LATERAL COHESION I N PROTEIN MONOLAYERS* LYMAN FOURT' Department of Zoology, Washington University, St. Louis, Missouri Received November 18, 19%
The formed structures of animal cells and tissues are composed primarily of protein and are presumably laid down by alteration of soluble protein. Each type of structure,-for example, membranes or fibrils,-may have its specific mode of formation and series of precursor compounds, as in fibrin clotting, yet the physical processes involved may be of very general occurrence throughout biological systems. Such a process is seen in the adsorption of protein molecules at an interface, with subsequent rearrangement of structure and the formation of bonds linking the units, which are themselves polymers of amino acids, in another degree of polymerization. To obtain information concerning the processes by which interfacial structures can be produced from solutions containing protein one may study the properties of protein monolayers. Such films offer the possibility of a great degree of control over the relation of molecules to each other and to the aqueous subsolution. Since the cohesion of molecules within the plane of the film is of particular interest from the point of view of membrane formation, a quantitative method was devised for studying the viscosity and elasticity of unimolecular films as an index of the lateral bonding of molecules. This paper presents results obtained by applying this method to unimolecular films of casein and of nerve axis cylinder protein. Findings with cholesterol are included because of the information yielded regarding the nature of the elastic effects. THEORY
Some general aspects of the theory of surface viscosity measurement and an experimental comparison of the results obtained for long-chain normal alcohols by oscillating discs or rings and oscillating vanes have already been published (6). The theory of the oscillating-vane method The material of this paper is from the thesis presented by the author in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Graduate Studies of Washington University. A preliminary description of this work has been published elsewhere (5). * Present address: Department of Chemistry, University of Chicago, Chicago, Illinois. 887
888
LYMAN FOURT
is less familiar than that for systems of circular symmetry presented by Langmuir and Schaefer (9) and is therefore outlined here. Oberbeck (13) compared the rate of damping of the oscillations of a vane, according as it was completely immersed in a liquid or as its upper edge approached and penetrated the surface. I n the present work the surface is penetrated by the lower edge of the vane only, so that the sensitivity to changes seated a t the air-water interface is increased. The essential difference between this work and that of Oberbeck is in the recognition of the presence of a film, deliberately caused to be present and quantitatively specified by the film-balance technique of Langmuir, Adam, and Gorter. Two conditions are postulated in the quantitative theory of the method: (1) that the differences between a clean surface and one covered with a film can be attributed entirely to the film, and (2) that the resistance of the film to an oscillating vane may be localized as rupture a t the ends of the vane. The first assumption requires that any resistance of water accompanying the film be included in the computed film resistance, but this is only a magnification of an effect essentially caused by cohesion within the film itself. The effective immersion of the vane must remain the same in the presence of the film. For films of many substances the resistance rkes to values a thousandfold greater than that of the clean surface. The differential equation dZe de I -dt2 + R - + fdte = O for the oscillation of a system with moment of inertia I, having a resisting de torque R - proportional to the angular velocity, and a restoring torque dt Te proportional to the angular displacement e, leads to the expressions
and A = -RP
21
(3)
in which P is the period and X is the logarithmic decrement. According de to the second condition, the torque R - arises from the rupture of the film dt a t the ends of the vane. It is convenient to obtain X in common logarithms, 'converting to base e by the factor 2.3. The resistance of the clean surface is to be subtracted, according to the first condition. Letting
LATERAL COHESION IN PROTEIN MONOLAYERS
889
I be the length of the vane and u the force in dynes required to rupture the film a t unit rate, we have u =
X 2.3
A($)i4
(4)
The quantity u has the dimensions of surface viscosity, the unit being the “surface poise”. If the film has no appreciable elasticity, the effect of increasing cohesion of its molecules is to increase R, and hence lengthen P , according to equation 2. But if elastic forces in the film add to the restoring torque of the suspension, the value of T is increased, hence shortening P. As a protein film is compressed, P is observed first to increase slightly, then to decrease greatly. These effects are illustrated in table 1 and in figure 2. The contribution, AT, of the film to the restoring torque can be computed:
The subscripts refer to the clean surface. A sufficiently accurate approximation in a form more convenient for calculation is obtained by neglecting the A’s: AT = 4x21(j$
-
&)
A logical first assumption as to the nature of the elastic force exerted on the vane by the film is that it is compressional. This was the view of Marangoni (ll),that the advancing vane piled up the film before it and left the water surface clean behind. However, one can have certain films (long-chain alcohols, cholesterol) under high pressure, without any elastic effects. To be manifest, a sweeping action would require either a very large viscosity or the presence of shear elasticity. The latter can be demonstrated in many films by the behavior of talc motes, and would itself contribute to the elastic effect. A theoretical analysis of the elastic effect resulting in an expression for the elastic constants (compressibility, modulus in shear) has not yet been accomplished. Trials have led to the empirical result that the effect of change in the length of vane can be taken into account by computing an elasticity index:
E = -AT
P
(7)
This index can be used to compare and characterize films of different substances.
