August, 1957
THICKNESS AND REFRACTIVE INDEX OF PLASMA ALBUMINFILMS ON STEARATE 1039
under the plug was examined. I n no case was the thickness under the plug less than the monolayer. The variation was very great. Table VI1 shows TABLE VI1 POST-RUPTURE THICKNESS DETERMINATIONS Material
Acrylic acid Methyl methacrylate Acrylonitrile
Stress, g./mm.2
Thickness,
A.
1060 Too thick t o measure 607 0-7 140 0-17.1 827(1) 16.2
the difference in the thicknesses of the area under the plugs. When a range is reported, the spread
was considerable over the many samples observed. Single values are averages of no less than nine samples. Conclusions.-Monolayers do not add appreciably nor detract markedly from the rupturestress values obtained when a droplet of the same liquid is deposited on a bare substrate. Such phenomena are in line with earlier considerations of cybotaxis. Utilizing present methods, it is not possible to rupture metalorganic bonds when monolayers are deposited onto the substrate. I n every case examination revealed cohesive rather than adhesive bond rupture.
THE THICKNESS AND REFRACTIVE INDEX OF PLASMA ALBUMIN FILMS ON STEARATE BY J. B. BATEMAN AND E. D. ADAMS Contribution jrom Chemical Corps, Fort Detrick, Frederick, Maryland Received January 10, 1967
Films of bovine plasma albumin (BPA) formed under a variety of conditions on barium stearate (Bast) surfaces by direct contact with solutions of the protein, followed by washing and drying, have been characterized as to thickness, refractive index and mass per unit area, by use of 9 new stepped interference reflector. Film properties are much dependent upon conditions of adsorption and upon subsequent treatment, ?a the following examples show: (a) 0.5% BPA in water, slide washed with water; refractive index, 72, 1.6, thickness, d , 34 A . ; (b) BPA in 0.15 ionic strength acetate buffer, p H 4.7, washed (c) BPA in HC1, p H 3; n = 1.24, d = 15 A. The various values of d are all compatwith buffer; n = 1.5,d = 16-22 ible with the dimensions and possible orientations of the BPA molecule without distortion or formation of a spread monolayer. Low values of n are interpreted in terms of incomplete coverage of the adsorbent and it is shown that following a second exposure of such films to BPA n approaches the close-packed value 1.5-1.6.
w.;
The experiments described in this paper may be said to stem from the work of Langmuir and Schaefer, who used barium stearate multilayer surfaces for the adsorption of proteins from solution, and of Blodgett,2 who devised a simple optical method which was applied by Langmuir and Schaefer in determining an apparent thickness of the protein film remaining after the multilayer exposed to the protein solution had been washed with water and dried. Subsequent workers have obtained much valid information concerning adsorbed protein layers by means of modified or new optical methods; however, the methods used have in common the limitation that the films are characterized by a single measurement, whereas the most elementary model of a thin film regarded as a plane homogeneous sheet demands the specification of a t least two properties corresponding to a metrical thickness and a density or refractive index. This limitation is partly overcome by introducing plausible assumptions concerning film density, but the need for such assumptions clearly precludes the use of these methods in cases where wide differences in film density and thickness may be encountered, as, for example, if it be desired to follow the process of occupation of vacant sites on the adsorbent by molecules which may be capable of assuming different intramolecular configurations and different orientations with respect to the surface.
