EXPANSION PATTERNS OF PROTEIN MONOLAYERS ON WATER' VINCENT J. SCHAEFER Research Laboratory, General Electric Company, Schenectady, New York
Received July 1.6, 1958
When a monolayer of a protein is formed on a water surface, it exists as a homogeneous, insoluble, reversibly compressible (8, 6), two-dimensional structure with certain properties characteristic of the specific proteins. Protein monolayers can be formed by a number of methods: for example, from a small amount of dried protein placed on the water surface ( 5 ) , from a small amount of dissolved protein ejected from a micropipet inserted parallel and half submerged in the water surface (4), from drops of a solution placed on a flat plate dipped into the water, by dissolving the protein in the bulk substrate followed by subsequent stirring, or by spreading the protein on a strong salt solution followed by removal to another tray ( 7 ) . As has been shown by many investigators, a protein spreads best a t or near its isoelectric point. Protein monolayers spread and deposited under low pressures show thicknesses ranging from 6 A. to 18 A,, depending on the specific protein used. This paper will describe a very simple technique for rendering surh films visible and will indicate the possibilities available for following changes in molecular structure, orientation, and other characteristics which are related to the fundamental properties of the native protein. The apparatus required for studying expansion patterns is exceedingly simple. A shallow tray with flat edges, several barriers for cleaning the water surface, suitable illumination, and a few drops of indicator oil comprise the total necessary equipment. Our trays are made of %-in.angle brass fastened to a &-in. flat sheet of the same material, the joints being water-proofed and the inside covered with black Bakelite paint. A mixture of carbon black and paraffin applied hot will serve satisfactorily. The black background whirh emphasizes the contrasting intensities may also be obtained by using a sheet of cobalt glass or Bakelite in the bottom of the tray or by coating the outside of a glass tray with black paint. Presented a t the Fifteenth Colloid Symposium, held at Cambridge, Massachu setts, June 9-11, 1938. 1089
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VINCENT J. SCHAEFER
Dr. K . B. Blodgett has previously described the preparation of indicator oil (2). Automobile oil oxidized by prolonged heating is mixed with pure mineral oil. When placed on clean water this mixture sweads to form thin films of a definite thickness, depending on the amount of the oxidized material added. For observinQ expansion patterns a mixture which produces a thickness of about 1500 A. and a first-order yellow film when viewed a t 45" has proved to be very satisfactory. Several lamps placed behind a sheet of opal glass (milk glass) proride a very fine source of diffuseillumination for viewing the patterns. Daylight interspersed by a sheet of translucent paper also works quite well. FORMATION OF AN EXPANSION PATTERN
In order to illustrate what is meant by the expansion pattern of a protein, let us use a sample of pepsin. The important steps are illustrated in figure 1. Distilled water at equilibriuni with the air at pH 5.8 is placed in the tray. The surface is cleaned by sweeping several times with barriers, and enough indicator oil is applied to cover three-quarters of the clean surface. A cleaned platinum wire transfers a small amount of pepsin in the form of powder to the center of the surface area. It spreads on the water, forming an invisible film whose advancing border will be seen as it drives the indicator oil ahead of it in radial directions. I n the case of pepsin, this advancing film will be seen to present a smooth rounded concentric edge, of a radius equal to the distance from the point of application. The outline of the advancing boundary of the monolayer is often characteristic of specific proteins and will be referred to from time to time. Care should be observcd that only enough protein is applied so that the remaining free surface is nearly covered with a monolayer. After the monolayer is spread, barriers are used to push the surrounding indicator oil into intimate contact with the outer edge of the monolayer, care being taken that the monolayer is not subjected to any degree of pressure. This is controlled by stopping compression before any change in color of the indicator oil occurs. A perceptible change in color equals a pressure of F = 1dyne per centimeter. After intimate contact is reached with the oil and the monolayer, a small drop of indicator oil is placed in the central portion, and will in the case of pepsin expand to produce a geometrical figure of a star-like form, with the indicator oil showing a peculiar gradation in color intensity, appearing thinner than normal a t the monolayer boundary. If other portions of the monolayer are treated in a similar way. patterns identical in every respect will be observed, indicating that the film possesses a structure which is uniform throughout, showing no memory of the point of formation. Because of their high compressibility, many proteins, if permitted, will spread on the water to build up pressures of more than 10 dynes per centimeter. Most proteins exhibit varied changes in expansion pattern as the
