~'4mg m-2
-~
_
?
(
FIG. 9. Schematic representation of the structure of adsorbed films of lysozyme at the air-water interface. See text for an explanation. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
436
GRAHAM AND PHILLIPS
1r-A curve at 8 mN m -1. As suggested by Bull (8) for ovalbumin films, such an inflection marks the point at which the adsorbing globular protein molecules are prevented from unfolding by the pressure of the molecules already in the film. The fact that the rr-A curve of the homologous and more labile ot-lactalbumin molecule has an inflection at a higher rr (Fig. 6) is consistent with this view, as is the observation that the ~rCu plot of reduced a-lactalbumin (Fig. 6) and the rr-A curve of denatured lysozyme (Fig. 4) do not have an inflection. If the surface denaturation of lysozyme is analogous to its denaturation in solution, the mixed film of native and denatured molecules (Figs. 9c, d) may approximate a two-state situation in that all the latter molecules could adopt a common form; whether or not the denatured molecules are unfolded to the same or different degrees, they are irreversibly adsorbed (A > 0.3 m 2 mg -1) whereas the native molecules can exchange with lysozyme in the substrate. Although we were unable to monitor the enzyme activity of lysozyme while in the surface films, the evidence from other studies (12, 14, 38) suggests that lysozyme molecules adsorbed when ~r > 8 mN m -1 would be active. Native lysozyme molecules continue to assemble in the monolayer until a limiting area of about 0.2 m 2 mg -~ is attained; this is close to the area expected for a close-packed array of ellipsoidal lysozyme molecules (5). At this point 7r is - 2 0 mN m-l; lysozyme does not coagulate readily on shaking (36) so it is unlikely that the film is overcompressed much beyond its 7re although it was difficult to measure the latter parameter (Table I). Further adsorption leads to formation of multilayers which are up to about 100/~ thick (Fig. 9). Since lysozyme dimerizes at pH > 4.5 (39) it is probably this entity which packs below the primary layer and exchanges with lysozyme in solution. When the temperature is above 50°C and the pH = 1, lysozyme is heat-denatured (27, 28), and it is apparent from Figs. 4 and Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
5 that this causes a dramatic change in the surface properties of the protein. The adsorption isotherm and ~--Cp plot in Fig. 5 now have the form reminiscent of that of the flexible, disordered B-casein molecule (cf. Fig. 1 in Part II). This increase in surface activity is a consequence of the exposure of hydtophobic amino acid residues that would normally be found in the interior of the globular native molecule. Denaturation of lysozyme leads to an expansion in its 7r-A curve so that at low rr, the form of the denatured lysozyme zr-A curve is similar to the Singer curve (Fig. 4). However, unlike the case of B-casein, the fit is probably fortuitous because (i) in the denatured state the disulfide bridges and a-helices are not removed (40) and (ii) the denatured molecules (pI = 10.5) are highly charged at pH 1 (net charge of +19) so that electrostatic interactions are likely to be significant. The ~'-A curve of denatured lysozyme becomes rapidly condensed at decreasing A and approaches the native lysozyme curve (Fig. 4). In addition, zr and F became saturated at levels ofTr - 13 mN m -1 and F = 2.9 mg m -2 (A - 0.35 m 2 mg-1). Hence, a further increase in ~r due to the increase in surface density of protein residues resulting from the adsorption of native molecules (this is the most efficient way of packing amino acid residues) cannot take place because there are no native molecules in the subphase. Consequently, this lends support to the model proposed above (Figs. 9c-e), that reversibly adsorbed, native molecules can coexist in the interface with surface denatured molecules. The adsorption of lysozyme is insensitive to pH in the range 1-12 (Fig. 7) indicating that at the Cp chosen the air-water interface is behaving similarly to a hydrophobic solid surface (cf. 41) and that the hydrophobic term is the main component in the adsorption free energy. In contrast, adsorption on hydrophilic solids is maximal at the pI (41, 42) because the electrostatic repulsion between adsorbed molecules is reduced, pH denaturation of the protein molecules in so-
PROTEINS AT LIQUIDINTERFACES--III lution can be a complicating factor, but this does not appear to apply to the lysozyme situation described in Fig, 7. Bovine serum albumin (BSA ). As depicted in Fig. 3, the 7r-A curve for an adsorbed BSA monolayer is more condensed than the theoretical curve for an all-train conformation obtained from Eq. [2] with z' = 4 and a0 = 15 ,~2 residue-l. This implies that BSA cannot adopt an all-train conformation with all residues in the surface, and for a fit between theory and experiment a value of p < 1 (the fraction of residues in direct contact with the surface) in Eq. [4] must be assumed; i.e., loops must exist at all 7r. If the physically realistic value of a0 = 15 is maintained, then with z' = 2.1,p = 2/3, or alternatively, a~ = 10 (Fig. 2). However, since a-helices are stable at the air-water interface (34), the simple loop-train model is likely to be inapplicable to BSA which contains 50% a-helix in the native state (Table I, Part I). A more realistic structure is probably something like that shown in Fig. 9a for lysozyme but with a greater proportion of a-helices lying in the interface. Since the 7r-A curve of BSA is more expanded than that of lysozyme (cf. Figs. 3 and 4), the degree of order and cohesion is less in the BSA film; this is consistent with the solution behavior of these proteins because BSA is more labile with respect to heat denaturation than lysozyme (Table I, Part I). The fact that the 7r-A curve for a spread monolayer of BSA (Fig. 5 in (43)) is more expanded than that for an adsorbed monolayer (Fig. 3), which is the reverse of what is observed with/3-casein, also suggests that the simple loop-train model is not realistic for BSA. The residual native structure causes rr for BSA to be lower than ~r for B-casein at all areas (cf. Figs. 1 and 3). The expansion in the ~--A curve of the spread film relative to that of the adsorbed film suggests that delivering all the molecules simultaneously to the surface with ~- - 0 allows more extensive unfolding than when the same molecules experience a positive 7r on arrival in
437
the surface, as in an adsorption experiment (cf. 43). The absence of an inflection in the 7r-A curves suggests that no native BSA molecules are retained i n the monolayer. The limiting area of about 0.4 mz mg-1 (~7 /~z residue-l) indicates that a large portion of the BSA molecule must be out of the plane of the interface (see Fig. 9a). The maximum 7r is about 17 mN m -1, which is the same as the 7re attained when crystalline protein is placed on the air-water interface (Table I) suggesting that, if sufficient time is allowed, the collapse phase of the BSA film may be the native molecule. This could be possible if the 17 disulfide bridges and 50% a-helix (Table I, Part I) are intact to control the refolding to the native state once the molecules leave the interface. Neither ~- nor F for BSA shows a maximum at the isoelectric point (pH - 4.5) indicating that, as with lysozyme, the hydrophobic contribution to the adsorption free energy is dominant at the air-water interface. The change in 7r at pH - 3 may reflect the N - F transition and acid expansion which is known to occur with BSA below pH 4 (44). The decrease in F at high pH is probably due to electrostatic repulsion because of the high net negative charge on the molecules under these conditions.