The recent development, in this Laboratory,a of a method by which two measurements can be resolved into values for film thickness and refractive index would seem to remove some of the limitations of previous optical studies. Miscellaneous experimental data already published4 have shown that in several instances the method yields results which are in conformity with bulk constants, with established molecular dimensions, and in some cases with values obtained by independent optical methods requiring the use of very thick films. The present report addresses itself to the study of films for which no wholly satisfactory independent characberization is possible. Bovine plasma albumin (BPA) was chosen because of its ready availability in sufficient quantity and because of the great amount of physico-chemical information available. However, there remains a large gap between the adsorbed film and the bulk material, and one is forced to use the rather tenuous criteria of general plausibility, both as to the validity of the physical method and the compatibility of the results with the established molecular properties of the protein. These criteria are applied in discussing tJhe results of measurements on films formed under widely differing conditions of bulk concentration and pH and having widely differing optical parameters. No attempt has been made in this
(1) I. Langmuir and V. J. Schaefer, J . Am. Chem. SOC.,S9, 1406 (1937). (3) Iliat esaminatioii of the r:Lw experimental data should give a correct impression of changes in film concenAXs in order to establish the condition of minimum reflected intensity. A similar phenomenon can be observed by tration. I t map be noted also by comparing equations 1, employing light polarized in the plane of incidenre a t an 2 and 4 that i n general the observations may be expected to angle of incidence p,, of about 81 if the compound reflector give a more accurate estimate of y than of the individual has about 38 double layers of Bast, the increased thickness parameters n and d . I t is important to remember that the met,hod can be exbeing required because the phase changes a t the Bast-Cr pected to give physically meaningful results only to the extent that t,he films behave as though they conformed to the Msxwellian boundary conditions used i n the theory. The point is illlistrated in Fig. 1 showing, in descending order, ( a ) the ideal plane-parallel homogeneous film envisioned i n the theory, ( b ) an homogeneous film of refractive index n and a roJgh surface varying about a median thickness d , ( r ) one of several possihle forms of “incomplete” film of confitant thickness d and refractive index n given by equation 3, ( d ) a film of constant thickness but with refractive index decreasing with distance from substrate, to which the film equations are not, applicable. Apparatus and Procedure for Optical Measurements.The s- and p-compound reflectors referred to above are combined in a Bingle chromium-plated glass slide covered with stepped multilayers of B a s t (Fig. 2). The wave lengths for minimum reflected intensity are determined by illuminating the stepped reflector with plane-polarized white light and scanning the spectrum of the reflected light with a photomultiplier tube (Fig. 3). After the initial observatioiis the reflector is either calibrated with additional double Fig. 1.-Film diagrams illustrating types of departure stepped layers of Bast, and values of a. and CY* ralculated,a or else, from ideal postulated in optical theory (see text). if acceptable average values of 017are available, is coated with interface for the 8 and p-rays differ by about 180”. The the “unknown” film and the measurements repeated. change of wave length needed for minimum reflected inten- In order to minimize the effect of local variations in the sity following addition of a thin film to the p-reflector is now reflector, 62 areas (each 2 X 0.6 mm.z) are examined, these AX,, and in general AXp # Ahs. The new method consists being arranged in 4 rows of 13 areas parallel to the s-p essentially in the measurement of the two quantities AXy, boundary and about 3 and 5 mm., respectively, from the where y stands for s or p, and its success depends upon the boundary on the s- and p-steps. The procedure is illusfact that because of the difference in form of the two Fresnel trated in Table I giving a complete protocol of an experireflection coefficients, the two equations expression AX7 as ment,. Adsorption of Bovine Plasma Albumin.-This paper deals functions of film thickness, d , and refractive index, n , are also different in form and sufficiently independent to permit solely with protein films formed by immersing the stepped simultaneous solution. There is no simple exact solution but reflector in a solution of the protein followed by thorough in t,his paper we shall require only an approximate solution washing first with pure solvent and finally with distilled which by a rearrangement of Mattuck’s equations 15 and 16a water. Preliminary experiments showed the difficulty of obtaining uniform films. In the final arrangement the can be written procedure was carried out in a cylindrical waxed Lucite vessel n = 1.5 ( E . / E , ) ’ / ~ (1) 1.5 inch deep and diameter 1.25 inch provided with an axial inlet tube of i.d. a/16” by which the vessel could be connected through a Fisher and Porter tapered bore flowmeter (“rotad = 0.8681 2.25 ssep(2) Eli - ep meter”) and water reservoir. The stepped reflector was attached to the slow drive shaft of a variable speed motor and where lowered into the empty vessel. The protein solution was €7 = ( u ~ A X ~ introduced with a syringe and the slide rotated (1 turn per and the constants ay characterize the Bast multilayer to be second) for the required time, 5 minutes unless stated otherdetermined by a calibration mentioned later. The numeri- wise. The protein solution was then either ( a ) removed c,d factors are values of functiyns of the air-Bast reflection with a syringe, and a stream of solvent passed through the vessel, or ( b ) displaced with a stream of solvent. There coefficients stated in Mattuck s equation 1 1 . 3 ( 5 ) K. B. Blodgett and I . Langmuir, Phye. Rea., 61, 961 (1937). (6) R. E. Hartman, R. S. Hartman, K. Larson and J. B . Bateman, J . Opt. SOC.Amer., 44, 197 (19.54).