EXPANSION P A m E R N S OF PROTEIN MOKOLAYERS
1091
surface pressure is increased. Egg albumin, for example, when subjected to a eompression of 35 dynes in one direction will show a fibrous structure when expanded by a high-pressure indieator oil, while still under pressure, with the fibrous sheets oriented parallel to each other a t right angles t o the direction of eompression. Such sheets may he lifted from the water surface as threads and when dried are nearly invisible
Fro. l. Formation of expunrimi ~ x t t t w n s . ii. :t~,plyingindieator oil; b, indicator oil spreading; e , applying protaiu rniinol;ayrr: (I, ,cxpnnding monolayer; e, pepsin expansion pattern. CLASSrFICATIOS OF EXPANSION PATTERNS
Expansion patterns of the proteins stidird may he roughly divided into three general groupings of geometrienl configuration. The terms “starlike,” “rough circular,” and “smooth rireular” will servr to describe these three classes. “Star-like” refers to patterns jt-hirh expand at lox pressure to produre
11392
VINCENT J. SCHAEFER
figures related in configuration to a star. Although five-pointed stars are often formed, the number of points observed varies from three to six, Typical examples of these figures may be found with pepsinogen, tobacco seed globulin, and trypsinogen, illustrated in figure 2. In certain cases, particularly with pepsin and trypsinogen, the star form produced by the expansion of the indicator oil is further modified by a peculiar gradation in the color of the indicator oil, producing a thinning out of the oil film which under normal conditions is exceptionally uniform. The protein monolayers which produce expansion patterns of the star-like form are, in general, of the type described by Hughes and Rideal as a gel structure. I t should be noted here, however, that the gel-like monolayers considered in this paper are formed spontaneously at very low pressures of the order of 0.5 to 1.0 dyne per centinieter and in general are found to be characteristic of the specific protein, regardless of the spreading method used. That higher pressure often produces a film of the gel type from a liquid monolayer may be observed if gliadin acetate is compressed to 15 dynes per rentimeter. Under this compression this protein, which is very liquid under low pressure, shoa s a typical star-like expansion pattern. While about half of the proteins studied show the general star-like expansion pattern, each one has its own modification of the general form. iiside from the question of p H and relation to the isoelectric point, there are other characteristics peculiar to the specific protein. Thus the advancing edge of the monolayer in the case of pepsin is smoothly circular, while with egg albumin it is very irregular, with the indicator oil forming peculiar wedge-like structures which divert the advancing edge of the monolayer to either sidr t o produce the irregularity mentioned. In the rough circular classification trypsin, papain, and 1% heat gliadin may be used as examples (figure 3). This form is characterized by an expansion pattern whosr general form is circular, but when examined locally the edge exhibits a roughened appearance, the extent of irregularity being characteristic of the specific protein used. Thuq trypsin produces a circular advancing edge but an internal slightly ragged outline, with the peculiar grading in indicator oil color observed with pepsin and trypsinogen. With whrat gliadin, on the other hand, both external and internal patterns are identical, the outline being very jagged. The smooth circular form is characteristic of such proteins a5 zein, gelatin, and protamine (figure 4 ) . and of films formed by proteins in an advanced stage of denaturation or by protein degradation products which dill form monolayers. EFFECT O F DEXATURATION O X EXPAXSION PATTERXS
Many investigatorb (1, 3, 9, 10) have shown that protein solutions, when subjected to treatment such as heating, ultraviolet radiation, and
EXPANSION PATTERNS OF PROTEIN MONOLAYERS
1093
: s 7 - 1 , .
4 I.:fiert of lieat denaturation of pcpsin. a , 2 mi". at 65°C.; b, 5 mi". at 6 5 T ;c.7min.nt65°C.;d,25min.ht65'C. l30. 5
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VIKCEKT J. SCHAEFER
shaking, tend to alter the protein in such a way as to modify its native properties. 