O i l - W a t e r Interface Studies of proteins at the oil-water interface have been limited, and little is known at the molecular level (for a review, see (14)). This study shows that although the general characteristics of adsorption are similar at the air-water and oil-water interfaces (Part II), replacing air by oil has distinctly different effects on /3-casein as compared to the globular lysozyme and BSA molecules. The ~--A curve of fl-casein is more condensed at the oil-water interface whereas the isotherms of lysozyme and BSA are expanded compared to the air-water curves (Figs. I, 3, and 4). Since the 7r-A curve for/3-casein at the Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
438
GRAHAMAND PHILLIPS
oil-water interface is condensed and does not have the inflection which is characteristic of the train to loop transition at the airwater interface, it is likely that the oil molecules eliminate the all-train conformation. Solvation of apolar amino acid side chains by the oil molecules leads to enhanced loop formation in the oil phase. The reduced number of residues in the surface gives a lower 7r. In order to fit Eq. [4] to the experimental ¢r-A curve (Fig. 1) a value ofp = V3 (or a~ = 5/~2 residue-i) must be assumed. Consequently, the adsorbed film at the oilwater interface can be considered as comprising molecules with about 140 of the 209 amino acid residues in looped configurations and about 70 residues in train configurations (Fig. 8). An increase in 7r may be due primarily to an increase in the surface density of fl-casein molecules rather than changes in the loop-train distributions of individual molecules. At 7rs~t, which is similar for both interfaces, it is interesting that the conformation of fl-casein at both interfaces seems to be similar, i.e., about 70 residues in trains and 140 residues in looped conformations. Of the 209 amino acid residues in a fl-casein A molecule, 106 are hydrophobic in nature, 47 are charged, and 56 are neutral (45). It is tempting to speculate that at "B'sat where the adsorbed protein has about 140 residues in loops (see Fig. 8) the hydrophobic and charged residues are looped into the oil and aqueous phases, respectively, with the neutral residues in trains in the surface. Unlike/t-casein, the Tr-A curves of lysozyme and BSA are more expanded at the oil,water than at the air-water interface. Furthermore, lysozyme and BSA give essentially the same rr-A curve at the oilwater interface (cf. Figs. 3 and 4). This suggests that both proteins adopt some similar and more extensively unfolded conformation at the oil-water interface. Presumably, solvation by oil molecules reduces the van der Waals cohesion between apolar side chains thereby causing more unfolding and expansion of the 7r-A curves. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
For ¢r < 10 mN m -1 the rr-A curves of both proteins are more expanded than the Singer equation (Figs. 3 and 4) which suggests that considerable segmental interactions exist. This is likely to be electrostatic repulsion because both molecules carry significant net charges (of opposite sign) at pH 7 as they are some 3 pH units away from their isoelectric points. At 7r > 10 mN m -1, the observed curves are more condensed than the Singer equation, but Eq. [4] can be used to achieve a fit. At ¢rsat where the limiting areas are about 0.4 m 2 mg -l, a large proportion of the protein molecule is out the plane of the interface. If this can be considered as loops and tails, then at 7tsar, if z' = 4, p = 0.3 (i.e., a~ - 4 ,~2 residue-i) whereas if z' = 2.1, p - 0.4 (a~ - 6/~2 residue-~). Consequently, in either case, there may be at least 60 (z' = 2.1) or 70% (z' = 4) of the amino acid residues in some close-packed, looped configuration and not in direct contact with the surface. CONCLUSIONS The structures formed by fl-casein, lysozyme, and BSA at the air-water and oilwater interfaces reflect the intrinsic flexibilities of these protein molecules. Thus, the surface pressure (¢r)-molecular area (A) curve of fl-casein can be described in terms of segments of a disordered polypeptide chain forming either trains of amino acid residues in the interface or loops and tails of residues protruding into the bulk phases. In contrast, the globular lysozyme and BSA molecules retain elements of their native structure when adsorbed so that their ~--A curves are relatively condensed. The flexible, disordered fl-casein molecule and the globular proteins respond differently when the apolar phase is changed from air to oil: the rr-A curve of fl-casein is condensed whereas the lysozyme and BSA curves expand. The Tr-A curves of adsorbed films of the proteins are not the same as those for spread monolayers because the degree of
PROTEINS AT LIQUID INTERFACES--III
unfolding in the surface is a function of film pressure. ACKNOWLEDGMENTS We thank Dr, R. A. Badley of this laboratory for providing the a-lactalbumin and Dr. J. de Feijter (Unilever Research Laboratory, Vlaardingen, The Netherlands) for valuable discussion. REFERENCES 1. Neurath, H., and Bull, H. B., Chem. Rev. 23, 391 (1938). 2. Bull, H. B., Advan. Protein Chem. 3, 95 (1947). 3. Cumper, C. W. N., and Alexander, A. E., Rev. Pure Appl. Chem. 1, 121 (1951). 4. Cheesman, D. F., and Davies, J. T., Advan. Protein Chem. 9, 439 (1954). 5. Yamashita, T., and Bull, H. B., J. Colloid Interface Sci. 27, 19 (1968). 6. Khaiat, A., and Miller, I. R., Biochim. Biophys. Acta 183, 309 (1969). 7. Gonsalez, G., and MacRitchie, F., J. Colloid Interface Sci. 32, 55 (1970). 8. Bull, H. B. ,J. Colloid lnterface Sci. 41,305 (1972). 9. Adams, D. J., Evans, M. T. A., Mitchell, J. R., Phillips, M. C., and Rees, P. M., J. Polym. Sci. Part C 34, 167 (1971). 10. Phillips, M. C., Evans, M. T. A., and Hauser, H., in "Proceedings. 6th International Congress on Surface Activity," Vol. 2, p. 381. Hanser Vedag, Munich, 1973. 11. Phillips, M. C., Evans, M. T. A., Graham, D. E., and Oldani, D., Colloid Polym. Sci. 253, 424 (1975). 12. Miller, I. R., in "Progress in Surface and Membrane Science" (D. A. Cadenhead, J. F. Danielli, and M. D. Rosenberg, Eds.), Vol. 4, p. 299. Academic Press, New York, 1971. 13. Miller, I. R., and Bach, D., in "Surface and Colloid Science" (E. Matijevic, Ed.), Vol. 6, p. 185. Wiley, New York, 1973. 14. James, L. K., and Augenstein, L. G., Advan. Enzymol. 28, 1 (1966). 15. Benjamins, J., de Feijter, J. A., Evans, M. T. A., Graham, D. E., and Phillips, M. C., Discuss. Faraday Soc. 59, 218 (1975). 16. Armstrong, J. McD., Hopper, K. E., McKenzie, H. A., and Murphy, W. H., Biochim. Biophys. Acta 214, 419 (1970). 17. Hopper, K. E., and McKenzie, H. A., Biochim. Biophys. Acta 295, 352 (1973).