(7) B . W. Low in “The Proteins.” edited by H. Neurath and K. Bailey, Vol. I, Part A , Academic Press, New York, N . Y., 1953, p. 294(8) P Doty and E. P. Geiduschek in ref. 7, p. 383.
THICKNESS AND REFRACTIVE INDEXOF PLASMA ALBUMINFILMS ON STEARATE 1041
August, 1957
TABLE I PROTOCOL OF EXPERIMENT WITH STEPPED REFLECTOR A-2 8
P
S
P
18-39 double layers 75 72 67 74 70 72 75 71
Bast, March 9, 66 64 65 64 73 72 73 74
1956 67 65 72 69
64 67 71 76
69 64 74 70
65 65 70 71
64 68 73 73
Bast, March 9, 1956 87 87 87 83 87 91 87 90 87 87 89 87
83 86 86 83
84 83 86 88
88 83 89 87
91 89 86 89
419 419 215 207
415 419 212 218
423 418 219 216
20-41 double layers Bast BPA, April 7, 1956 39 36 38 42 41 43 39 41 40 40 37 43 39 41 19 21 17 19 17 20 22 21 17 17 21 18 17 20
39 38 18 18
40 42 18 19
38 40 13 18
55 55 55 54 32 31 30 34 n = 1.39 d = 15.8A.
52 59 29 32
57 51 av. 52.4
4772 80 4980 80
66 89 83 79
73 73 77 72
71 70 74 76
5194 93 5187 87
92 02 87 91
91 89 91 88
20-41 double layers 91 90 91 85 89 94 87 87 88 86 84 84
422 413 207 207
426 413 202 212
418 416 214 216
5245 45 5221 21
44 41 19 18
41 40 21 20
51 52 38 34
52 49 32 27
AX S
P
419 417 214 208
419 420 215 213
416 421 423 418 217 214 211 213 as = 3.234 aP =
423 426 215 216 6.305
420 418 218 218
427 av. 419.6 421
ii: av
213.4
+
S
P
Ah S
P
51 53 48 50 45 52 51 52 33 32 34 29 34 31 36 32 cs = 3.234 X 52.4 = 169.5 ep = 6.305 X 31.3 = 197.3
54 56 30 30
52 49 32 32
TABLE I1 WASHING OF BPA FILMS ADSORBED O N Bast Slide
Wash time t , min.
703 620 570 538 109 605
2 5 10 15 20 40
Vol. of water, cc.
500 1230 2260 3930 5100 9400
AAs
137.6 140.7 153.3 155.5 150.8 150.0
FROM
49 58 31 34
i i a v . 31.3
0.1% AQUEOUS SOLUTION
AAP
€8
ZP
n
a, A.
7, mdm.'