890
LYMAN FOURT
PROCEDURE
A film balance and trough of the Langmuir-Adam type furnished pressure-area data. Proteins were spread from solution by Gorter’s technique. From 30 to 60 min. were allowed to insure relatively complete spreading before compression was begun. The stepwise compression of the film extended from 1 to 4 hr. In the pressure-area plots only the equilibrium points are shown; equilibration was not so extensive as that observed for egg albumin (7). Cholesterol was dissolved in redistilled benzene and applied dropwise. The trough wm 14 cm. wide and 1.8cm. deep. To obtain maximum symmetry, the oscillating vane was suspended 7 cm. in front of the float which divides the clean surface from that which is covered with film. A box with suitable doors and windows inclosed the apparatus, preventing disturbances from drafts. The oscillating system was a brass cylinder, split in half along the axis and held together by brass screws. The phosphor bronze suspension wire was soldered into a small brass plug, which screwed into the center of the upper end of the cylinder. A microscope cover glass clamped symmetrically in the lower end of the cylinder served as the vane. The glass was cleaned with hot chromic-sulfuric acid before each experiment. A pair of air jets directed a t the ends of the vane served to control oscillation in either direction. The lower edge of the vane should parallel the water surface, and in its resting position the vane should be parallel to the direction of compression. Under these conditions the vane suffers no displacement during the compression of a strongly coherent film, since the sharp edge can cut the film. At the same time, the vane acts like a screwdriver blade, and does not break loose from the film, as a circular object has frequently been observed to do (14, 15, 12). These considerations indicate the special applicability of the vane to highly rigid films. An oscillating ring system was not satisfactory for this type of film, because it showed a marked tendency to be carried along as the film (casein) was compressed. The moment of inertia in all of the experiments was 105 g. cm.*; the period in the clean surface was about 12.2 sec., except in some preliminary experiments in which it was 4.05 sec. Period and decrement were independent of amplitude, for the films studied here; any trend with amplitude was so slight as to be obscured by random variations. Hence five or ten swings were used whenever possible, to facilitate computation of XI,,. The average of two or three sets a t a given area and pressure is presented as a single point. MATERIALS
The cholesterol was from the Eastman Kodak Company (Rochester, New York). The casein was “Labco” brand, vitamin-free, from the Casein Corporation of America (New York City). Casein solutions were
LATERAL COHESION IN PROTEIN MONOLAYERS
89 1
made up by dissolving the powder in 0.1 M sodium hydroxide, then neutralizing the solution with acetic acid, the final solution being 0.01 M in sodium acetate, and containing about 1 mg. of casein per milliliter. Nerve proteins were simple extracts of minced leg and claw nerves of lobsters in isotonic phosphate buffer, pH 7.4, clarified by centrifuging and filtering. This extract has been shown by Bear, Schmitt, and Young (3) to contain as its chief protein constituent the protein complex of the axis cylinder, designated by them as neuronin. EXPERIMENTAL RESULTS
Cholesterol
