Calcium binding in stearic acid monomolecular films

Calcium binding in stearic acid monomolecular films

Calcium Binding in Stearic Acid Monomolecular Films 1'2 R O N A L D D. N E U M A N a The Institute of Paper Chemistry, Appleton, Wisconsin 54911 Rece...

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Calcium Binding in Stearic Acid Monomolecular Films 1'2 R O N A L D D. N E U M A N a The Institute of Paper Chemistry, Appleton, Wisconsin 54911

Received May 28, 1974; accepted June 30, 1975 Stearic acid monomolecular films on 10-4 M CaC12 subsolutions were characterized at a surface pressure of 31 mN m -1 over the pH range 2-9 by film area, surface potential, surface viscosity, monolayer uniformity, and stability measurements. Characteristic property changes were observed at pH 4.2, 6.4, and 8.0. A new model of Ca 2+ binding satisfactorily explained the monolayer behavior over the entire pH range. The surface film primarily consisted of unionized stearic acid molecules in the pH range 2.04.2 and then behaved as a mixture of stearic acid and calcium distearate molecules with increasing subsolution pH. But upon exceeding pH 6.4, a structural rearrangement occurred as the ionized molecules began to associate into calcium stearate surface micelles. The close-packed monolayer, although uniform below pH 8.0, became nonuniform at higher pH values. Electron micrographs of deposited monolayers and multilayers suggested that the average surface micelle diameter was approximately 5-6 nm. Surface micelle formation and its physiological implications are discussed. INTRODUCTION T h e importance of Ca 2+ to the structure and function of biological systems is well known. M a n y investigators (e.g., 1-5) have a t t e m p t e d to clarify the n a t u r e of the Ca 2+ interactions with the anionic sites in biological m e m b r a n e s b y studying f a t t y acid monolayers at the a i r water interface. B u t even in this simple lipid system, Ca 2+ binding is not understood clearly. I n the present investigation, the effect of Ca 2+ on f a t t y acid surface films was investigated further as a function of the subsolution pH. T h e surface pressure (II), area per molecule (A), surface p o t e n t i a l (AV), surface viscosity (~), uniformity and stability of 1A portion of a thesis submitted in partial fulfillment of the requirements of The Institute of Paper Chemistry for the degree of Doctor of Philosophy from Lawrence University, Appleton, Wisconsin, June 1973. 2 Presented at the 77th National Meeting of the American Institute of Chemical Engineers, Pittsburgh, Pennsylvania, June 2-5, 1974. 3Present address: Department of Forest Products, College of Forestry, University of Minnesota, St. Paul, Minnesota 55108.

C a - H - S t monolayers, i.e., stearic acid monomolecular films on subsolutions containing calcium, were measured. The structure of deposited C a - H - S t films also was examined b y electron microscopy using replica techniques. EXPERIMENTAL A pparatus

The a u t o m a t i c a l l y recording L a n g m u i r - t y p e film balance described b y M a j o r (6) was modified for constant surface pressure operation. A schematic d i a g r a m of the a p p a r a t u s is shown in Fig. 1. T h e film balance consisted of a Lucite trough (50.2 X 13.4 X 1.2 cm), containing a dipping well, m o u n t e d rigidly to a brass support. Lucite compression and sweeping barriers, along with t h e trough edges, were covered with polyfluorocarbon t a p e (3M No. 549). Purified paraffin wax (Sun Oil No. 5512) was applied to the remainder of the trough. The surface pressure was measured with a Cenco torsion head, paraffin-coated mica float, 161

Copyright ~ 1975 by AcademicPress, Inc. All rights of reproductionin any form reserved.

Journal of Colloid and Interface Science, Vol. 53, No. 2, November 1975

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FIG. 1. Schematic diagram of film balance for insoluble monolayer and Langmuir-Blodgett deposition studies. and 0.061-ram Teflon film end loops. The sensitivities of the surface pressure measurements and the constant pressure operation were both ~ 0 . 2 m N m-L A glass probe thermistor was used to measure the subsolution temperature. The dipping apparatus for transferring monomolecular films by the Langmuir-Blodgett method (7, 8) has been described elsewhere (9). The film balance assembly, which was enclosed by a Lucite cabinet, and the barrier drive mechanism were mounted on individual tables, employing Unisorb pads to reduce vibrations further, in a dust-free constant-temperature atmosphere. Materials Stearic acid (Fluka AG, purity 99.6%) spreading solutions were prepared in calibrated glassware. The n-hexane was twice distilled and slowly percolated through an adsorption column of alumina and silica gel. The 10.4 M CaCI2 (Suprapur) subsolutions prepared from triply distilled water, the second distillation being from alkaline permanganate, were varied over the p H range 2-9 by the addition of HC1, KHCOa, or K H C Q (10-a M) plus K O H of AR quality. Water-saturated filtered air was bubbled through the subsolutions having p H values > 5.8.