63.9 55.8 65.1 70.5 67.2 66.5
425.3 434.9 473.9 480.7 466.1 463.7
378.6 330.6 385.7 417.7 398.2 394.0
1.58 1.70 1.64 1.60 1.61 1.61
25.0 20.3 24.3 27.1 25.7 25.4
3.14 2.97 3.34 3.50 3.37 3.35
Regression equations n = 1.634 - 0.0007t; 95% confidence limit of slope =k 0.0007 0.069t; 95% confidence limit of slope f 0.164 d = 23.58 y = 3.136 0.0072t; 95% confidence limit of slope f 0.0125
+ +
was no indication that the results depended upon (a) or ( h ) , and (a) was sometimes preferred when it was desired to conserve protein solution. I n the experiments with acetate buffers, 200 cc. of buffer was used, which was then displaced with distilled water flowing a t about 200 cc./minute and the washing continued for 10 minutes. When the solvent was dilute HCl, washing with 2000 cc. of dilute HCl took the place of the final washing with distilled water. The washed slide was then removed, attached horizontally to the fast axle of the motor, and rotated until dry. Reproducibility and Sources of Error.-The preparation of adsorbed protein films has been difficult to standardize to the point a t which the sensitivity of the optical method3 can be used to fullest advantage. The procedure just described usually guarantees films free from obvious flaws but the results of duplicate experiments may show discrepancies not attributable to variations in any of the factors obviously under control. Little attention was given to temperature changes and t o variations in the pH and possible casual contamination of the wash water, obtained from a Barnstead 3-stage metal still, so that the importance of these
factors cannot yet be assessed. In one experiment dried protein films washed a second time with distilled water showed significant increases in mass. That the time allowed for washing (10 minutes) was sufficient is shown in Table 11. Much random variation probably arises from unequal deposition of protein on the s- and p-steps because of irregularities in the washing and drying procedures. It is suspected also that under some circumstances unsymmetrical deposition may be the cause of systematic error; SchaeferO has noted that the character of the Bast multilayer surface varies in a periodic manner with thickness, and our observations suggest that the properties of the 8- and p-surfaces as adsorbents for protein may not be identical, since the s- and p-steps sometimes differ considerably as to the pattern and rate of drainage of the water films adhering t o the washed prot,ein coated slide when it is removed for drying. The apparent thickness, d , and refractive index, n, of asymmetric films of true thickness and refractive index d y and n y can be calculated from the equations (9) V. J. Schaefer, THISJOURNAL, 45, 881 (1941).
J. B. BATEMAN AND E. D. ADAMS
1042
Vol. 61
Materials.-The BPA was an Armour crystalline preparation; i t was not dialyzed nor was any attempt made to remove decanol.
*”
40
thin added film barium stearate double layers chromium
~~
Fig. 2.-Chromium plated glass slide with stepped multilayer of mixed barium stearate-stearic acid.
/-\
constant deviation
Iiarium stcaratc on clironiiuni
Fig. 3.-Apparatus for determining wave lengths of minimum reflection from stepped interference reflector using incident beam of plane polarized white light.
I
I
I
I
I
600
400 ui
200
0.05
c, %.
5%
Fig. 4.-Values of E for films formed by 5 minutes immersion in BPA solutions of concentrations shown on abscissa. Open circles, s-ray; closed circles, p-ray. Lower set of circles films deposited from BPA in acetate buffer, ionic strength 0.15, p H 4.7, followed successively by lvashing with 200 cc. buffer and 2000 cc. water. Upper set of circles, films from BPA in H20, washed with 2000 cc. HzO.
(7) For the case d, = d,, n p = 1.3 and n. = 1.5, one finds n = 1.74; conversely, if np = 1.5 and n. = I .3, n = 1.11. Random asymmetry may thus account for occasional unrealistic values such as 1.7 for the apparent refractive index; systematic asymmetry may cause significant error but there is no reason to believe that the main trends reported in the next section are entirely due to artifacts requiring the use of equations 6 and 7. Pending further study of other types of stepped reflector, quantitative evaluation of this source of error is not possible.
Results Films Adsorbed from Solutions of BPA in Water and Acetate Buffer: Changes with BPA Concentration.-Two series of experiments were done with BPA solutions in distilled water of unadjusted p H ; decimal dilutions of BPA. were used over the range 0.005 to 501, and duplicate stepped reflectors were used a t each concentration in each series. Two similar series were done using BPA in acetate buffer of ionic strength 0.15, p H 4.7. The over-all consistency of the results can be judged from the plots of E against BPA concentration given in Fig. 4, from which also the following observations can be made: (a) The films in the acetate buffer series contain consistently less protein per unit area than those in the water series; (b) the refractive index values of the films deposited from acetate buffer are not significantly different from 1.5; (e) a t concentrations 0.01 to 0.5% BPA the film refractive index in the water series is consistently about 1.6; (d) the mass of protein per unit area deposited in 5 minutes in the water series is concentration-dependent, with a pronounced maximum a t 0.5%; (e) a much less pronounced maximum is suggested in the acetate buffer experiments. Figure 5 contains values for n, d and y calculated from the average values of E . The maximum in y for films obtained from 0.5% BPA is seen to be due principally to a maximum in the film thickness,1° the refractive index remaining substantially independent of concentration except for the dubious value a t 0.005% where the stepped reflector was imperfectly wetted by 5 minutes immersion and the drying was unsymmetrical. The less pronounced maximum thickness in the acetate buffer series may well be questionable since the corresponding value of n is low and random asymmetry may be suspected; on the other hand, in a series in acetate buffer a t p H 5.5 (not reported here) both the rise of d and the depression of n a t this concentration were again apparent. Some selected numerical values for the film constants which seem justified by the data are given in Table 111. TABLE 111 Model
Film data YI
Y,
d
A’. Parallelepiped, on side 49 Parallelepiped, flat 22 Prisms, over lap22 ping Priams, end-toend 15
mgi/ m. 6.67
Solvent
A.