439
18.-Riordan, J. F., and Vallee, B. L. in "Methods in Enzymology" (C. W. Hirs, Ed.), Vol. 11, p. 545. Academic Press, New York, 1967. 19. Phillips, M. C., and Hauser, H., J. Colloid Interface Sci. 49, 31 (1974). 20. Singer, S. J., J. Chem. Phys, 16, 872 (1948). 21. Motomura, K., and Matuura, R., J. Colloid Sci. 18, 52 (1963). 22. Frisch, H. L., and Simha, R.,J. Chem. Phys. 2,4, 652 (1956). 23. Frisch, H. L., and Simha, R., J. Chem. Phys. 27, 7O2 (1957). 24. Silberberg, A., J. Phys. Chem. 66, 1884 (1962). 25. Silberberg, A. ,J. Polym. Sci. Part C 30,393 (1970). 26. Hoeve, C. A. J.,J. Polym. Sci. Part C 34, 1 (1971). 27. McKnight, R. P., and Karasz, F. E., Thermochim. Acta 5, 339 (1973). 28. O'Reilly, J. M., and Karasz, F. E.,Biopolymers 9, 1429 (1970). 29. Evans, M. T. A., Mitchell, J., Mussellwhite, P. R., and Irons, L., in "Surface Chemistry of Biological Systems" (M. Blank, Ed.), p. 1. Plenum Press, New York, 1970. 30. Hamaguchi, K., J. Biochem. (Tokyo), 42, 705 (1955). 31. Yamashita, T., and Bull, H. B., J. Colloid Interface Sci. 24, 310 (1967). 32. MacRitchie, F., and Alexander, A. E., J. Colloid lnteface Sci. 18, 453,458,464 (1963). 33. Bull, H. B.,J. Colloidlnterface Sci. 41,305 (1972). 34. Malcolm, B. R., in "Progress in Surface and Membrane Science" (D. A. Cadenhead, J. F. Danielli, and M. D. Rosenberg, Eds.), Vol. 7, p. 183. Academic Press, New York, 1973. 35. MacRitchie, F., and Owens, N. F.,J. Colloidlnterface Sci. 29, 66 ('1969). 362 Henson, A. F., Mitchell, J. R., and Mussellwhite, P. R., J. Colloid Interface Sci. 32, 162 (1970). 37. Loeb, G. I.,J. Colloid lnterface Sci. 31,572 (1969). 38. Cheesman, D. F., and Schuller, H., J. Colloid Sci. 9, 113 (1954). 39. Hampe, O. G., Eur. J. Biochem. 31, 32 (1972). 40. Tanford, C.,Advan. Protein Chem. 23, 121 (1968). 41. MacRitchie, F., J. Colloid Interface Sci. 38, 484 (1972). 42. Buli, H. B. ,Biochim. Biophys. Acta 19,464(1956). 43. Mitchell, J., Irons, L., and Palmer, G. J.,Biochim. Biophys. Acta 200, 138 (1970). 44. Peters, T., Jr., in "The Plasma Proteins" (F. W. Putnam, Ed.), 2rid. ed., Vol. 1, p. 133. Academic Press, New York, 1975. 45. Swaisgood, H. E., Crit. Rev. Food Technol. 3, 375 (1973).
Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
Most o f the earlier work on proteins at
fluid interfaces has been involved with spread films so that while there is much information available on spread protein films at the airwater interface (for reviews see Refs. (1-4)) the literature on adsorbed protein films is very limited (see Refs. (5-11) for some isolated papers and (12-14) for reviews). The molecular details inferred from surface pres1 Present address: New Technology Division, BP Research Centre, Sunbury on Thames, Middlesex, TW16 7LN, U. K. 2 Present address: Department of Physiology and Biochemistry, The Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, Pennsylvania 19129, U.S.A.
sure (Tr)-molecular area (A) curves obtained in these earlier studies have led to the following two concepts of protein films. (i) In dilute films, the protein molecules are completely unfolded and spread out at the surface so that no protein tertiary or secondary structure remains. (ii) In concentrated films, native and completely unfolded molecules coexist at the surface. Four detailed models have been derived (for a review see (14)). Two of these models depict the surface as being covered with a primary layer of completely unfolded protein molecules and an additional layer of protein molecules packed either immediately above, or below the primary layer. The other two models depict the protein films as containing native molecules
427
Journal of Colloidand Interface Science, Vol.70, No. 3, July 1979
0021-9797/79/090427-13502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.
428
GRAHAM AND PHILLIPS
together with either completely unfolded molecules or molecules having different degrees of unfolding. Because most of the studies mentioned above were performed before the three-dimensional structures of proteins were known, the above models for protein films are necessarily vague about the molecular details. Here we attempt to improve the situation by using the surface pressure and adsorption isotherms for fl-casein, BSA, and lysozyme presented in Part II to construct rr-A curves for films of these proteins adsorbed at the air-water and oil-water interfaces. This work extends our earlier study of the adsorption of caseins at the air- water interface (15). The molecular area and film thickness data (Part II) are used to deduce the steady-state conformations of the adsorbed protein molecules. In addition, the isotherms are compared with those of linear flexible polymers which have been calculated from surface equations of state based upon statistical theories of polymers. In this way, the conformations of the different protein films at both the air-water and oil-water interfaces can be evaluated in terms of protein segments lying in the surface (i.e., in trains) or being solvated by either bulk phase (i.e., loops or tails), and in terms of residual native protein structure. EXPERIMENTAL Materials
Most of the materials have been described in Parts I and II. A crude a-lactalbumin (B variant) was prepared from the milk of a single cow by the method of McKenzie (16). Following large scale (5 g) fractionation on Sephadex G-100 in water at pH 7 the protein was further purified by applying 200- to 500-mg portions to a 2.5 x 20-cm column of DEAE-Bio-Gel A (Bio-Rad, Bromley, U. K.) equilibrated with 5 mM Tris buffer at pH 8.1. A linear gradient of NaC1 (0-0.1 M) eluted the proJournal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
tein; the material obtained was free ofglycoa-lactalbumin and the F fractions described by Hopper and McKenzie (17). N-Ethylmaleimide a-lactalbumin (NEM-a-lactalbumin) was prepared from reduced a-lactalbumin following methods indicated by Riorden and Vallee (18). Methods
In order to monitor the adsorption of lysozyme at 50°C, the procedure described in Part II for the experiments at 22°C was modified as follows. The sealed cabinet which housed the microbalance, gas flow counter, and Teflon trough was heated to 50 _ 2°C by circulating hot water through coils mounted inside the back of the cabinet. In addition, submerged glass capillary heating coils maintained the 100-ml aqueous phase in the trough at 50 __+0.5°C. The atmosphere in the cabinet was saturated with water vapor from constantly moistened fibers, thus minimizing evaporation from the trough subphase. The aqueous phase (0.1 M HC1) and glass Wilhelmy plate were equilibrated at 50°C before the interface was cleaned, the gas flow counter was positioned over the trough, and the protein solution, which had been equilibrated similarly at 50°C for 20 minutes, was injected into the subphase. The subphase was stirred continuously during protein adsorption. The effects of pH on protein adsorption were measured using the methods of Part II but with substrates containing appropriate levels of NaCI, NaOH, and HC1. The equilibrium spreading pressures (We) of the proteins were determined using a procedure described previously (19). Briefly, finely powdered, solid protein was introduced on to the aqueous substrate and left until no further increase in rr was noted; further solid was then added. This procedure was repeated until no increase in pressure was noted on the addition of further solid. This final pressure was taken as 7re.