The Brazilian Ruby mica and collodioncovered microscope slides have been described previously (9). The paraffin substrates were prepared by immersing and withdrawing a freshly cleaned microscope slide (75 X 50 ram) from molten paraffin (Sun Oil No. 5512) three times under carefully controlled conditions. The paraffin wax in the molten state was purified by passage through a silica gel column. -Procedures The spreading solution was deposited uniformly with a calibrated Agla syringe on the clean surface of subsolutions maintained at 20.5 ~ 0.3°C. Six minutes were allowed for reaction before compression at an average rate of 0.01 nm 2 molecule-1 min-L The surface pressure was maintained constant at 31 naN m - I for the determination of the interracial proFerties and the deposition of C a - H - S t monolayers. The H-A isotherms generally were duplicated, and the results were superimposed. The maximal error due to surface-active contaminants was shown to be only +0.0001 nm 2 at 1 naN m -~ using Robbins and La Mer's procedure (10). Film area corrections were determined from photographs of the end-loop areas and subsolutlon meniscus. The estimated

Journal of Colloid and Interface Science, Vol. 53, No. 2, N o v e m b e r 1975

163

CALCIUM BINDING IN FILMS errors in II and A were 4-0.1 m N m -1 and 4-0.001 nm 2, respectively. Surface potentials were measured by the ionizing electrode method (11) using a potentiometer (L & N Type K), electrometer (Cary Model 32), aged 6-mm square gold air electrode (5 ~Ci 226Ra, Radiation Developments) positioned 4 m m above the subsolution surface, and a miniature calomet electrode. The ionizing air electrode, shielded to obtain stable readings, was mounted to a X-17 combination of Unislide assemblies. The reported AV values, averages of eight or more measurements at different monolayer locations, were corrected for reference electrode drift and were reproducible within 4-5 inV. The slit viscometer method (12) was used to determine surface rheological changes. A 2.54-cm long Teflon insert containing a slit was transversely mounted in a Teflon compression barrier. The flow rate below p H 5 was corrected for barrier movement due to monolayer c311apse (13, 14). The surface viscosity, calculated from Joly's equation (15), was reproducible within 4-10%. The monolayer stability was determined by i

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Fro. 3. A 3t-pH curve for Ca-H-St monolayers. measuring, as a function of time, the change in the film area. The percentage that the film area decreased in 15 rain was selected as the basis for comparing the relative stabilities. Direct carbon replicas, either unshadowed or preshadowed, of deposited C a - H - S t surface films were prepared in the manner previously described (16). However, the unshadowed replicas on mica could be floated off by tl:e stripping technique. The replicas were examined and photographed at a magnification of about 22000 diameters at 50 kV in a calibrated RCA EMU-3F electron microsccFe equipped with high-voltage fine focusing. Measurements were obtained from Ortho film enlargements made using a Durst S-45EM Enlarger. A Jeol JSM-U3 scanning electron microscope equipped with an Ortec X ray spectrometer and Quanta/Metrix E D X 80 energy dispersive X ray analysis system was used to obtain X ray spectra of the replicas. Sample areas of 1 m m 2 were scanned at 25 kV for 1000 sec.

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Fro. 4. Comparison of the stabilities of Ca-H-St monolayers at 31 mN m-~ and a compression time of 15 min.

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FIG. 8. The uniformity of AV within C a - H - S t monolayers at 31 m N m -I.

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The II-A isotherms of the C a - H - S t monolayers are shown in Fig. 2. The formation of calcium salts results in a gradual transition from a liquid-condensed film to a solid film. Fig. 2, in addition, shows that the average area per molecule at high surface pressures is not constant with increasing subsolution pH. The area at 31 inN m -1 (A31) above pH 6.4 appears to be reduced by as much as 2.3%, as shown in Fig. 3. An increase in the reaction time from 6 to

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Journal of Colloid and Inlerface Science, Vol. 5.3, No, 2, November 197.5

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FIO. 10. Electron micrographs of Pd-preshadowed replicas of Ca-H St bilayers deposited on paraffin at 31 mN m -1. X33 000.

