Oct., 1954
847
MONOLAYERS OF BOVINE PLASMA PROTETNS
MONOLAYERS OF BOVINE PLASMA PROTEINS BYJ. L. SHERESHEFSKY AND MAURICE E. KING’ Contribution from the Chemistry Department, Howard University, Washington, D. C . Received March 6, 1064
Surface pressure-area measurements were made on monolayers of bovine plasma protein fractions of albumin, @- and yglobulins and fibrinogen. These measurements were made at different values of pH and salt concentration of substrates. Molecular weights calculated from an analog of Amgat’s gaa equation are lower than those obtained from osmotic pressure or sedimentation measurements, and vary with pH and salt concentration. The data on albumin were also fitted to an equation which includes a force constant, and the value of the constant is at a minimum a t the isoelectric point and decreases with increasing cation concentration of the substrate. The molecular weights are higher than those obtained from the Amagat equation and are approximately a multiple of a Svedberg unit.
Introduction I n 1945 Bull2 reported the results of his studies of monolayers of egg albumin and /?-lac toglobulin on concentrated salt solutions using the Wilhelmy type film balance. By applying an analog of the Amagat gas equation in two dimensions to the low pressure regions of the force-area curves, values were obtained for the molecular weights which agreed with those found from osmotic pressure and sedimentation data. It was considered desirable to apply this method of molecular weight determination to crystalline bovine aIbumin which has become available and to study the effect of pH and salt concentration on molecular weights obtained in this manner. Experimental
face of the substrate with an Agla micrometer syringe and three to five minutes allowed for the film to spread. The area of the film was then slowly compressed and the corresponding surface pressure measured. At the end of each
TABLE I SUMMARY OF CONSTANTS FOR ALBUMIN FROM COMPOSITE FORCE-AREACURVES Substrate 0.2 M Acetate 0.2 M Phosphate 0 . 1 m NaCl 0 . 1 6 m NaCl 0 . 2 m NaCl
pH 4.4 4.8 5.9 6.8 7.2 7.3 7.3 7.3
Limitinz area (ex(Islopeof trap.), curve), e ~ m. . per mg. 0.94 0.70 1.00 .73 0.94 .75 1.04 .60 1 .oo 1.07 0.96 0.79 .36 1.04 .75 1.00
nRT, ergs 2 . 2 x 103 3 . 4 x 103 2 . 2 x 103 3 . 6 x 103 3 . 2 X 108 2.0 x 103 3 . 4 x 103 2 . 3 x 103
Mol. wt. 11,320 7,340 11,320 6,920 7,780 12,450 7,340 10,800
TABLE I1 The film balance used in this study was of the type used in the Colloid Science Laboratory and as previously de- SUMMARY O F CONSTANTS FOR ?-GLOBULINFROM ~ O M P O S l r P ~ scribed.3 FORCE-AREA CURVES The bovine plasma protein fractions, albumin, @-globulin, Limiting area y-globulin and fibrinogen were obtained from the Armour (ex- (slope of Packing Company in the solid form. The albumin fractrap.), curve), nRT, Mol. tion was the crystalline material prepared by this company Substrate pH sq. m. per rng. ergs wt. and used as a standard in osmotic pressure measurement^.^ 1,OMAcetate 4 . 8 1.00 0.69 1.0 X l o 3 24,900 Stock solutions containing 1 mg./ml. of the fractions were 5.5 1.06 .63 1 . 6 X IO3 15,600 0.2 M prepared by dissolving albumin in water, the @-globulinand .57 2 . 4 X 108 10,380 Phoe6.8 0.78 the y-globulin in 2 M sodium chloride and the fibrinogen phate 7.2 1.01 .89 4 . 0 X IO3 6,240 in 0.05 M sodium citrate. Spreading solutions6 were pre7.3 0.60 .50 1 . 6 X 103 15,600 0 . 1 m NaCl pared by mixing two ml. of stock solution with pro yl alco0 . 1 6 m NaCl 7.3 .70 2 . 0 X 108 12,450 0.95 hol and distilled water to produce a total volume often ml. 0.2mNaCl 7.3 0.61 .47 1 . 