Surface activity of urea

Surface Activity of Urea D. F. SEARS Department of Physiology, School of Medicine, Tulane University, New Orleans, Louisiana 70112

Received May 23, 1968 The effects of high concentrations of urea to increase surface pressure and alter surface potentials of stearic and palmitic acid mmiolayers were examined at temperatures between 15 ° and 40°C. Results of the investigation show that penetration of urea into an interracial region depends upon the presence of molecules at the interface, where dipole-dipole interactions with the urea can occur. Such interactions allow the concentration of urea to increase to equal the bulk concentration. In the absence of such molecules urea is desorbed from the interface.

INTRODUCTION Hemolysis of red blood corpuscles by urea and other solutes has been attributed to alterations of intra- and extracellular water lattice structure (1). Hemolysis occurred more rapidly when corpuscles were exposed to structure-breaking solutes (urea) than when they were exposed to structure-making solutes (thiourea). However, additional sites exist where urea could disrupt membranes: (a) partitioning of urea as an inert molecule into membranes independent of any specific interaction with membrane molecules to expand the membrane; (b) dipole-dipole interactions of urea with polar groups of molecules forming cell membranes to change intermolecular associations; (c) formation of inclusion complexes of urea about the hydrocarbon chains which comprise the hydrophobic portion of the membrane; (d) denaturation and loosening of surface-adsorbed protein networks which are responsible, according to Mitchison and Swarm (2), for strength of cell membranes; or (e) any combination of the above. Both structured water and the sites listed are located in the interracial region between a cell and the external medium. It has been well documented that interfaeial regions have unique properties not present in the bulk phase of a medium (see, for example, Davies and Rideal (3)). Two earlier studies of interfacial regions (4, 5) have examined

the effects of cations and of heavy water on properties of stearic acid-water interfaces. These studies indicated that water molecules play an important role at interfaces. This supports the idea of water structure involvement in hemolysis in agreement with Good. This paper reports experiments designed to examine the action of urea in interfacial regions. The results give information concerning the first three sites of molecular interaction listed above. The data presented are for monolayers of stearie acid and palmitic acid on substrates containing high concentrations of urea, and, in some experiments, 0.5 M NaOH and KOH, and 10 -4 M Ca(OH)2. iV[onolayers of phospholipids or mixed monolayers of phospholipids, cholesterol, and proteins would more nearly approximate specific interactions of urea with cell membranes, and such monolayers can be examined. However, interpretation of such a study requires preliminary examination of simpler systems such as the fatty acid monolayers. As will be shown in this work, the surface activity of urea depends upon the nature of the interface; urea is desorbed from an airwater interface but is adsorbed at a hexanewater interface. At a fatty acid-water interface, dipole-dipole interaction between urea and the fatty acid occurs and urea is adsorbed. This adsorption changes the monolayer spacings of the fatty acid. If the area per fatty acid molecule is held constant, urea

Journal of Colloid and Interface Science, VoL 29, No. 2, February 1969

288

SURFACE ACTIVITY OF UREA produces changes in the surface pressure. W i t h this three-component s y s t e m of water, urea, and f a t t y acid, it is possible to use the Gibbs adsorption isotherm and determine the surface concentration of urea. T h e results of these calculations have been interpreted to mean t h a t changes in the monolayer surface pressure due to increasing concentrations of urea in the substrate can be attribu t e d to the kinetic energy of the urea molecules which partition into the interfacial region. MATEP~IALS AND METIIODS Surface pressure (II)-area (A) relations were measured f r o m a Teflon t r o u g h with an a u t o m a t i c a l l y recording Wilhelmy balance a t t a c h m e n t . Simultaneous surface potential (AV) measurements were obtained with an air electrode. Details of equipment design and of experimental procedure have been presented previously (4). Regulation of temperature, both of the air ! i

15°C

60- -

Stearic acid HCl M Urea

0.01N

....

50-

.... ~-300 i

4055-

50260

.....

25-

/~...

~..

/H/

j~

/

',\ '.,4"" /

k240

....

~_

). ...

15-

......

io

t200

",,\i~"

50

"x.'.~[.£............. i

18

i

25°C Sfearic acid

55-

~-400

. . . . 1.0 M Urea - - " 5.0 M Urea ........ 5.0 M Urea

50i..:>~. 45-

550 ~

~-

1300

4035~30

I!,i. . . .

"~25-

~,V.. , £ 9 . . . . .

-250

, .2"/

20

":2;

15-

230

/...., "\L..