111 the case of pepsin this modification generally parallels loss in activity of its enzymatic properties. When 0.1 per cent solutions of pepGii are subjected to such denaturation processes, the loss of activity is 5trlkingly demonstrated by the change in expansion pattern. I n the case of pepsin the .tar-like pattern gives way to a circular form, the pattern shoning a rapid transition, the firqt changes appearing before the loss of activity becomes readily detectable by the change in the power of pepsin to clot milk (6). When a 0 1 per cent solution of pepsin (Lilly), held in a quartz tube 15 cm. from an ultraIioiet lamp of 1.2 amperes and 145 volts, emitting \ i a ~ d e n g t h sof 2536 and up, wab irradiated for 5 min , no change in activity or expansion pattern was found. When irradiated for 10 min., the pattern was conipletely changed, nhile the protein had lost but 4 per cent of it.. original activity. The pattern had become entirely circular after 15 min. with 25 per cent loss in activity, although the area which a given amount of protein covered remained about the same. After 30 min. of irradiation, the activity showed a 68 per cent loss, and now the actual area covered had decreased to less than half of that covered by the same ainouiit of iolution irradiated for 15 min. The alteration in expansion pattern M hen a solutiaii of pepsin 13 iubjected to a vigorous qhakiiig will non be considered. One hundred cubic centimeters of a 0.1 per cent pepsin solution in a half-liter bottle ma5 placed in a niachjrie -rThich produces 400 movements a minute, the distance of travel for each stroke being 3 cm. Samples of the solutio11 were withdramn a t definite time intervals, and the same amount of solution used for producing a monolayer on water. The transition in expailsion pattern showed a considerable chaiige after only 1 min. of shaking, the alteration in pattern again shifting from the itar-like to the circular form, as nith the deactivation by ultraviolet irradiation. The ability of the p e p i n to clot milk after 1 miii of shaking had decrea>ed hy more than 60 per ceiit. -4ftei 5 min. of qhaking. the area of fiini produced qho.ived about a 50 pel cent decrea.e nith the activity decreased by 83 per cent. Xftei 10 inin. of %hakingthe area of film had decreased still more, x i t h the activity decrea-ed by 88 per cent with no further change in expansion pattern. TJ’hen a solution of pepsin at pH 5 8 is d j e c t e d to an elevated teniperatiire, it rapidly lose? it- enzymatic properties. which again is indicated by the change in pattern A 0.1 ppr ceiit iolrition of pepsin n a s heated at 65°C for definite priodc of time; then it n a. chilled to room temperature and the expansion patterns oliieri ed. Heating for 2 inin failed to produce any apparent change in pattein, and actiyity ~iieasiir~~rieiits h o n e d the prp-in retiiried it. tu11 poner to clot milk n’hrn heated for 3 rnin ,
-x.
EXPANSION PATTERNS OF PROTEIN MONOLAYERS
1095
however, the pattern indicated a slight change, and the milk-clotting test showed a 16 per cent decrease in activity. After 5 min. the activity had decreased 28 per cent, and the pattern changed to a very broad star form. After 10 min. the pattern was an irregular figure approaching a circle with
F m 6. l’rpsin denaturation a, irradiated with ultraviolet light for 10 mi”.; b, irradiated wit11 ultraviolet light for 30 min.; e, shsken for 1 “in.; d, shaken for 10 mi”.
Fro. 7. EKeet oi dissolved salts on expansion patterns. a, insulin; b, insulin 41 0 P M zinc ohloride; e , insulin 10-4 M cupric chlmidc; (1, gliadin acetate; e, gliadin acetate 1 0 P M cupric chloride.
+
+
a decrease in activity of 38 per cent; after 25 min. the pattern was a perfectly smooth circle, and the pepsin then showed a loss in activity of 62 per cent. The alterations in pattern and area doe to the various types of denaturation are sholvn in figures 5 and 6.
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VISCEXT J. SCHAEFER
ion11 that the addition of a T ery small amount of pepsin to solutions of protein- grratly increase5 their tendency t o form monolayers. When a solutioii ut cgg albiunin containing 10 mg. of the protein, partly coagulated by hmtiiig, n-w iiioculatcd with 0.01 ing. of pepsin and tlir p H of the solution hcld at 4.6, the monolagcr formed from a given amount of protein coreicd *P\W time3 the area of the control. The expansion pattern was of t h r smooth circular type, and gradually reverted to t h r star-like forin a$ the film remained on the mater. When the same procedure was followed v i t h the pH of the inoculated ~olutioiiheld a t 2 0.
TBBLE 1 General classification of e x p a n s i v n patterns PATTERN F O R l I AT F 0.5 TO PER CENTIMETER
1.0
DYNE
PitOTEIN
Star-like
g?:fjr
1
circular Smooth
- _-I- ___ ____ Egg tLih:imin . . . . . . . . . . . . . . . . . . . . . . . . . .' X Pepsin ........................... X Pepsinogei! . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X Trypsin... . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x l Trypsinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I' x i LTrease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' x ! Edestin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x Tobacco seed globulin, . . . . . . . . . . . . . . . . . .' . x Tobacco mosaic virus*. . . Wheat gliadin,.. . . . . . . . . . . . . . . . . . . . . . . , x Gliadin acetate, . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zeiii . . . . . . . . . . . . . . . . . x Papain . . . . . . . . . . . . . . . x~ Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 s I Protamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' ' X Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X Protamine-insulin. . . . . . . . . . . . . . . . . . . . . . ' I X Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 X _ _ _ _ _ _ _ _ _ _ ~ ~ _ _ ~ ~ ~ _ _ ~ . _ _ * Formed on saturated ammonium sulfate solution. ~
...