Cholesterol is perfectly fluid to talc, as noted by Adam and Rosenheim (1). The period of the pendulum and the quotient, Xlo/P,which is proportional to the viscosity, show almost no change from the largest areas to the collapse of the film. The resistance is slightly less in the presence of the film than for the clean surface; in terms of viscosity the maximum decrease is 0.01 surface poise. This apparent negative viscosity is probably due to decrease of effective immersion of the vane combined with an extremely small vi~cosity.~Decrease of surface tension lessens the capillary rise along the vane. This is borne out by an experiment with palmitic acid in which the vane was not quite level. .4t large surface pressures the higher end was released from contact with the water. The cholesterol experiment shows the independence of viscosity, and especially elasticity, from the film pressure, since the film was compressed to collapse without significant change. Casezn
The pressure-area graphs of five films are shown in figure 1. The uniformity of these films with respect to compression was the basis on which they were selected for testing equations 4 and 7. Other films, not so uniformly spread, showed similar relations. Comparisons between the oscillating vane and talc observations were made in order to correlate the results with those of Hughes and Rideal (€9,who noted the appearance of elasticity a t a certain stage in the compression of gliadin films. Table 1 shows that the elasticity becomes apparent to talc at about the same step in compression as the first shortening of the period. That is, the vane method, as used here, has about the same sensitivity as the talc observation method, but has the advantage of permitting numerical comparison of degrees of elasticity. In figure 2 are shown the data for the computation of elasticities and Langrnuir, Schaefer, and Sobotka (IO) record the surface viscosity of cholesterol as less than 0 002 surface poise, the limit of sensitivity of their measurement$.
892
LYMAN FOURT
viscosities. The open symbols represent results with a 5-cm. vane; the solid symbols are for a 3.5-cm. vane. Figure 3 shows the extent to which
24t
I
24
b\
16 CL W
a v) v) Y
a
a
8
0
a0
0.8
0.4
5
1.2
AREA
0.0
ao
0A
s
0.8
1.2
AREA
FIG.1 FIG.2 FIG.1. Pressure-area relations of casein on 0.01 M hydrochloric acid. Ordinate, surface pressure in dynes per centimeter; abscissa, area in square meters per milligram, F I ~2. . Data from casein films for testing equations 4 and 7. Open symbols represent measurements with a vane 5.0 cm. long; solid symbols with a vane 3.5 cm. long. Ordinates: period in seconds, quotient XIOIP. Abscissa, area in square meters per milligram. TABLE 1 Comparison of observations with the vane and with talc VANE IN
Film Talc indication Fluid
Labco casein: Period, in seconds. . . . . . . . . . 12.21 Decrement, X l o . . . . . . . . . . . . . 0.003 Area, m*. per milligram.. . . . Pressure, dynes per centimeter. . . . . . . . . . . . . . . . . . .
Crude casein: Period, in seconds. . . . . . . . . 4.02 Decrement, X,O. . . . . . . . . . . . . 0.001
12.23 0.026
4.06 0.012
-
1
Fluid
Rmiatant I Elastic
12.34 0.035 1.58
12.30 0.034 1.35
12.35 0.039 1.11
12.14 0.156 0.87
1.71
2.78
3.74
8.07
4.06 0.013
4.06
0.021
agreement is obtained between the viscosities measured with different vanes, using equation 4. This is taken as evidence favoring the validity of the equation. At low pressures the casein films are fluid; the point of
893
LATERAL COHESION IN PROTEIN MONOLAYERS
I2
>
c 3
a
t
v)
0 V
2
5
4
0
a4
s
OB
I2
AREA
AREA
FIG.3
FIQ.4
FIG.3. Viscosity of casein. The open and solid symbols represent measurements with vanes of different lengths brought to agreement by equation 4. Ordinate, viscosity in surface poises; abscissa, area in square meters per milligram. FIG.4. Elasticity of casein. The open and solid symbols represent measurements with vanes of different lengths brought to agreement by equation 7. Ordinate, elasticity index; abscissa, area in square meters per milligram.
5
*
1
4 0
k
0
z3 -
52 >
Y 2
-
0
4
I 0 0
0
A
.
A
t o
3
0
l -
I
:Po .
.
-
p .
I
-
'
A
A.
-I
0
'
A
V
m
.