I

~,,, .\...........

i o-

50

/ f 7.f

-

~ \ -

~

18

i

22

i

26

""" J

,

30

i

,

34

J

i

38

i

i

42

,

7m-

Area (~.a/motecule)

FIG. 2. II-A (bold lines), AV-A (light lines), and ~-A (light lines) curves for stearie acid on 0.01 N tIC1 (----), 1.0 M urea (- - - -), 3.0 M urea (. . . . . ), and 5.0 M urea ( . . . . . ) at 25°C. Area scale is from 18 to 42 A~ per molecule. All urea solutions were titrated to pH 2.0. Compression rate was 3 A 2 per molecule per minute.

•' 50 M Ucea

i

45-

~

289

t--i

20

,

22

,

j

,

i

,

,

24 26 28 Area (,~ 2/molecule)

,

,

30

,

i--

32

Fro. 1. II-A (bold lines), A V - A (light lines), and ~-A (light lines) curves for stearie acid on

0.01 N HC1 ( ), 1.0 M urea ( - - - - ) , 3.0 M urea (- . . . . ), and 5.0 M urea ( . . . . . ) at 15°C. Area scale is from 18 to 32 A 2 per molecule. All urea solutions were titrated to pII 2.0. Compression rate was 3 A ~per molecule per minute.

above the monolayers and of substrata solutions, was accomplished b y placing the equipment in a c o n s t a n t - t e m p e r a t u r e r o o m and b y circulating water from a Precision Scientific Co. water b a t h about the trough. T e m p e r a t u r e variation was less t h a n 40. I°C during the course of an experiment. Stearie a d d was reerystallized f r o m methanol and ether. T h e melting point was 68 ° . 69°C. Palmitie acid was obtained f r o m K & K Laboratories and h a d a melting point of 60°-61°C. Urea, N a O H , K O H , Ca(OH)~, and HC1 were " B a k e r A n a l y z e d " reagents. After preparation and a d j u s t m e n t of pH, all solutions were passed t h r o u g h a column of activated charcoal to remove a n y surfaceactive contaminants. Water was Pyrex distilled. Continuous compression was obtained by a motor-driven barrier and rates of compression are given in the legends of the figures. RESULTS Some preliminary stalagmometer determinations were made of the surface tensions

Journal of Colloid and Interface Science, VoL 29, No. 2, February 1969

290

SEARS

of solutions of urea in water at 37°C. I n the concentration range of 1 to 5 M urea, there was a progressive increase in surface tension with increasing urea concentration. At 5 M urea the tension was 0.56 d y n e / c m greater than the surface tension measured for water (70.38 dynes/era). This increase in surface tension indicates desorption of urea from the solution surface. Interracial tensions were measured for the urea solutions against hexane. For 5 M urea, the interracial tension decreased 2 dynes/era below the value 44.5 dynes/cm measured for the hexane-water

-

~:

45 -

-400

1,

-500

40-

"~

,',.

55-

-200

~

~3o 1 100

25

15

\ "\

-0 N.

I

',

\

-

37°C Siearic acid ~-400 - - 0 . 0 1 N HCI .... 1.0 M Urea - - " 5.0 M Urea -350 "....... 5.0 M Urea

55 50-

-~-.~'~, i ~ iit" "'"'~:~'~:....

45-

-3oo

,,\''V..

4o-

:::1

25o "~

X

55-

%:',. . . . .

200

",( '.....

50~

~ ~,

50

interface at 37°C. Urea was adsorbed at this interface. Thus the surface activity of urea is dependent upon the nature of the interface. Effect of Urea on Stearic Acid Monolayers. II A, AV - A, and surface dipole (~) A curves for stearic acid monolayers spread on 0.01 N HC1 and on high concentrations of urea at 15 °, 25 °, and 37°C are shown in Figs. 1, 2, and 3, respectively. I n comparing these figures it is important to note the change in scale for areas per molecule. All urea solutions were titrated to p H 2.0 with 10 N HC1.

Palmitic acid 00[ N HCI - - 280°C 52.5% --37Aoc

55-

25 --

)I/'~ I: . I

...... I ......

.....

t

15

z

\ ...... 50

270

F -250

....