~~~
~
~
I
~
1
I
thp area covered twice that of the control, sho1Yiiig a siiiooth cirrular rxpansion pattern 1% hich remained unchanged with time. EFFECT O F DISSOLVED SALTS ON CXPAXSIOS PBTTEKKS
The expariiioii pattern of pure insulin TI hen spread oii di+tilled nater a t pH 5.8 xvxasfound to be circular in form. When a inolar solution of cupric chloride was added to the water, the expansion pattern of insulin spread in the same manner showed a major change, exhibiting the star-like pattern somewhat similar to pepsin. With the same concentration of zinc
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EXPANSION PATTERNS OF PROTEIN MONOLAYmS
chloride a different form was found, showing much less molecular cohesion than was exhibited by insulin spread on water containing cupric ion. In order to determine whether cupric ion might cause the same alteration in pattern with other proteins, gliadin acetate was spread on the cupric chloride solution which produced the striking change in the insulin pattern. No alteration in the normal circular pattern of gliadin acetate could be found. It would thus appear that the insulin molecule has reactive groups which readily combine in a specific manner with certain divalent ions to produce a definite change in the monolayer. These effects are shown in figure 7. TABLE 2 Expansion patterns of. .proteins
-
PATTERN FORM AT F 0.5 TO DYNE PER CENTIYETER
1.0
PROTEIN
Star-like
Rou h
c,rc$r
Smooth circular
Pepsin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pepsin denatured by ultraviolet irradiation. . . . . . . . . . Pepsin denatured by h e a t . . . . . . . . . . . . . . . . . . . . . . . . . . . Pepsin denatured by shaking . . . . . . . . . . . . . . . . . . . . .
Insulin
+ 10-4 M zinc chloride . . . . . . . . . . . . . . . . . . . . . . .
Gliadin acetate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gliadin acetate 3. 10-4 M copper chloride. . . . . . . . . . . Wheat Wheat Wheat Wheat
gliadin.. . . gliadin gliadin gliadin
+ + +
..........
Egg albumin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Egg albumin 0.01 N hydrochloric acid. . . . . . . . . . . . Egg albumin 0.01 N ammonium hydroxide. , . . , . , ,
+ +
EFFECT OF P H ON EXPANSION PATTERN
Wheat gliadin spread in dry form on distilled water a t pH 5.8 exhibited an expansion pattern of jagged outline, both internally and externally, and
was very different from gliadin acetate, which produced a circular pattern in both respects. When the gliadin was spread on 0.01 N acetic acid (pH 3.4), the pattern was identical with that of the gliadin acetate. Since 0.01 N hydrochloric acid (pH 2.0) also produces a circular pattern, it is quite probable that the pH is a factor in producing the change in
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VINCENT J . SCHAEFER
pattern. I t should be noted, however, t'hat a change in pH does not have the same result with a protein such as egg albumin, which retains its starlike pattern at pH 5.8, 3.4, 2.0, and 10.6 with only slight modifications. When the gliadin was spread on 0.01 N ammonium hydroxide (pH 10.6), it had a pattern uearly the same as that found a t pH 5.8. Wheat gliadin dissolved in 70 per cent ethyl alcohol produced a monolayer ident,ical with that of gliadin acetate in its pattern. Table 1 is a general classification of observed expansion patterns of proteins spread on the cleaned surface of distiiled water a t pH 5.8. While it is realized that proteins spread best' a t or near their isoelectric points, it is significant that no change in the observed patterns could be found when special buffered solutions were used at t'he isoelectric points of specifir proteins. The only difference was t'he greater area of monolayer fornicbd for a given amount of protein when spread a t the isoelectric point. This observation is due probably to the fact that the pH 5.8 is not very far from the isoelectric point of most of the prot,eins used. 'Tack 3 records a number of alterations in the expansion patterns of Ppecifk proteins by changes in the composition of the substrate solution or by alterafioris in the protein before spreading. REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
M. L., AND MIRSKT,A, E.: J. Gen. Physiol. 13, 121 (1930). BLODGETT, K. B.: J. Optical SOC. Am. 24, 313 (1934). GORTER,E. K.: Trans. Faraday SOC. 33, 1125 (1937). GORTER, E. K., AND GRENDEL, F.: Trans. Faraday Soc. 22, 477 (1926). HUGHES, A. H., AND RIDEAL,E , : Proc. Roy. SOC.(London) A137,62 (1932). L.4NGJlUIR, I., AND SCR.4EFER, \'. J.: J. Am. Chem. SOC. 60, 1351 (1938). LANGMUIR,I., AND SCHAEFER, V. J.: J. Am. Chem. SOC., t o be published. LANGMUIR, I., SCHAEFER, V. J., AND WRINCE,D.: Science 86, 76 (1937). NEURATR, 11.: J. Phys. Chem. 40,361 (1936). PHILLIPI:On the Natxre of Proteins; Thesis, Amsterdam, 1936. ANSON,