w.?/ m.
n
d,
Water
1.6
34
.5
Acetate, pH4.7 HC1, pH
1.5
16-22
.8-2.3
1.24
15
.2
3.00 2.64 2.07
3
Adsorption from 0.1% BPA Solutions in HC1.The data in Fig. 6 show how the character of the BPA films formed by 5 minutes contact with a 0.1% solution varies with the p H of the solution. (10) We have been unable to substantiate the lower values of d given in an earlier paper.4 Since there is considerable change with pH in the isoelectric region (q.v.) we think t h a t the discrepancy may be due to differences in the pH of the wash water
August, 1957
THICKNESS AND REFRACTIVE INDEX OF PLASMA ALBUMIN FILMS ON STEARATE 1043
TABLE IV EFFECT OF 0.1% BPA SOLUTION ON PROPERTIES OF COMPLETE” BPA FILMS Slide
A-21 A-24 A-2 A-13 A-12 A-29
X
ns
n1
1.35 1.29 1.39 1.39 1.28 1.20 1.27
1.61 1.63 1.67 1.57 1.47 1.52 1.48
dl
15.6 22.6 15.8 22.5 13.6 19.9 12.9
a2
18.4 19.4 17.1 24.5 26.3 22.7 26.6
dl
-
“INa1
2.8 - 3.2 1.3 2.0 12.7 2.8 13.7 Av. 4.6
40
30
d There is a striking decrease in mass/unit area as 20 -the pH is lowered from 4.75, while the crossing and :separation of the E curves suggests a systematic ,:decrease of film refractive index. The changing #characterof the BPA film was noted in the draining 10 :and drying behavior; a t pH 3 the slides emerged -wet after washing but the water broke quickly from the edges of the slide, leaving the greater part of the surface quite dry. At pH 2.5 to 3 this be‘h,aviorwas exaggerated and below pH 2.5 optical -memurementbecame impossible because of fogging (of the Bast multilayer. The film constants are ;shown in Fig. 7, from which it is apparent that .,the mer-all decrease in y with decreasing pH re0.05 5% :suits from a rather sudden decrease in film thickc, %. ness combined with a consistent decrease of re5.-Values of film refractive index, n, thickness, d, .fracitive index from the bulk value 1.6 a t pH 5 to andFig. mass per unit area, y , derived from averaged data of about 1.2 at pH 3. Fig. 4. Closed circles, acetate buffer solvent; open circles, Adsomtion of BPA by BPA Films of Low Re- HzO solvent. fractive -Index.-The BPA films of low apparent refractive index might be spongy structures conataining enclosed air pockets, or they might form a mosaic of BPA and uncovered areas of Bast. Purther information was obtained by exposing BPA films to 0.1% BPA in water for 5 minutes, followed by the standard washing and drying. From a total of 18 experiments, the second exposure to 400 BPA cauwd the calculated film refractive index to increase in 14 cases; in two cases (n = 1.46 and 1.58) the values remained unchanged, and in two cases where the initial refractive index was uni usually high (1.66) there was a slight decrease. The regression equation for n2, the final refractive index, on the initial value nlwas 200 n2
= 1.232
+ 0.234n1
so that the final value of n2 for the “saturated” film is pot quite independent of the initial value; however, the 95% confidence limits for the slope are f 0.213 so that zero slope would not be inconceivable. Values of n1 ranged from 1.20 to 1.66 and n2from 1.46 to 1.67. 0 0 3 4 5 This result has some force in indicating the physical reality of the low refractive index values and PH. Fig. 6.-Values of E for films formed by 5 minute immerin pointing to a mosaic structure of the “incomsion in 0.1% BPA in HCl solution of pH shown on abscissa. plete” BPA films. It is complicated, however, by Films were washed with 2000 cc. of solvent a t the same pH. the fact that the expected increase of n was some- Small open circles, s-ray, small closed circles, p-ray; large times accompanied by an apparent increase in film circles, airorages. thickness; the result seemed to depend upon the conditions of formation of the first BPA film, and primary films which had been formed from acid there was also an inverse correlation between in- (HC1) solutions of BPA (Table IV); in these the crease of refractive index and increase of thickness. increase of film thickness is slight except in two The nearest approach t o a simple “fill-in” behavior cases (slides A-12 and X) where the increase of n is shown in the results of seven experiments with was much less than anticipated. More pronounced
J. B. BATEMAN A E. D. ADAMS
1044
L~~
4 L
30
-
’ti20
-
10
-
1.6
6 1.4 1.2
I
I
I
I
I
-
t L
I
I
I
I
3
I
5
4
PH.