I ,

5

,

J

15

FIG. 3. II-A

,

"\

, ,

,

~2,o ~

,

j

""... ,

,

,

25 35 45 55 Area(A 2/molecule)

(bold lines), A V - A

,

65

,

75

(light lines),

and #-A (light lines) curves for stearic acid on 0.01 N IIC1 ( ), 1.0 M urea (- - - -), 3.0 M urea (- . . . . ), and 5.0 M urea ( ..... ) at 37°C. Area scale is from 5 to 75 AS per molecule. All urea solutions were titrated to pH 2.0. Compression rate was 3 ASper molecule per minute.

0 /

,

5

i

i

15

,

,

25

,

t

,

,

i

i

35 45 55 Area (~,Z/molecule)

,

,

65

J

i

75

FIG. 4. H-A (bold lines) and AV-A (light lines) curves for palmitic acid on 0.01 N HC1 at 28°C ( ), 32.5°C ( - - - - ) , and 37.4°C (- . . . . ). Compression rate 5 ASper molecule per minute. Stearic acid monolayers have solid and liquid condensed regions which can be characterized from the II - A curve by calculating the surface compressional moduli (3). Both increasing the concentration of urea in the substrate and increasing the temperature progressively expanded the liquid condensed regions of the stearic acid monolayer. Neither urea nor temperature produced any marked alteration in the solid region of the monolayer. Transition from liquid condensed to solid monolayers occurred at higher surface pressures and at larger areas per molecule as the urea concentration in the substrafe increased. However, these changes were slight. Collapse areas were little affected by the concentration of urea, and collapse pressures were similar for monolayers on HC1 and urea solutions at 37 ° and 25°C, but at 15°C these pressures were 3 to 10 dynes/cm higher on the urea solutions. Since stearic acid monolayers in the solid state and the coareas per molecule were little affected by urea in the substrate, no inclusion complexes between urea and the hydrocarbon portion of stearic acid monolayer were formed. If urea had formed inclusion complexes about the

Journal of Colloid and Interface Science, Vol. 29, No. 2, February 1969

SURFACE ACTIVITY OF UREA hydrocarbon chains, areas of closest packing would have increased. Therefore the expansion of the monolayer due to urea is produced through interactions with either the COOH groups or with water or both. In support of the involvement of the polar groups, AV and were lower on urea solutions at areas less than about 28A 2 per molecule.

Palmiticacid 55-

5.0 MUrea(pH2.01 -400 - -

PNmiticacid

55

400

3.0 M Uree(pH 2.0) - - 25.5% .... 503% - - . 367oc

50 45-

300

40-

278oc

. . . . 31.7oc - - . 355oc ........ 59.5oc

50-

[ 3@45" 4o

~ - / %~ , % ,

Effect of Urea on Palmitic Acid Monolayers.Palmitie acid monolayers were spread

on substrates containing 3 and 5 M urea at temperatures from 25 ° to 39°C. The data are shown in Figs. 4, 5, and 6. Palmitie acid contains 16 carbons, 2 less than stearie acid. In terms of the surface pressure-area curves, reducing the length of the hydrocarbon chain is equivalent to increasing the temperature; according to Adamson (6) each CH~ group removed is roughly equal to a 5°C temperature increase. Curves for paimitic acid are more expanded than for stearie acid at the same temperatures. Transition pressures where the palmitic acid monolayers change from liquid condensed to solid are sharply defined on the pressure-area curves. Adam (7) and Langmuir (8) considered the liquid condensed monolayer as a semisolid film having water between the polar heads.

291

L

-3O0

~q

~\~"

"~'--,'2:~"/24 ........

20-

" "'~ "

15-

"i\ "'..

"0

,(\ ......

IO-

\~\ k..

~ 0

...... ,oo

~

~~..Z. . .15. . . .25. . . 55 45

.......

......

• .~-~, 7 ,

55 65 Areo (/~ 2/molecules)

75

FIG. 6. Sm'face pressure--and surface potential --area curves for palmitie acid on 5.0 M urea (pH 2.0) at 27.8°C, 31.7°C, and 35.5°C. Compression rate was 5 A~per molecule per minute. With compression the water is squeezed out until the film becomes solid. If only water is coplanar with the carboxyi groups of the palmitic acid when the monolayer is spread on urea-containing substrates, the pressure required to remove the water should decrease with increasing urea concentration as the chemical potential of water decreases. If urea is present eoplanar with the earboxyl groups, the pressure required to squeeze the urea out against its concentration gradient would increase with increasing urea concentration in the bulk phase. The plot of transition pressure against temperature at 0, 3, and 5 M urea concentrations is shown in Fig. 7. Transition pressures increase with increasing urea concentration within the temperature range examined. The areas at which the transition occurred were between 35 and 40 A? per molecule and were not directly related to the bulk urea concentration. Surface potentials of palmitic acid monolayers decreased at areas less than 35 A ~ per molecule on substrates eontaiuing urea. This is further evidence for the presence of urea in the interface, eoplanar with the polar groups,

li°°

55-

252015I0-

"',, "\ ,k\ , .\,

55

15

25 .35 45 55 AreG(~ Z/molecule)