15 min produces no significant changes, thus demonstrating that the decrease in Aal is not due to monolayer losses during the reaction time. The area decrease also does not appear to be attributable to losses during film compression because the results presented in Fig. 4 show that the close-packed Ca-H-St monolayer is very stable on neutral and alkaline subsolutions. The dependence of r~ on the subsolution pH is presented in Fig. 5. The surface viscosity is plotted on a linear scale over a limited pK range in Fig. 6 in order to define changes in the rheological properties more clearly.

The AV-pH curve is shown in Fig. 7 with the results of Sanders and Spink (17) presented for comparison. The 95% confidence limits, calculated from the dxV measurements within a given monolayer, were employed as a measure of the monolayer uniformity. The Ca-H-St monolayer is observed to be nonuniform at areas (>0.25 nm 2) corresponding to very low surface pressures. Fig. 8 shows that the monolayer becomes uniform during compression below pH 8.0, but not at higher pH values. The increasing degree of nonuniformity and its large scale nature are shown also in Fig. 9. Although the non-

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RONALD D. NEUMAN

Fro. 11. Electron micrographs of Pt-preshadowed replicas of (a) Ca-H-St monolayer deposited on collodion at pH 8.3 and 31 mN m -~, and (b) blank showing the collodion sabstrate with no film N21 800.

uniformity decreases with surface aging, the fihn remains nonuniform even after 1 hr of compression.

were employed. Upon shadow-casting with platinum (Pt), for example, numerous microholes are present in deposited monolayers (Fig. lla), bilayers (Fig. 12a), and trilayers Electron Microscopy (Fig. 12b). The microholes, presumably arising Figure 10 shows representative electron from the evaporation of film molecules during micrographs of deposited Ca-H-St bilayers the Pt shadow-casting procedure (16), have shadow-cast with palladium (Pd) metal. All a very uniform diameter of about 5-6 nm. electron micrographs are positive prints with Microholes having larger diameters are not the shadows appearing dark. Whereas the observed; however, paired microholes, i.e., two molecular aggregates 4 on the bilayers in the adjacent microholes, commonly are present. pH range 2.0-4.0 are of varying size and The large hole ( ~ 5 nm deep) in the trilayer, lenticular-shaped, the molecular aggregates furthermore, appears to be lined with calcium present above pH 8.5 appear to be about 5-6 stearate aggregates. Unshadowed carbon replicas of deposited nm in diameter and demonstrate a tendency Ca-H-St films should be transparent to the to line up in a pearl-string formation. In electron beam. At high values of the subsoluaddition, the calcium stearate aggregates tion pH, however, electron-dense (white) appear to be close to 5 nm or two long-chain regions having a diameter of 5-6 nm are stearate molecules in height, or integral observed, as shown in Figs. 12c and 12d. multiples thereof. Electron probe microanalysis shows that the This diameter (approx. 5-6 nm) appears to be a characteristic dimension of deposited unshadowed (or preshadowed) carbon replicas Ca-H-St films at high pH values, observed contain calcium, e.g., see spectrum 3 of Fig. 13. not only under Pd shadow-casting conditions, The electron-dense regions appear to correbut also when other replication techniques spond to embedded film species, presumably calcium stearate aggregates in the case of Fig. These molecular aggregates presumably arise from 12c. Larger electron-dense regions also are the slow monolayer collapse process which concomitantly occurs during the multilayer deposition of steafic present in Fig. 12d. The radiant energy associated with carbon replication apparently acid monolayers under acidic conditions (14). Journal of Colloid and Interface Science, VoL 53, No. 2, November 1975

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Fro. 12. Electron micrographs ef (a) Pt-preshadowed replica of a Ca-H-St bilayer deposited on paraffin at pit 8.9 and 31 mN m-Z; (b) Pt-preshadowed replica of a Ca-H-St trilayer deposited on mica at pH 8.3 and 31 mN rn-~ and 250 nm underfocused; (c) carbon replica of a Ca-H-St bilayer deposited on paraffin at pH 9.0 and 31 mN m-Z; and (d) carbon replica of a Ca-H-St monolayer deposked on mica at pH 8.6 and 31 mN m-1. a, X21 000; b, X86 400; c, X56 700; d, X21 600. causes some of the monolayer molecules to evaporate and others to restructure.

Proposed Model The classical hypothesis of Ca 2+ binding is a single divaIent calcium ion reacting with two fatty acid anions to produce a disoap molecule (18-20). A copolyrneric lattice structure alternatively has been proposed (2, 19, 21). Characteristic changes, summarily illustrated in Fig. 14, occur in the surface properties of the closepacked C a - H - S t films at p H 4.2, 6.4, and 8.0.