3 X 103 19,150 One ml. of 1.7 M sodium acetate was added to the globulin solutions to prevent coagulation. TABLE I11 The buffered substrates were prepared according t o FROM COMPOSITE Green.6 The acetic acid-sodium acetate system was used SUMMARY OF CONSTANTS FOR @-GLOBULIN for solutions of pH 4.4 and 4.8 and the monopotassium FORCE-AREA CURVES phosphate-dipotassium phosphate system for the higher Limiting area (ex- (slope of values of BH. trap.), curve), nRT, Mol. For the’ study of the effect of salt concentration on the Substrate ~ € 1 sq. m. per mg. ergs wt. protein films, a stock solution was prepared containing 1 . 0 MAcotate 4 . 8 0.55 0.40 0.8 X 103 31,150 54.560 g. of sodium chloride, 7.760 g. of disodium phos0.2M 5.5 0.7G .54 1 . 8 X IO3 13,650 phate and 1.162 g . of monopotassium phosphate per liter. Phos6.8 1.17 .77 4 . 0 X 103 G,210 This solution was one molal with respect to uni-univalent phate 7.2 .73 4 . 0 X lo3 6.240 1.07 ion concentration and contained the phosphate buffers t o 0.1 mNaCl 7.3 0.62 .40 1 . 4 X lo3 17,800 maintain a pH of 7.3. Hypotonic, isotonic and hypertonic 0.16mNaCl 7.3 0.62 .46 1 . 7 X lo3 14,030 solutions were made by diluting, respectively, 100, 160 and 0.2mNaCl 7.3 0.66 .56 1 . 3 X 103 19,150 200 ml. of the stock solution to a volume of one liter. The trough of the film balance was filled with the approTABLE IV priate substrate until the liquid was one mm. above the edges. Surface contamination was removed by sweeping on both SUMMARY OF CONSTANTS FOR FIBRINOGEN FROM COMPOSITE sides of the mica float until the zero position remained conFORCE-AREACURVES stant. The spreading solution was deposited on the surLimiting area
-
(1) Based on a thesis submitted by Maurice E. King in partial fulfillment of the requirements for the Master’s degree. (2) H. B. Bull, J . Am. Chem. SOC.,67, 4 (1945). (3) J. L . Sheresliofsky and A . A. Wall, ibid.,66, 1072 (1944). (4) G . Scatchard, A . Batchelder, A . Brown and M.Zosa, ibid., 68, 2G10 (194G). (5) S. Stalberg and T. Teorell, Trans. Paraday Soe., 36, 1413 (1939). (G) A. A. Green. J . A m Chern. 900.. 65, 2331 (1033).
Substrate 1.0 M Acetate 0.2 M Phosphate 0 . 1 m NaCl 0.16 mNaCl 0 . 2 mNaCl
pH 4.8 5.5 6.8 7.2 7.3 7.3 7.3
(ex- (slope of trap.), curve), sq. m. per rng. 0.95 0.77 0.95 .G8 1.18 .80 1.08 .go 0.7G .5G 0.93 .76 0.00 .57
nRT, ergs 1 . 2 X IO3 2 . 2 X lo3 2 . 4 X 103 2 . 4 x 103 2 . 6 X 103 1 . 8 X 103 1 . 9 X 103
Mol. wt. 20,750 11,320 10,380 io,38n 9,600 13,850 13,100
J. L. SHERESHEFSKY AND MAURICE E. KING
848
run, the film was swept to the end of the trough and removed with a suction pipet. In each case at least three runs were made and composite surface pressure-area curves drawn from the data.
Vol. 58 TABLE V
SURFACEPRESSURES OF BOVINEALBUMIN MONOLAYERS Area,
Results and Discussion The type of force-area curves obtained for the four different protein fractions js shown in Fig. 1, and a typical FA-F curve is shown in Fig. 2.
-pH
sq. rn.
per nig. 1.0 1.2 1.5 2.0
4.4 0.76 .52 .35 .24
Surface pressure (dynes per om.) p H constant (7.8) VariableConcn. PH 0.10 0 . 1 6 0.20 4.8 5.9 6.8 7.2 m m m 0.94 0.85 1.44 1.40 0.77 1.22 0.70 .61 .58 0.82 0.73 .57 0.77 .48 .38 .38 .46 .50 .35 .52 .34 .28 .28 .36 .40 ,24 .42 .24
TABLEVI SURFACE PRESSURES OF BOVINE^/-GLOBULIN MONOLAYERS Area,
aq. m.
per
mg.
0.80 0.90 1 .oo 1.20 1.50 2.00
&
Surface pressure (dynes erConstant om.)
-pH 4.8 0.68 .46
.37 .31 .28
.25
Variabl5.5pH6.8 0.86 0.88 .64 .65 .53 .56 .44 .45 .37 .36 .32 ,33
7.2 3.20 1.50 1.03 0.56 .51 .38
0.10 m 0.64 .57 .55 .52 .50 .50
(7.3) Concn. 0.16 0.20 m
m
2.20 0.92 .67 .45 .32 .26
0.56 .49 .25 .22 .21 .20
1
TABLE VI1 SURFACE PRESSURES OF BOVINE ?-GLOBULINMONOLAYERS Area,
sq. rn.
per
mg. 0.80 1 .oo 1.50 2.00
Surface pressure (dynesp & erConstant cm.) -pH
4.8 0.42 .38 .34 -31
Variable-
pn
5.5 0.79 .55 .37 .35
6.8 5.00 1.60 0.56 0.40
7.2 1.70 0.60 0.44
0.10 m 3.00 0.58 .25 .22
(7.3) Concn. 0.16 0.20 m m 0.49 0.47 , -40 .34 .24 .23 .24 .20
TABLEVI11 SURFACE PRESSURES O F BOVINE FIBRINOGEN MONOLAYERS Area, sq. m. per mg.
01 0
I
Fig. 1.-Typical
I
1
I
I
1 2 3 A , sq. m./mg. curve of surface pressure us. area for plasma proteins.