I
I
I
FIG.5. Computed bulk viscosity of casein. The ordinate numbers times 10' are the viscosity in poises; the abscissa is the thickness of the film in Angstrom units.
solidification, about 5 dynes and 0.95 square meter per milligram, is marked S in the figures. The elasticity index computed according to equation 7 is shown in figure 4.
894
LYMAN FOURT
Surface viscosity can be compared with bulk viscosity if we divide by the thickness of the film. I n computing this, a density of 1.33,corresponding to the specific volume of protein in solution (4), was assumed for the material in the film. Figure 5 presents the results of this operation. The computed bulk viscosity is not constant, although a constant viscosity would be expected for increasing thicknesses of the same material, but increases, an indication that the structure of the film is different a t each step in compression. The large magnitude of these viscosities rules out comparison with the viscosities of ordinary liquids. Since the films are in fact elastic solids, it is with the flow characteristics of solids that comparison should be sought.
Nerve proteins A single preparation of nerve protein was used for all of the experiments a t pH 5.0, another for those at pH 4.3, and two for those at pH 2.0. Data TABLE 2 Spreading of nerve protein at p H 6.0 TRIAL
CONDITIONS
I Aqueous protein solution on dilute buffer
Aqueous protein solution on dilute buffer Alcoholic protein solution on dilute buffer Alcoholic protein solution on dilute buffer Aqueous protein solution on 0.5 M sodium chloride Aqueous protein solution on 0.5 M sodium chloride
VOLUME OF SOLUTION
AREA OP FILM
cm.1 pa
0.1415 0.2885 0.1400 0.0852 0.1410 0.0650
mi.
1230
1920 4800 4640
4600 4900
are lacking by which to compare the area relations between the different preparations, but for a particular preparation pressure-area comparisons in terms of area per milliliter of solution can be made. At each pH these relations had the general form shown by casein, that is, above the lowest pressures a straight line, intersected a t a higher pressure by another straight line of lesser slope. Table 2 indicates the manner of spreading on 0.0033 M acetate buffer a t pH 5.0. Applied directly, as in trials 1 and 2, the spreading was not quantitative, as the area (obtained by extrapolating the lower linear part of the plot to zero pressure) shows. However, by the addition of alcohol, diluting four volumes of nerve protein solution with one of 47.5 per cent alcohol, quantitative spreading was obtained. The same area was found for spreading a t this pH on acetate buffer made 0.5 M in sodium chloride. The lower straight-line portion of the pressurearea curve began a t about 2 dynes pressure; the intersection of the two linear parts came a t 15 dynes per centimeter.
LATERAL COHESION I N PROTEIN MONOLAYERS
895
Anothkr preparation of nerve protein showed a similar type of pressurearea relation on 0.0033 M acetate buffer a t pH 4.3. Here the lower pressure-area line also began a t about 2 dynes per centimeter; the intersection of the two linear portions at 20 dynes per centimeter. On 0.01 M hydrochloric acid, pH 2.0, only the first linear portion was observed, commencing a t about 5 dynes per centimeter. The experiments only extended to 16 dynes per centimeter. Figure 6 presents viscosity as a function of surface pressure for nerve protein on different subsolutions. This relation should be independent of 20
I
I
I
I
I A
b
A
15
0
> IO
t ro
0 V
Y 1
0
5
IO
I5 20 SURFACE PRESSURE
25
30
FIG.6. Viscosity of nerve protein. Ordinate, viscosity in surface poises; abscissa, pressure in dynes per centimeter. Curves A, B, and D are for films on subsolutions of pH 5.0, 2.0, and 4.3, respectively.
the actual amount of protein on the surface. In curve A, which shows the values a t pH 5.0, the results of trials 1 and 2 of table 2, in which spreading was incomplete, are represented by x’s. Here, although all of the protein released did not go onto the surface, that which did is exactly like other films in which all of the protein was spread. This similarity is likewise shown by the elasticity relations presented in figure 7. The viscosity-surface pressure relations are nearly linear but show a slight upward curvature, continuing smoothly to pressures above that of the break between the two linear parts of the pressure-area curves.