Fig. 7.-Values
of n, d and
y,
from averaged data in Fig. 6.
d I
I
-3
I
I
-1
1
log c. Fig. 8.-Values of y for various BPA films. Abscissa, log BPA solution concentration. Open circles, data from Fig. 5 for residual film of BPA deposited on B a s t from aqueous solution; closed circles, data from Fig. 5 for residual film of BPA deposited on B a s t from acetate buffer, 0.15 p H 4.7, and washed i n buffer. Double circles data of Ihsk16 for residual film of BPA deposited on B a h from acetate buffer, 0.15, pH 4.9, and washed with water. Small heavy circles, data of Bull14 for primary film of BPA deposited on Pyrex particles from acetate buffer, 0.05 ionic strength, p H 4.66.
increases of thickness were found with primary films of relatively high refractive index formed from aqueous solution and from acetate buffer, and here the formation of second molecular layer must be considered possible. Discussion Primary and Residual Fiims.-The stepped reflector method in its present form can be used only in the study of dried films and its success in the foregoing experiments depends upon the existence of an equilibrium which favors the adsorbed state
Vol. 61
to such an extent that no reversal occurs even during prolonged washing with water. This is also a limitation, since the “irreversible” reaction actually studied may be only one of two or more reactions occurring at the solid-liquid interface. The film actually measured is a “residual” film from which an undetermined fraction of protein may have been eluted; it is also a “secondary” film in the sense that changes of structure may have OCcurred during drying. The distinction between “reversibly” and “irreversibly” adsorbed protein occurs in the literature, reviewed by Neurath and Bull”” and by Zittle,”b and it is of some practical importance in adsorption fractionation techniques. Polarimetric measurement of the optical thickness of “primary” films and their reactions has been made by TrurnitI2; it is assumed that the stepped reflector can likewise be used for such studies and calculation of the appropriate optical conditions will be undertaken shortly. Mass of Protein in “Isoelectric” Films.-We refer here to the data given in the top part of Fig. 5 and draw attention to the following points: (1) Films formed from acetate buffer, ionic strength 0.15, pH 4.7, and washed in buffer, contain consistently less protein than those formed from aqueous BPA; the values range from 1.8 to 2.3 mg./m.2 compared to 3.0 to 4.5 mg./m.2. Studies made by other methods throw some light upon these results. Lindau and Rhodius13 found that after placing quartz particles in a concentrated solution of egg albumin and washing with distilled water the residual film contained 4.2 mg./m.2. BuIl,’4 on the other hand, found the primary film deposited on Pyrex glass from o.5yO BPA in 0.05 ionic strength acetate, pH 4.66, t o contain about 4.5 mg. BPA/m.2, whereas the residual film after exhaustive washing with buffer contained only about 1.3 mg./m.2 (estimated from Bull, Fig. 2 and 5). These data suggest to us that the primary films formed from acetate buffer and from water solutions of BPA are closely similar, and that the difference in the residual films noted in our experiments is due to elution of some of the primary film during washing with acetate buffer, which does not occur during washing with water. Intermediate protein film values (2.6-3.9 mg./me2)were obtained by FiskI6 who apparently washed primary BPA films formed in acetate buffer with distilled water, omitting the preliminary washing with buffer (Fig, ,8). (2) Films formed from aqueous *BPA contain more protein the higher the BPA concentration up to 0.5y0 and less thereafter. A similar phenomenon has been noted by Bull14for primary films of BPA on Pyrex formed in acetate buffer; his data and ours are