0 Fig. 2.-Typical
4.8 1.00 0.50 .34 .30
Surface pressure (dynes per om.) pH Constant (7.3) Variable----. Concn. 0.10 0.16 0.20 5.5 p H 6 . 8 7.2 m m m 1.05 3.60 0.75 0.76 0.54 0.77 2.35 4.00 .63 .60 .48 .48 0.64 0.50 .62 .35 .33 .40 0.42 0.32 .32 .26 .29
Tables I-IV was obtained by extrapolating the F-A curve to zero surface pressure, F. The ‘(area constant” given in the fourth column was obtained from the slope of FA-F curve, and in the fifth column are given the intercepts of this plot when extrapolated to zero pressure, F. The molecular weights given in the sixth column were calculated from the intercept by equating it to nRT, where n is the number of moles in 1 mg., R the gas constant and T the absolute temperature. Bovine plasma albumin and fibrinogen show complete or nearly complete spreading under the various conditions of p H and salt concentration with the exception that fibrinogen is incompletely spread on 0.1 m sodium chloride with a pH of 7.3. The average limiting area for albumin is 1.0 i 0.04 sq. m. per mg. and for fibrinogen, 0.96 f 0.09 sq. m. per mg. 7-Globulin and @-globulindo not spread as readily as albumin OF fibrinogen. Their limiting area when spread completely is about 1sq. m.per mg. Tables V-VIII show that the surface pressure of the protein films is greatly affected by the hydrogen ion and cation concentration. The surface pressure increases with the pH of the substrate, but is
.
t
01
0.90 1 .oo 1.50 2.00
I
-pH
I
1
I
,
I
I
1
2 3 ‘4 F , dynes/cm. curve of FA us. F for plasma proteins.
In Tables I-IV are given the characteristic values for the various protein films on the several substrates of different p H and salt concentrations. In Tables V-VI11 are given a t several constant areas, the surface pressures a t different p H . The “limiting area” given in the third column of
s
Oct., 1954
MONOLAYERS OF BOVINE PLASMA PROTEINS
affected irregularly by the salt concentration. Thus albumin and yglobulin show maximum surface pressures on an isotonic substrate, while for P-globulin and fibrinogen, the surface pressure decreases with salt concentration. Similarly, but to a lesser extent, the specific area of the protein fractions a t constant surface pressure seem to increase with the p H of the substrate. The calculation of the molecular weights was based on the two-dimensional analog of Amagat’s gas equation F A =,nRT + BoF where Bois the difference in area per n moles of the real and ideal monolayer. The value of Bo which is also taken as the area of the molecules in the monolayer is less than the “limiting area” obtained by extrapolation of the F-A curve. This equation accounts for the osmotic factor and the area, but fails to account for the attractive forces. The values of the molecular weights obtained from this equation are low, and vary with the p H or salt concentration. The low values of the molecular weights and their variation with p H and salt concentration, and the variation of the area with these factors a t constant surface pressure, strongly support the presence of denat~ration.~ The variation of the surface pressure with p H and salt concentration of the substrate is indicative of strong interaction forces between the protein molecules in their different ionized states. It was, therefore, of interest to try to apply to at least part of the present data an equation of state that includes a force constant. The equation (7) E. Mishuck and F.Eirich, 12th International Congress of Pure and Applied Chemistry, September 10-13, 1951, Paper #31, “Surface Films of Synthetic Polypeptides.”
(F
849
- Fo)(A - AO) = nRT
was thus applied to the data for albumin at several pH’s and salt concentrations and the constants FO, Ao and the molecular weights evaluated. The Ao values are higher than the Bo values obtained from Amagat’s equation, and approach closely the values of “the limiting areas.” The FDvalues are positive and vary with p H ; at p H 4.8,the isoelectric point for bovine albumin, it is a minimum. At constant p H these values decrease with salt concentration. The molecular weights are higher than those obtained from the analog of Arnagat’s equation. At p H 4.8 the value is approximately 67,000, and a t higher p H it varies from 20,000 to 23,000.
DISCUSSION HERBERT L. DAvm-Are your results compatible with the suggestion that in these proteins charge and hydration play roles comparable to those known to apply in dispersion? Thus, for albumin! increasing pH results in increasing pressure and this might reflect increasing hydrate volume. Likewise, salt increases charge through a maximum as it is known to do in sol stability. If these be justified, it will be necessary to distinguish how solvation here gives increased volumes, and in the sedimentation findings of Dr. Ross gives decreased volumes. F. M. FOWKEs.-It appears that the authors have used the “Amagat” equation incorrectly for the determination of molecular weight. As discussed by N. K. Adam (“Physics and Chemistry of Surfaces”), the “Amagat” lot of F A us. F for monolayers goes through a minimum wxen the film is in the li uid expanded state; a gaseous monolayer is obtained only a t much lower film pressures and at larger values of F A . The intercept at F = 0 of F A for gaseous films may be several times the intercept obtained by extrapolation of FA from the high pressure portion OI the plot as was done in this paper. The use of the Langmuir equation J . Chem. Phys., 1, 1 (1933)) for liquid expanded films F’) (A A ’ ) = nRT appears novel for determination of molecular weight and may prove very useful.
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(h -