PROTEINS
AT LIQUID
THEORY
Several theories relating the surface concentration (F) and the conformation of the adsorbed molecules to the surface pressure 7r have been developed. Singer (20) derived an expression (Eq. [1]) for rr for flexible macromolecules adsorbed flat to a surface; he utilized a statistical-thermodynamic treatment of these surface polymers which is analogous to that of Flory and Huggins for polymers in solution. x - 1 z' 7F=
7'/"0
x
2
~ro = kT/ao, k is the Boltzmann constant, T is the temperature, ao is the limiting, condensed area per statistical unit of the linear polymer, z' is a number closely related to the lattice coordination number, 0 is the degree of surface coverage given by the ratio ao/a where a is the observed area per statistical unit of the polymer (0 is also defined asAo/A where A0 is the condensed area per linear polymer molecule and A is the observed area per molecule), and x is the number of statistical units per polymer molecule. In the case of the proteins discussed here, x is large and (x - l)/x ~ 1. The coordination number of the surface lattice has been designated (20) to be a value between 4 (when the polymer is completely flexible and can sample any adjoining latttice site on the surface) and 2 (when the polymer is rigid and has a very limited choice of lattice site). For proteins where all segments are in trains in the surface, Eq. [1] can be written 7r= rr0 ~ - l n
1-
-In(l-
0)
[21
Equation [21 predicts that the surface pressure ~r increases with 0 and becomes infinite when 0--~ 1; it is therefore clear that Eq. [2] can only explain the low-surface-pressure region of the 7r-A curve. It must be borne in mind that proteins are more corn-
429
INTERFACES--III
plex than the macromolecules treated in the above theory because they are not homopolymers, and they contain a large variety of different amino acid residues, have more internal structure, and are also charged. The Singer model of a polymer on a surface lattice neglects both segment-segment interactions and loop formation, and so it is not surprising that the theoretical and experimental zr-A curves can deviate for proteins. The model by Singer has been extended by Motomura and Matuura (21) to account for segment-segment and segment-solvent interactions, and by Frisch and Simha (22, 23), Silberberg (24, 25), and Hoeve (26) to account for loop formation when some of the polymer segments are not in direct contact with the surface. Frisch and Simha's (22) equation for zr (see Eq. [3]) when only part of the polymer is in contact with the surface is similar to that of Singer. Assuming that the amino acid residues can be equated to the polymer segments discussed by Frisch and Simha it follows that (S v-1 In(1 rr = 7ro 2 v(~ 1/t) X (1-~)pO)-ln(l-pO)
2 S 1 ,
[3]
where zr0 = kT/ao and a0 is the limiting area per amino acid residue and also the actual area of a statistical site on the surface, 0 = ao/a where a is the observed area per amino acid residue, t is the number of amino acid residues per protein molecule of which v are in direct contact with the surface and t - v are in looped configurations, p is the ratio v/t, and S (~z') is a number close to the two-dimensional surface lattice coordination number. For the case of proteins, t -> 1, Eq. [3] reduces to Sv-1 ,r = fro 2 v xln(1
- - ~2- p 0 ) -
In
(1--pO)]
[4]
Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
430
GRAHAM AND PHILLIPS 3t3
25 'E 2 0
:
i
g, 5--
0
L
L
I
I
I
I
J
I
I
0-5
I
I
I'0
1"5 Areo / m 2 mg-I
I
I~
I
I
t
I
~
~
2"0
*
I
I
25
FIG. 1. Surface pressure-molecular area isotherms for adsorbed films of//-casein at the air-water (phosphate buffer, pH 7) interface (O, steady-state values; A, values during the adsorption process) and oil-water interface at 22°C (©, steady-state values): . . . . , theoretical isotherm calculated from Singer's Eq. [2] with z' = 4 and ao = 15 A2 residue-k F o r the specific case when all the amino acid residues are in trains, then v = t,p = 1, and Eq. [4] b e c o m e s identical to the Singer Eq. [2]. Equation [4] can effectively describe a linear p o l y m e r which adopts both loop and train conformations. Consequently, agreem e n t b e t w e e n theory and e x p e r i m e n t can be induced by arbitrarily choosing values of e i t h e r p or a~ (where a~ is the effective condensed area per amino acid residue and e q u a l s p "a0 such t h a t p ~< 1), and by assuming that the protein has a fixed flexibility so that the lattice coordination number, z', can be taken as a constant. RESULTS
7r-A Curves Derived from Adsorption Isotherms of B-Casein at Air-Water and Oil- Water Interfaces The steady state adsorption ( F - C p ) and surface pressure (¢r-Co) isotherms h a v e been given in Part II for B-casein at the a i r w a t e r (Fig. 1, Part II) and o i l - w a t e r (Fig. 5, Part II) interfaces. By combining the respective isotherms, z r - A (whereA = F -~) curves can be deduced for steady-state a d s o r b e d B-casein films at the two interfaces (see Fig. 1). Journal of Colloid and Interface Science,
Vol.
70,
No.