896
LYMAN FOURT
On the subsolutions of pH 5.0 and 4.3 the films were elastic within a few seconds after spreading; even a t zero pressure, with unoccupied surface available, they formed elastic membranes of definite shape. On acid solution, pH 2.0, however, the nerve protein resembled casein in remaining fluid a t low pressures for several hours. On standing overnight under pressure on a solution of pH 2.0 the nerve protein underwent a change, in that upon reexpansion it was no longer fluid, but weakly elastic. This
SURFACE P R E S S U R E
FIG.7. Elasticity of protein films. Ordinate, elasticity index (on a tenfold larger scale in the lower portion); abscissa, pressure in dynes per centimeter. Curves A, B, and D are for nerve protein on subsolutions of pH 5.0, 2.0, and 4.3, respectively; curve C is for casein on 0.01 M hydrochloric acid. In curve C t h e open and solid symbols represent measurements with vanes of different lengths.
type of increase of elasticity with time was observed by Hughes and Rideal (8) with gliadin, and has been encountered with casein as well. This shows that the differences in elasticity a t zero pressure are essentially differences in the rate of formation of bonds in the film. The elasticity indices of the films spread on subsolutions of different reactions are shown in figure 7. For these films from one-half to one hour of aging after spreading was allowed. The plot shows that on acid solution both casein and
LATERAL
COHESION IN PROTEIN MONOLAYERS
897
nerve protein have vanishingly sinall elasticities a t low pressure#, while a t the higher pH values the nerve protein has a marked elasticity down to the lowest pressures. Although the nerve protein is knit together rapidly after spreading on solutions of pH 5.0 or 4.3, and shows elasticity even a t zero pressure, there is an initial period in which the surface is mobile. This could be demonstrated by sprinkling the surface wtth talc before applying the protein, to make the film boundary visible. If the amount of protein and the area available for its spreading are so adjusted that some surface is always unoccupied, the patch of film is rounded, and this rounded outline is retained after the release of compression. However, if the film is spread on a restricted area, in which a pressure of a few dynes develops, and the barrier is then displaced to greater areas, the straight edge of the patch of film is retained. That is, during an early interval in which the film is fluid it can be moulded, after which it has a definite set. DISCUSSION
Bonding of the protein molecules within the plane of the film, as evidenced by elasticity and viscosity, takes place either spontaneously with the passage of time or can be brought about by compression. For relatively short experiments of only a few hours duration, the elasticity and viscosity manifested upon compression are largely reversible. This suggests that the changes in elasticity and viscosity with compression arise rather simply from the felting together and mechanical interference of the separate protein molecules. The elasticity exhibited a t zero pressure, whether soon after spreading or as a very slow development, seems better explained by relatively permanent bonds within the film. Presumptively these are of the same type as the labile bonds of native proteins. It has been recognized, as pointed out by Cohn (4), that some kind of an unfolding must take place to account for the change of dimensions from the round shape of a dissolved protein molecule to the unimolecular film of much less thickness. The details of this unfolding are still a matter of speculation (see 2 and 16),but it is evident that the exposure of reactive groups capable of forming a plane polymer may be involved. The nature of these reactive groups should be revealed by the effects of substances dissolved in the subsolution upon the tendency of the film to form a membrane spontaneously. The pH dependence encountered here suggests that ionizable amino acid side chains are largely responsible; repression of carboxyl ionization by high acidity prevents intermolecular bridge formation, while a t more nearly neutral reaction carboxyl groups of one molecule may unite with amino groups of another to form links between units. This bonding of the protein to form an elastic membrane requires some time, even if the hydrogen-ion concentration is favorable. The initial
898
LYMAN FOUKT