Neither model, however, satisfactorily explains the monolayer behavior over the entire p H range. The electron microscopic results collectively suggest the existence of a fundamental molecular grouping within the deposited films of C a - H - S t monolayers on alkaline subsolutions. Caution must be exercised in attempting to relate the structure of deposited films to that of the original monola.ver. However, the C a - H - S t surface properties are consistent with the view that two-dimensional molecular

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FIG. 13. X ray spectra of (l) blank graphite holder and carbon replicas of Ca-H-St monolayers deposited on mica at (2) pH 6.2 and (3) pH 8.6 and 31 nan m-1. The replica was draped over the graphite holder. Note: Base lines shifted for clarity. aggregates exist in close-packed C a - H - S t monomolecular films, and as such, a new structural model of Ca ~+ binding is proposed. i

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The surface micelle model is illustrated schematically in Fig. 15. The monolayer primarily consists of unionized stearic acid molecules in the p H range 2.0-4.2. The calcium salts that form with increasing subsolution p H are believed to be calcium distearate molecules. But upon exceeding a critical Ca 2+ concentration in the film at p H 6.4, corresponding to a degree of dissociation of about 62°fo, the ionized molecules begin associating into surface micelles. The calcium stearate surface micelles, being 5-6 nm in diameter, contain 100-150 stearate anions and are envisaged, as illustrated in Fig. 16, to be coordination complexes.

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The C a - H - S t monolayer composition was estimated, in part, from the study of Bagg, et al. (22). Ionization appears to become appreciable at about p H 4.2, the degrees of dissociation are approximately 62% at p H 6.4 and 90% at p H 8.0, and the film is ionized completely only at p H values above 9.0. The decrease in the average molecular area, beginning at p H 6.4, does not appear to be due to monolayer losses by dissolution, 1975

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evaporation, or collapse, but rather, is attributed to a structural rearrangement of the ionized molecules into surface micelles. A staggering of the stearate anions presumably permits a closer packing of the hydrocarbon chains. The surface viscosity remains constant over the pH range corresponding to unionized stearic acid. The viscosity increase between pH 4.2 and 6.4 is due to the increasing concentration of calcium distearate molecules while the large increase from pH 6.4 to 8.0 is attributed to the formation of surface micelles. The surface potential may be represented by an expanded form of Schulman and Hughes’ expression (23, 24). The decrease in AV between pH 2.0 and 4.2 is assumed to be due to the initial dissociation of stearic acid. However, the AV decrease between pH 4.2 and 6.4 is relatively minor even though about 62y0 of the stearic acid molecules form the calcium salt. The sharp decrease above pH 6.4 is attributed to the reduction in the surface dipole moment associated with the calcium stearate surface micelles. The absence of electron-dense regions in the electron micrographs of unshadowed carbon replicas of Ca-H-St monolayers at pH 6.2, in contrast to those present at pH 8.6, also is consistent with the surface micelle model. Journal

It generally has been accepted that monolayers are homogeneous at high surface pressures. However, the close-packed Ca-H-St films are nonuniform above pH 8.0. Other property changes also occur as shown in Fig. 14. The subsolution pH decreases, albeit slowly ( (0.001 pH unit/min), at these higher alkalinities. Carbonldioxidexabsorptionf and bicarbonate buffer are known to influence monolayer properties (2.5) and may play some role in the observed film behavior. Another

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FIG. 16. Schematic drawing of proposed lattice structure of a calciumstearate surfacemicelle.

of Colloid

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Interface

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RONALD D. NEUMAN

interpretation, as shown in Fig. 15d, is that the surface micelles tend to aggregate into large clusters when they become sufficiently numerous. The incomplete juxtaposition of these clusters during compression gives rise to less dense regions, which are either monolayer-free or contain film molecules in some other phase, being interspersed throughout the surface film. With continued micelle formation, the monolayer becomes increasingly nonuniform, AV and ~ decrease, and the average molecular area remains approximately constant. Surface Micelle Formation

Examination of the reported studies on the reactions of other metal ions with stearic acid monolayers reveals that characteristic surface property changes also occur at bulk p H values where the monornolecular film consists of approximately 62% stearate anions. Studies to investigate further whether surface micelle formation may be a general physicochemical phenomenon are in progress. This phenomenon may be particularly relevant to interfacial processes occurring in biological systems. The formation of surface micelles in the lipid bilayer regions of biological membranes may be closely associated with the structural changes (26) and entropy decrease (27) reported to occur during membrane excitation. CONCLUSIONS