3, July
1979
It is clear from Fig. I that the B-casein film is more condensed at the o i l - w a t e r than at the a i r - w a t e r interface. Another significant difference is that the z r - A curve at the a i r w a t e r interface has an inflection at 7r - 8 m N m -a which is not a p p a r e n t in the m o r e condensed o i l - w a t e r curve. H o w e v e r , the two z r - A curves tend to collapse at similar ~- (23.5 ___ 0.5 m N m -a) and A (0.35 ___ 0.03 m 2 mg -1 or 6.6 ___ 0.5/~2 residue-i). When the monolayer of protein becomes saturated, zr tends to reach a saturation value; in the case of B-casein this occurs at both interfaces when Cp = 1 × 10 -4 wt% (see Figs. 4 and 5 in Part II), and both zr and F remain constant o v e r the Cp range 10 -4 to 10-2 wt%. At Cp > 1 × 10 -2 wt%, F continues to increase while zr remains constant, and this is depicted on the ¢r-A curve as the " c o l l a p s e . " The collapse pressures shown ~n Fig. I are close to the equilibrium spreading pressure of 22 m N m -~ obtained by placing solid B-casein on the a i r - w a t e r interface ( s e e Table I). In the case o f B-casein, a flexible protein which a p p r o x i m a t e s to a r a n d o m coil, the molecule can be considered to be a linear sequence o f amino acid residues, and z' for the lattice model can be taken to be 4. Theoretical ~--A curves have b e e n calculated
PROTEINS AT LIQUID INTERFACES--III TABLE I Equilibrium Spreading Pressure rre for Proteins on W a t e r (pH 5.5) at 21°C Protein
"a'e(mN m i)
fl-Casein BSA Lysozyme fl-Lactoglobulin Ovalbumin Conalbumin
22 17 15a 19 20 17
a This value m a y be low b e c a u s e spreading occurred very slowly and equilibrium m a y not h a v e b e e n attained.
from Eq. [2] with z' = 4 for values of a0 in the range 5-30/~2 residue-1 (Fig. 2); in addition, two curves for z' = 2.1 are presented. The 7r-A curves become more condensed as the values of a0 or z' are decreased.
~r-A Curves for BSA and Lysozyme Adsorbed at the A i r - W a t e r and Oil-Water Interfaces The 7r-A curves for native BSA and lysozyme adsorbed at the air-water and oil-
30
(a)
(f) (b)
431
water interfaces are presented in Figs. 3 and 4. The adsorption and surface pressure isotherms of lysozyme denatured in 0.1 M HCI at 50°C are given in Fig. 5, and the resulting ~r-A curve is compared with that of the native form in Fig. 4. The denaturation temperature of lysozyme in 0.1 M HC1 (pH 1) is 45°C (27) so that with the subphase at 50°C the lysozyme has undergone an unfolding transition which is 80% reversible on cooling (28). It is clear that the 7r-A curves of the native globular proteins at the air-water interface are more condensed than that for flcasein at the air-water interface with lysozyme being more condensed than BSA (cf. Figs. 1, 3, and 4), In contrast to fl-casein, lysozyme and BSA form relatively expanded films at the oil-water interface. Prior denaturation of the lysozyme molecule by heating also leads to expansion (Fig. 4). It is interesting that lysozyme at the air-water interface exhibits an inflection in the 7r-A curve at ~- = 8 mN m -1 whereas BSA does not. Lysozyme, in particular, has been studied by many workers using both spread (29, 30, 31) and adsorbed (5, 29, 32) films, but
(9) (e)
(d)
(e)
2~
-rE
20
eJ 15
o
I0
0"5
I'0
15
2'0
Area / m2 n~-I
FI~ 2. Surface p r e s s u r e - m o l e c u l a r area i s o t h e r m s calculated from Singer's Eq. [2]. (a) a0 = 5 A2/ residue, z' = 4; (b) ao = 10 A2/residue, z' = 4; (c) a0 = 15 AS/residue, z' = 4; (d) a0 = 20 A2/residue, z' = 4; (e) ao = 30 A~/residue, z' = 4; (f) ae = 10 A2/residue, z' = 2.1; (g) ao = 15 A2/residue, z' = 2.1. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
432
GRAHAM AND PHILLIPS 3o
I /
25
~
--
zo
\
-
5
0"5
I-0
1"5
2"0
I
2"5
Area /m2mg "1
FIG. 3. Surface pressure-molecular area isotherms for adsorbed films of bovine serum albumin (BSA) at the air-water (V) and o i l - w a t e r interface (V) at 22°C. The theoretical curve is the same as in
Fig. l. the inflection has h i t h e r t o b e e n u n o b s e r v e d a l t h o u g h there is s o m e e v i d e n c e for it in a partial i s o t h e r m (5). A similar inflection at 7r = 12 m N m -1 has b e e n o b s e r v e d b y Bull (33) for a d s o r b e d o v a l b u m i n films, and we h a v e f o u n d that a - l a c t a l b u m i n , w h i c h is h o m o l o g o u s to l y s o z y m e , exhibits the s a m e effect at rr = 12 m N m -1 w h e n a d s o r b e d at the a i r - w a t e r interface a l t h o u g h r e d u c e d N E M - a t - l a c t a l b u m i n exhibits no such inflection (Fig. 6). It is a p p a r e n t f r o m Fig. 4 that heating o r replacing the air p h a s e with tolu-
30t "rE
z5
ene r e m o v e s the inflection in the 7r-A c u r v e of lysozyme. T h e effects o f c h a n g i n g p H on the a d s o r p tion b e h a v i o r o f fl-casein, B S A , and lysoz y m e are s u m m a r i z e d in Fig. 7. DISCUSSION
Air- Water Interface fl-Casein. In o r d e r to i n d u c e Eq. [2] to fit the e x p e r i m e n t a l z r - A c u r v e s , the value o f either a0 or z' can be c h o s e n arbitrarily.
I /
-
-
"C
~, 10-
0"5
1.0
{'5
2.0
2.5
Area / m2mQ-I
FIG. 4. Surface pressure-molecular area isotherms for adsorbed films formed at the air-water interface from native (I-/, phosphate buffer (pH 7) substrate at 20°C) and denatured (©, 0.1 M HCI (pH 1) substrate at 50°C) lysozyme. II, Native lysozyme at the oil-water interface. The theoretical curve is the same as in Fig. 1. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
433
PROTEINS AT LIQUID I N T E R F A C E S - - I I I 6
~4 Q
Q
0 10-6
10-5
10-4 i0-~, 10-2 Initial Substrate Protein Concentration /wt %
10-1
FIG. 5. Adsorption and surface pressure isotherms for a heat-denatured form of [l-l~C]acetyl lysozyme adsorbed at the Mr-water interface. The substrate was 0.1 M HCI (pH 1,I = 0.1) at 50 +- 0,50C. e , surface concentration; ©, surface pressure; A, surface pressure obtained with unlabeled lysozyme.