step of protein spreading is a fluid surface film. However, its condition need not be the same as the condition of the films that remain liquid a long time on acid solution. There the average thickness of the film, computed from area per milligram, implies an extensive change of molecular structure. In the first stage of spreading a film could be fluid with no change of molecular structure a t all,-being in fact a thin layer of almost unchanged protein solution. For example, the average amount of protein solution used per film in these experiments was 0.08 ml., which would cover the area usually available (14x 25 cm.) to the depth of 2 X lo4 cm. This is 500 times the diameter, 40 A., of a small protein molecule in solution. The conception of the process of protein spreading as having two steps, (1) distribution as a layer of protein solution and (8) adsorption to the surface, with change of molecular shape and possible new linkage, permits an explanation of some of the methods of securing quantitative spreading. On acid solutions, or on others on which the film remains fluid, quantitative spreading is readily secured. Here the adsorption of a small amount of protein can bring about the distribution of the whole by forces conventionally described as surface tension. Although the adsorbed protein molecules are altered in structure and become insoluble, they are not bonded together and so offer no obstacle to continued spreading. When an elastic membrane is formed, however, it tends to restrain the distribution and prevent full spreading of the part of the protein solution last applied. Alcohol acts to prevent this by accelerating the first step,distribution as solution. To some extent, the effect of increasing the electrolyte content of the subsolution may be the same, by increasing the surface tension difference. However, it is possible that the electrolyte may also act to favor adsorption of the protein to the surface, and promote spreading more by lowering the surface tension of the protein solution than by increasing that of the clean surface. SUMMARY
1. The surface viscosity of unimolecular layers of proteins and other
substances has been determined, subject to the fulfillment of certain conditions, from the damping of a vane oscillating in the film. This method is particularly useful in working with substances like proteins which form rigid films. 2. A quantitative index of surface elasticity is obtained from the shortening of the period of the oscillating vane. This effect is not due merely to the sweeping action of the vane but is related to the intrinsic elastic properties of the film materials. 3. Expressions relating the surface viscosity and elasticity to the lengtii of the vane are given. 4. The pH of the subsolution is found to be an important factor in de-
LATERAL COHESION I N PROTEIN MONOLAYERS
899
termining the viscosity and elasticity of a protein film. The pH dependence of rapid bond formation within the film suggests that these links are ionic. 5. Quantitative spreading into the unimolecular film is observed when the processes which bring about distribution of the film substance precede or overbalance the restraining tendencies of rapid bond formation. The author is much indebted to Professor F. 0. Schmitt for aid and advice throughout the course of this work. REFERENCES (1) ADAM,N.K.,A N D ROSENHEIM, 0 . : Proc. Roy. SOC.(London) A M , 25 (1929). (2) ASTBURY, W. T.:Kature 137, 803 (1936). (3) BIAR, R. S.,SCHMITT,F. O., A N D YOUNG,J. Z.: Proc. Roy. SOC.(London)
Bl23, 520 (1937). (4) COHN,E. J.: Ann. Rev. Biochem. 4, 93 (1935). (5) FOURT, L.: Am. J. Physiol. 119, 310 (1937). (6) FOURT,L., AND HARKINS, W. D.: J. Phys. Chem. 42,897 (1938). (7) FOURT,L., A N D SCHMITT, F. 0.: J. Phys. Chem. 40,989 (1936). (8) HUGHES,A. H., AND RIDEAL,E. K.: Proc. Roy. SOC.(London) A137, 62 (1932). (9) LANGMUIR, I., A N D SCHAEFER, V. J.: J. Am. Chem. SOC.69,2400 (1937). (10) LANGMUIR, I., SCHAEFER, V. J., AKD SOBOTKA, H.: J. Am. Chem. SOC.69, 1751 (1937). (11) MARANGONI: Kuovo cimento [2]6-6, 239 (1872). Cited from Rayleigh (14). (12) MOQUIN,H., AND RIDEAL,E. K.: Proc. Roy. SOC.(London) A114, 690 (1927). (13) OBERBECK, A.: Weidermanns Annalen 11, 534 (1880). (14) RAYLEIGH (STRUTT,J. W.): Scientific Papers 3,351,363, Cambridge University Press; Proc. Roy. Institution 13, 85 (1890);Proc. Roy. Soo. (London) 48, 127 (1890). (15) S C H ~ T K.: T , Ann. Physik [4] 13,712(1904). (16) WRINCE,D.M.:Nature 137,411 (1936).