A new model of Ca 2+ binding in fatty acid monomolecular films is proposed in order to explain the interfacial behavior of close-packed Ca-H-St monolayers. A structural change appears to occur when a critical degree of surface dissociation is exceeded. The ionized film species are believed to associate into two-dimensional or surface micelles having a diameter of about 5-6 nm. ACKNOWLEDGMENT

The author wishes to express his appreciation to Prof. J. W. Swanson who served as thesis

advisor and Dr. R. H. Atalla for his helpful discussions ; Mr. K. W. Hardacker for designing the servomechanism system for the film balance; Messrs. M. D. Filz, Jr. and P. F. Van Rossum for constructing the film balance; and Mrs. H. M. Kaustinen for the electron micrographs. A Graduate Fellowship received during the course of this work also is gratefully acknowledged. REFERENCES 1. WEBB, J., AND DANIELLI, J. F., Nature (London) 146, 197 (1940). 2. DEAMER, D. W., AND CORNWELL, D. G., Biochim. Biophys. Acta 116, 555 (1966). 3. HAUSER, H., AND DAWSON, R. M. C., Eur. J. Biochem. 1, 61 (1967). 4. ABOOD, L. G., AND RUSHMER, D. S., Advances in Chem. Set. 84, 169 (1968). 5. GOERKE, J., HARPER, H. H., AND BOROWITZ, M., in "Advances in Experimental Medicine and Biology" (M. Blank, Ed.), Vol. 7, p. 23, Plenum Press, New York, 1970. 6. MAJOR, E. I-I. JR., Ph.D. thesis, The Institute of Paper Chemistry, Appleton, Wisconsin, 1969. 7. LANG?eIrJIR,I., Trans. Faraday Soc. 15, 62 (1920). 8. BLOI)OET:r, K. B., J. Amer. Chem. Soc. 56, 495

(1934); 57, 1007 (1935). 9. NEU-MAN,R. D., J. Colloid Interface Scl. 50, 602 (1975). 10. ROBBINS,M. L., ANDLAMER, V. K., J. ColloidSci. 15, 123 (1960). 11. GAINES, G. L., JR., "Insoluble Monolayers at Liquid-Gas Interfaces," p. 73. Interscience, New

York, 1966. 12. DREHER,K. D., AND SEARS,D. F., Trans. Faraday Soc. 62, 741 (1966). 13. NEVMAN,R. D., Nature (London) 250, 725 (1974). 14. NEIJMAN,R. D., J. Colloid Interface Sci., submitted for publication. 15. JoL¥, M., Kolloid-Z. 89, 26 (1939). 16. NEU~AN, R. D., J. Microscopy, in press. 17. SAXDE~S,J. V., AND SPINK, ~. A., Nature (London) 175, 644 (1955). 18. SASAKI, T., AND MA~UIJRA, R., Bull. Chem. Soc. Japan 24, 274 (1951). 19. HARI~INS, W. D., "The Physical Chemistry of Surface Films," p. 153. Reinhold, New York, 1952. 20. KAVANAtr, J. L., "Structure and Function in Biological Membranes," p. 139. Holden-Day, San Francisco, 1965. 21. ARCltER, R. J., ANDLA MER, V. K., J. Phys. Chem. 59, 200 (1955).

Journal of Collold and Interface Science, Vol. 53, No. 2. November t975

CALCIUM BINDING IN FILMS 22. BAoc, J., ABRAMS0N,M. B., FICHHAN,M., HABER, Jr. D., AND GR~GOR,H. P., J. Atner. Chem. Soc. 86, 2759 (1964). 23. SCHUL~AN,J. H., AND HUCHES, A. H., Proc. Roy. Soc. (London) Set. A. 138, 430 (1932). 24. BE¢TS, ]. J., AND PE~mCA, B. A., Trans. Faraday Soc. 52, 1581 (1956).

171

25. GOODAI~D,E. D., S~T~, S. R., ANn KAO, 0., Y. Colloid Interface Sci. 21, 320 (1966). 26. TASAKI, I., WATANABE, A., SANDLIN, R., AND CARNA7, L., Proc. Nat. Acad. Sci. US 61, 883 (1968). 27. I-IOWARTH,J. V., I~EYNES, R. D., AND RITCHIE, J. M., J. Physiol (I.ondon) 194, 745 (1968).

Journal of Colloid and Interface Science,

VoI. 53. No. 2. November 1975