A reasonable value f o r a 0 seems to be 15 A 2 because it has been shown that the mean interfacial area of an amino acid residue of a protein molecule is 15 A s when in either a close-packed a-helical or B-sheet conformation (34), and also when in an all-train configuration with the amino acid side chains oriented more-or-less perpendicular to the plane of the surface (48). With this physical significance in mind and since the data in Fig. 2 suggest that the calculated value of 7r is more sensitive to ao than z', we use a0 = 15/~2 residue-~ and z' = 4 for the following tests of Eq. [2]. The curves in Fig. 1
show that on this basis Eq. [2] describes the experimental curve for /3-casein when ~r < 8 mN m-'. This fit implies that under these conditions, the monolayer consists o f /3casein molecules flattened into the surface in an all-train configuration (see Fig. 8a); i.e., most residues are located in the plane of the surface in the classical structure o f a dilute film. It is apparent from Fig. 1 that neither Eq. [2] nor Eq. [4] withp = 1 fit the experimental /3-casein 7r-A curve when 7r > 8 m N m -1 (F-' = A < 1 m 2 mg-a). Since a0 = 15 /~2 residue-' (or A = 0.8 m 2 mg-') corre-
25
2O
5
o'
io-5
~o-4
io-3
Lnilio[ Substrate Protein Concentration/wt %
FIG. 6. The variation of surface pressure at the air-water (phosphate buffer, pH 7) interface with initial substrate concentration of e , bovine c~-lactalbumin; II, NEM-reduced form of bovine ct-lactalbumin; and A, lysozyme at 22°C. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
434
GRAHAM AND PHILLIPS
20
(o)
o
o
R
"rE 16
~6
8
4 l
3-0
I
1
I
I
1
I~
12
(b)
% ~' 2-0
) p.c
I
2
I
4
i
6 ~ Substrate pH
I
14
FIG. 7. The pH dependence of the (a) surface pressures and (b) surface concentrations of the proteins (Cp = 1 x 10-4 wt%) adsorbed at the air-water interface at 22°C. The substrate pH was obtained with HCI or NaOH, and the ionic strength was adjusted to 0.1 with NaCI. (a) [],/3-casein; O, lysozyme, and A, BSA. (b) l , fl-casein; O, lysozyme; and A, BSA.
contain more loops and tails because there is insufficient time for molecular rearrangement to occur before compression. This is consistent with the kinetic data in Part I since, although it was shown that the molecular processes involved in adsorption of/3casein molecules to steady-state ~- conditions are fast (T on the order of 1 hr) compared to the overall rate of adsorption, they are slow compared to the overall rate of spreading (usually minutes). As 7r is increased progressively above 8 mN m -1 more loops and tails form, and in order to fit Eq. [4] to the experimental 7r-A curve in Fig. 1, the proportion of residues in trains (p) has to be decreased. For instance, at 7r = 12 mN m -1, the required value ofp = z/3(a~ = I0/~e residue -~) so that about 140 residues are in loops and tails, and only about 70 residues are in trains or direct contact with the air-water interface. Consequently at F s a t , the film is - 5 0 ,~ thick and consists of amino acid residues in Surface Pressure and Concentration
Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
Oil-water Interface
]]'¢8mN m-I
b)
sponds to the minimum possible area for the close-packed all-train structure, loop and tail formation must occur at lower areas (see Figs. 8b, c). It has been suggested (10, 11, 15) that the inflection in the air-water •~r-A curve (Fig. 1), which is also observed in the ~--A curve of the spread monolayer (29) and during continuous compression of an adsorbed film with a barrier, marks the point at which loop and tail formation becomes dominant. The 7r-A curve for an adsorbed B-casein film is more expanded at most rr than that for a spread monolayer (see data in Fig. 1 and Fig. 3 of (29)) although the convergence at higher rr indicates that this is not due to protein loss from the surface in the spreading experiment. The spread film may
Air-water Interface
~:lmg
m-2
]J. =SmNrn-
-,-.,_,.~--~...r~,..~.'x~,-,--.
c] Fsat>r> I mg rn-2 ~
.
.
.
.
.
['r,sat>~>8 mNm-I
17=/-[sot
e)
>rsat 11= IIsat
F
~
~
~
FIG. 8. Schematic representation of the structures of adsorbed films of fl-casein at the air-water and oilwater interfaces. See text for an explanation.
PROTEINS AT LIQUID I N T E R F A C E S - - I l l
loops and trains in the ratio 2:1 (see schematic structure in Fig. 8c). The limiting area is - 0 . 4 m 2 mg-1 (-7/~2 residue-l) at which point about half the residues are in loops and tails (Fig. 8d) and 7r = 24 mN m -1 (Fig. 1). This value of ,r is similar to the eqUilibrium spreading pressure (Tre)of solid/3-casein (Table I) suggesting that the collapse phase is in the same state as freeze-dried protein and that overcompression of the film has not occurred. It is necessary to attain a pressure of 27 mN m -~ in order to induce coagulation of the strongly coagulating ovalbumin molecule (35), and this 1r is significantly above the 7re of 20 mN m -1 (Table I). Since/3-casein does not adsorb to zr > 7re this protein does not coagulate strongly on shaking (36), and gross overcompression with a barrier is required to create coagulum. When the substrate concentration of/3casein is raised sufficiently, 7r does not increase further, but F increases again (C, > 10-2 wt%) and the film thickness becomes > I00 A (Part II). This effect appears to be independent of the state of aggregation of the protein molecules in the substrate because the same result is obtained at 4 and 22°C and is presumably due to fl-casein molecules stacking below the primary layer to form a multilayer (see Fig. 8e). These molecules are reversibly adsorbed and can exchange with /3-casein molecules in the substrate. Both ~r and F for/3-casein are independent of substrate pH in the isoelectric region (pH - 5 ) indicating that at neutral pH the hydrophobic contribution to the adsorption free energy is dominant. The surface concentration of/3-casein is reduced only at pH values sufficiently high (Fig. 7b) to induce a large net negative charge on the protein molecules so that mutual electrostatic repulsion inhibits adsorption; the decreases in 7r and F at high pH are reasonably consistent with the 7r-A curve for B-casein (Fig. I). Lysozyme. The 7r-A curve for native lysozyme is the most condensed of the iso-
435
therms studied, and the deviation from the curve expected from Eq. [2] for an all-train conformation is very large, even at low 7r (Fig. 4). This indicates that the lysozyme molecules are not fully unfolded (see Fig. 9a); the elements of native structure remaining are likely to include disulphide bridges and a-helices (cf. 34, 37). It seems that more unfolding occurs when lysozyme is spread (all the molecules are delivered to the surface when 7r = 0) on 3.5 M KCI because their 7r-A curve is more expanded (cf. 9, 31), The degree of unfolding is likely to be a function of the 7r against which the lysozyme molecules have to unfold rather than the ionic strength of the substrate because a partial 7r-A curve for adsorbed lysozyme on I M KC1 (5) is very similar to that in Fig. 4 for phosphate buffer (I = 0.1). T h e fact that conformational changes in the adsorbed protein molecules are sensitive to 7r probably explains the inflection in the lysozyme Surface Pressure and Concentration a) F ~ 2rag m-2 < 8raN m-I
rI~SmN m-I /
l~ > 8mN m - '
d) r=rsa~'4mg m-2
-~
_
?