compared in Fig. 8 which illustrates the close similarity between his primary acetate buffer films and our secondary distilled water films. The explanation of the maximum remains obscure, but since according to Fig. 5 it is (11) (a) H. Neurath and H. B. Bull, Chem. Revs., 23, 391 (1938); (b) C . A. Zittle, Advances an En;ymoloey, 14, 319 (1953). (12) N.J. Trurnit, Arch. Biochem. Biophus., 47,251 (1953). (13) G. Lindau and R. Rhodius, Z. physzk. Chcm., A178,321 (193.9, cited in ref. 10 and 11. (14) H. B. Bull. Biochim. Biophys. Acta, 19,464 (1956). (15) A. A . Fiak, Proc. Null. Acad. Sci., 36, 518 (1050).
e
August, 1957
THICKNESS AND REFRACTIVE TNDEX
brought about by increased film thickness, the refractive index remaining constant, some sort of redistribution of molecular orientations would seem to be involved without change in density of packing. Refractive Index of BPA Films.-The BPA films formed from aqueous solutions had refractive indices very close to 1.60, in agreement with the value 1.599 calculated from solution refractive increment and partial specific volume.* When the films were formed and washed in‘ acetate buffer, the refractive index was about 1.5, suggesting a more open structure, but the films were also much thinner. One can perhaps visualize a closepacked primary film formed so rapidly that vertical crowding of anisodiametric molecules occurs at the expense of protein-adsorbent contacts, resulting in a rather thick but loosely bound film; if however protein is removed from the solution and electrostatic attractions are diminished by electrolyte, some protein will be released and the remainder will become reoriented in a more energetically favorable, but no longer close-packed, arrangement. Protein Adsorption and pH.-A decrease in protein adsorption a t low ionic strengths on both sides of the isoelectric point often has been reported and no attempt will be made to review the earlier work. Our own results (Fig. 7) are in qualitative agreement with those of Bull14for BPA adsorbed on Pyrex, except for our finding that y decreases rather sharply below pH 4.8 while Bull’s data show a zone of constant adsorption which, for a 0.1% solution, would extend from pH 4 to 4.8. This difference we once again attribute to the difference in technique, assuming that the small concentrations of HC1 used in the wash water are sufficient to remove protein from the primary film. The decreased adsorption from acid solutions is seen (Fig. 7) to result in films of much lower apparent refractive index than dry protein in bulk. Further uptake of BPA by these films exposed to an isoionic solution and the resultant increase in apparent refractive index constitutes a convincing reason for thinking that low refractive index values point to the presence of a dilute or “incomplete” film. Such films might conceivably be formed if the adsorption reaction is greatly retarded in acid solution. We have not investigated this possibility but can find in the literature no evidence that more prolonged exposure to acid BPA solutions would result in further adsorption. It is more likely that an end-point has been reached and that incomplete coverage is due either (a) to formation of a close-packed film of swollen which shrink upon drying or (b) to formation of a mosaic in which the final molecular density is determined by electrostatic repulsions. The second mechanism is the more plausible and we may note in support of it that a slide dipped in BPA-HC1 at pH 2.5 and withdrawn dries very rapidly by “rounding (16) J. T. Yang and J. F. Foster, J . A m . Chem. Soc., 7’7, 2374 (1955). (17) C. Tanford, S. A. Swanson and W. 9. Shore, ibid., 77, 6414 (1965). (18) C. Tanford, J. G. Bussell, D. G. Rands and 8. A. Swanson, ibid., ‘77, 0421 (1955).
OF
PLASMA ALBUMIN FILMS ON STEAR.ITE 1045 8 = 0” d = 158. A,