(
FIG. 9. Schematic representation of the structure of adsorbed films of lysozyme at the air-water interface. See text for an explanation. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
436
GRAHAM AND PHILLIPS
1r-A curve at 8 mN m -1. As suggested by Bull (8) for ovalbumin films, such an inflection marks the point at which the adsorbing globular protein molecules are prevented from unfolding by the pressure of the molecules already in the film. The fact that the rr-A curve of the homologous and more labile ot-lactalbumin molecule has an inflection at a higher rr (Fig. 6) is consistent with this view, as is the observation that the ~rCu plot of reduced a-lactalbumin (Fig. 6) and the rr-A curve of denatured lysozyme (Fig. 4) do not have an inflection. If the surface denaturation of lysozyme is analogous to its denaturation in solution, the mixed film of native and denatured molecules (Figs. 9c, d) may approximate a two-state situation in that all the latter molecules could adopt a common form; whether or not the denatured molecules are unfolded to the same or different degrees, they are irreversibly adsorbed (A > 0.3 m 2 mg -1) whereas the native molecules can exchange with lysozyme in the substrate. Although we were unable to monitor the enzyme activity of lysozyme while in the surface films, the evidence from other studies (12, 14, 38) suggests that lysozyme molecules adsorbed when ~r > 8 mN m -1 would be active. Native lysozyme molecules continue to assemble in the monolayer until a limiting area of about 0.2 m 2 mg -~ is attained; this is close to the area expected for a close-packed array of ellipsoidal lysozyme molecules (5). At this point 7r is - 2 0 mN m-l; lysozyme does not coagulate readily on shaking (36) so it is unlikely that the film is overcompressed much beyond its 7re although it was difficult to measure the latter parameter (Table I). Further adsorption leads to formation of multilayers which are up to about 100/~ thick (Fig. 9). Since lysozyme dimerizes at pH > 4.5 (39) it is probably this entity which packs below the primary layer and exchanges with lysozyme in solution. When the temperature is above 50°C and the pH = 1, lysozyme is heat-denatured (27, 28), and it is apparent from Figs. 4 and Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
5 that this causes a dramatic change in the surface properties of the protein. The adsorption isotherm and ~--Cp plot in Fig. 5 now have the form reminiscent of that of the flexible, disordered B-casein molecule (cf. Fig. 1 in Part II). This increase in surface activity is a consequence of the exposure of hydtophobic amino acid residues that would normally be found in the interior of the globular native molecule. Denaturation of lysozyme leads to an expansion in its 7r-A curve so that at low rr, the form of the denatured lysozyme zr-A curve is similar to the Singer curve (Fig. 4). However, unlike the case of B-casein, the fit is probably fortuitous because (i) in the denatured state the disulfide bridges and a-helices are not removed (40) and (ii) the denatured molecules (pI = 10.5) are highly charged at pH 1 (net charge of +19) so that electrostatic interactions are likely to be significant. The ~'-A curve of denatured lysozyme becomes rapidly condensed at decreasing A and approaches the native lysozyme curve (Fig. 4). In addition, zr and F became saturated at levels ofTr - 13 mN m -1 and F = 2.9 mg m -2 (A - 0.35 m 2 mg-1). Hence, a further increase in ~r due to the increase in surface density of protein residues resulting from the adsorption of native molecules (this is the most efficient way of packing amino acid residues) cannot take place because there are no native molecules in the subphase. Consequently, this lends support to the model proposed above (Figs. 9c-e), that reversibly adsorbed, native molecules can coexist in the interface with surface denatured molecules. The adsorption of lysozyme is insensitive to pH in the range 1-12 (Fig. 7) indicating that at the Cp chosen the air-water interface is behaving similarly to a hydrophobic solid surface (cf. 41) and that the hydrophobic term is the main component in the adsorption free energy. In contrast, adsorption on hydrophilic solids is maximal at the pI (41, 42) because the electrostatic repulsion between adsorbed molecules is reduced, pH denaturation of the protein molecules in so-
PROTEINS AT LIQUIDINTERFACES--III lution can be a complicating factor, but this does not appear to apply to the lysozyme situation described in Fig, 7. Bovine serum albumin (BSA ). As depicted in Fig. 3, the 7r-A curve for an adsorbed BSA monolayer is more condensed than the theoretical curve for an all-train conformation obtained from Eq. [2] with z' = 4 and a0 = 15 ,~2 residue-l. This implies that BSA cannot adopt an all-train conformation with all residues in the surface, and for a fit between theory and experiment a value of p < 1 (the fraction of residues in direct contact with the surface) in Eq. [4] must be assumed; i.e., loops must exist at all 7r. If the physically realistic value of a0 = 15 is maintained, then with z' = 2.1,p = 2/3, or alternatively, a~ = 10 (Fig. 2). However, since a-helices are stable at the air-water interface (34), the simple loop-train model is likely to be inapplicable to BSA which contains 50% a-helix in the native state (Table I, Part I). A more realistic structure is probably something like that shown in Fig. 9a for lysozyme but with a greater proportion of a-helices lying in the interface. Since the 7r-A curve of BSA is more expanded than that of lysozyme (cf. Figs. 3 and 4), the degree of order and cohesion is less in the BSA film; this is consistent with the solution behavior of these proteins because BSA is more labile with respect to heat denaturation than lysozyme (Table I, Part I). The fact that the 7r-A curve for a spread monolayer of BSA (Fig. 5 in (43)) is more expanded than that for an adsorbed monolayer (Fig. 3), which is the reverse of what is observed with/3-casein, also suggests that the simple loop-train model is not realistic for BSA. The residual native structure causes rr for BSA to be lower than ~r for B-casein at all areas (cf. Figs. 1 and 3). The expansion in the ~--A curve of the spread film relative to that of the adsorbed film suggests that delivering all the molecules simultaneously to the surface with ~- - 0 allows more extensive unfolding than when the same molecules experience a positive 7r on arrival in
437
the surface, as in an adsorption experiment (cf. 43). The absence of an inflection in the 7r-A curves suggests that no native BSA molecules are retained i n the monolayer. The limiting area of about 0.4 mz mg-1 (~7 /~z residue-l) indicates that a large portion of the BSA molecule must be out of the plane of the interface (see Fig. 9a). The maximum 7r is about 17 mN m -1, which is the same as the 7re attained when crystalline protein is placed on the air-water interface (Table I) suggesting that, if sufficient time is allowed, the collapse phase of the BSA film may be the native molecule. This could be possible if the 17 disulfide bridges and 50% a-helix (Table I, Part I) are intact to control the refolding to the native state once the molecules leave the interface. Neither ~- nor F for BSA shows a maximum at the isoelectric point (pH - 4.5) indicating that, as with lysozyme, the hydrophobic contribution to the adsorption free energy is dominant at the air-water interface. The change in 7r at pH - 3 may reflect the N - F transition and acid expansion which is known to occur with BSA below pH 4 (44). The decrease in F at high pH is probably due to electrostatic repulsion because of the high net negative charge on the molecules under these conditions.
O i l - W a t e r Interface Studies of proteins at the oil-water interface have been limited, and little is known at the molecular level (for a review, see (14)). This study shows that although the general characteristics of adsorption are similar at the air-water and oil-water interfaces (Part II), replacing air by oil has distinctly different effects on /3-casein as compared to the globular lysozyme and BSA molecules. The ~--A curve of fl-casein is more condensed at the oil-water interface whereas the isotherms of lysozyme and BSA are expanded compared to the air-water curves (Figs. I, 3, and 4). Since the 7r-A curve for/3-casein at the Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
438
GRAHAMAND PHILLIPS
oil-water interface is condensed and does not have the inflection which is characteristic of the train to loop transition at the airwater interface, it is likely that the oil molecules eliminate the all-train conformation. Solvation of apolar amino acid side chains by the oil molecules leads to enhanced loop formation in the oil phase. The reduced number of residues in the surface gives a lower 7r. In order to fit Eq. [4] to the experimental ¢r-A curve (Fig. 1) a value ofp = V3 (or a~ = 5/~2 residue-i) must be assumed. Consequently, the adsorbed film at the oilwater interface can be considered as comprising molecules with about 140 of the 209 amino acid residues in looped configurations and about 70 residues in train configurations (Fig. 8). An increase in 7r may be due primarily to an increase in the surface density of fl-casein molecules rather than changes in the loop-train distributions of individual molecules. At 7rs~t, which is similar for both interfaces, it is interesting that the conformation of fl-casein at both interfaces seems to be similar, i.e., about 70 residues in trains and 140 residues in looped conformations. Of the 209 amino acid residues in a fl-casein A molecule, 106 are hydrophobic in nature, 47 are charged, and 56 are neutral (45). It is tempting to speculate that at "B'sat where the adsorbed protein has about 140 residues in loops (see Fig. 8) the hydrophobic and charged residues are looped into the oil and aqueous phases, respectively, with the neutral residues in trains in the surface. Unlike/t-casein, the Tr-A curves of lysozyme and BSA are more expanded at the oil,water than at the air-water interface. Furthermore, lysozyme and BSA give essentially the same rr-A curve at the oilwater interface (cf. Figs. 3 and 4). This suggests that both proteins adopt some similar and more extensively unfolded conformation at the oil-water interface. Presumably, solvation by oil molecules reduces the van der Waals cohesion between apolar side chains thereby causing more unfolding and expansion of the 7r-A curves. Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979
For ¢r < 10 mN m -1 the rr-A curves of both proteins are more expanded than the Singer equation (Figs. 3 and 4) which suggests that considerable segmental interactions exist. This is likely to be electrostatic repulsion because both molecules carry significant net charges (of opposite sign) at pH 7 as they are some 3 pH units away from their isoelectric points. At 7r > 10 mN m -1, the observed curves are more condensed than the Singer equation, but Eq. [4] can be used to achieve a fit. At ¢rsat where the limiting areas are about 0.4 m 2 mg -l, a large proportion of the protein molecule is out the plane of the interface. If this can be considered as loops and tails, then at 7tsar, if z' = 4, p = 0.3 (i.e., a~ - 4 ,~2 residue-i) whereas if z' = 2.1, p - 0.4 (a~ - 6/~2 residue-~). Consequently, in either case, there may be at least 60 (z' = 2.1) or 70% (z' = 4) of the amino acid residues in some close-packed, looped configuration and not in direct contact with the surface. CONCLUSIONS The structures formed by fl-casein, lysozyme, and BSA at the air-water and oilwater interfaces reflect the intrinsic flexibilities of these protein molecules. Thus, the surface pressure (¢r)-molecular area (A) curve of fl-casein can be described in terms of segments of a disordered polypeptide chain forming either trains of amino acid residues in the interface or loops and tails of residues protruding into the bulk phases. In contrast, the globular lysozyme and BSA molecules retain elements of their native structure when adsorbed so that their ~--A curves are relatively condensed. The flexible, disordered fl-casein molecule and the globular proteins respond differently when the apolar phase is changed from air to oil: the rr-A curve of fl-casein is condensed whereas the lysozyme and BSA curves expand. The Tr-A curves of adsorbed films of the proteins are not the same as those for spread monolayers because the degree of
PROTEINS AT LIQUID INTERFACES--III
unfolding in the surface is a function of film pressure. ACKNOWLEDGMENTS We thank Dr, R. A. Badley of this laboratory for providing the a-lactalbumin and Dr. J. de Feijter (Unilever Research Laboratory, Vlaardingen, The Netherlands) for valuable discussion. REFERENCES 1. Neurath, H., and Bull, H. B., Chem. Rev. 23, 391 (1938). 2. Bull, H. B., Advan. Protein Chem. 3, 95 (1947). 3. Cumper, C. W. N., and Alexander, A. E., Rev. Pure Appl. Chem. 1, 121 (1951). 4. Cheesman, D. F., and Davies, J. T., Advan. Protein Chem. 9, 439 (1954). 5. Yamashita, T., and Bull, H. B., J. Colloid Interface Sci. 27, 19 (1968). 6. Khaiat, A., and Miller, I. R., Biochim. Biophys. Acta 183, 309 (1969). 7. Gonsalez, G., and MacRitchie, F., J. Colloid Interface Sci. 32, 55 (1970). 8. Bull, H. B. ,J. Colloid lnterface Sci. 41,305 (1972). 9. Adams, D. J., Evans, M. T. A., Mitchell, J. R., Phillips, M. C., and Rees, P. M., J. Polym. Sci. Part C 34, 167 (1971). 10. Phillips, M. C., Evans, M. T. A., and Hauser, H., in "Proceedings. 6th International Congress on Surface Activity," Vol. 2, p. 381. Hanser Vedag, Munich, 1973. 11. Phillips, M. C., Evans, M. T. A., Graham, D. E., and Oldani, D., Colloid Polym. Sci. 253, 424 (1975). 12. Miller, I. R., in "Progress in Surface and Membrane Science" (D. A. Cadenhead, J. F. Danielli, and M. D. Rosenberg, Eds.), Vol. 4, p. 299. Academic Press, New York, 1971. 13. Miller, I. R., and Bach, D., in "Surface and Colloid Science" (E. Matijevic, Ed.), Vol. 6, p. 185. Wiley, New York, 1973. 14. James, L. K., and Augenstein, L. G., Advan. Enzymol. 28, 1 (1966). 15. Benjamins, J., de Feijter, J. A., Evans, M. T. A., Graham, D. E., and Phillips, M. C., Discuss. Faraday Soc. 59, 218 (1975). 16. Armstrong, J. McD., Hopper, K. E., McKenzie, H. A., and Murphy, W. H., Biochim. Biophys. Acta 214, 419 (1970). 17. Hopper, K. E., and McKenzie, H. A., Biochim. Biophys. Acta 295, 352 (1973).
439
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Journal of Colloid and Interface Science, Vol. 70, No. 3, July 1979