G5

75

FIG. 5. Surface and surface p o t e n t i a l - - a r e a isotherms for palmitic acid on 3.0 M urea (pH 2.0) at 25.5°C, 30.3°C, and 36.7°C. Compression r a t e was 5 A 2 per molecule per minute.

Journal of GolIoid and Interface Science, VoL 29, No. 2, February 1969

292

SEARS

and the subsequent removal and reorientation of urea beneath the C O O H groups when the monolayer was compressed above the transition pressure to produce solid monolayers. Effect of Urea on Soap Monolayers. Cations such as N a or K in association with the stearic acid mono]ayer cause expansion of the monolayer (4). This earlier study presented evidence t h a t this interfacial region is highly hydrated b y water in close association with the cations. Stearic acid monolayers were examined on substrates of 5.0 M urea prepared in aqueous solutions of 0.5 M N a O H or 0.5 M K O H . The p H of these solutions was greater than 13.5. Stearic acid monolayers were also spread on substrates containing 5 M urea and 7.8 mg of Ca(OH)~ per liter; the p H was 10.7. Results of these experiments are presented in Fig. 8. Urea did not expand the solid calcium stearate monolayers. Pressure-area curves of stearate on N a and K O H with 5 M urea were slightly expanded b y about 4 A 2 per molecule as compared with the curves presented by Sears and Schulman for monolayers on hydroxide solutions without urea. Surface compressional moduli for monolayers on N a and K O H with or without urea were those which characterize liquid expanded monolayers. There was no difference in surface potentials for these monolayers compared to monolayers on hydroxide solutions without urea.

Surface Concentration of Urea. Guggent

Paimitic acid 0.01 N He[ M Urea [] 50 M Urea /

•

25

o 3.0

50

LI~,"

45

~", E

40

Stearic acid

- - 0 . 0 1 N HCl ---- 5.0 M Urea(pH ZO) - - - 5.0 M Urea& Ca(OH)2 ~-500 (pH 10.7) ........ 5.0 M Urea~ 0.5 N KOH

~ [

(pH 15.5)

[

35

--

20 t

,

i

,

,

50

'~,,~,,.,,~ ............

;

~

50

i

i

27

J

,

,

i

i

,

,

i

29 31 33 35 Temperature (%)

J

J

~

- -

37

FIG. 7. Transition surface pressure--temperature curves for palmitie acid on 0.01 N HC1, 3.0 M urea, and 5.0 M urea. The surface pressure is that which corresponds to the transition from liquid condensed to solid monolayers.

,

,

,

,

,

70 90 IlO Area (A 2/molecute)

loo

, , , 130 150

(~n)/(Ax)].

r~/x :

F

~

-50

helm (9) presented the thermodynamics of an interfacial region between two bulk phases where each phase contains the same two components. The approach here follows from that of Guggenheim where he treated with the liquid-vapor interface. The interfacial region contains two independent variable components, water and urea, and stearic acid the surface concentration of which can be held constant. By measuring the change in surface pressure (AII) for different bulk concentrations of urea, the surface concentration of urea was calculated from:

I

25

12oo

FIG. 8. Surface pressure--and surface potential --area curves for stearic acid on 5.0 M urea in 0.5 M NaOIl, 0.5 M I(Og, and 0.0001 M Ca(OH)~ . Compression rate was 7 A e per molecule per minute. Temperature 25°C.

=

0/

.................................

............ ~

)L , I0

/

.?,

i

(I/RT)

e

(pH~35)

t

~ 3o

0 /

5.0 M Urea8~0.5 N NaOH

-

r,/(~

-

1)

1/x(1 -- x),

where AII is the change in surface pressure due to the change in mole fraction of urea (Ax), x is the mole fraction of urea R is 8.3 X 107 ergs per mole, T is the temperature in degrees absolute, r~ is the surface concentrations of water in moles per square centimeter, F2 is the surface concentration of urea in moles per square centimeter, and A is the area of stearie acid, held constant. I is

Journal of Colloid and Interface Science, VoI. 29, N o . 2, F e b r u a r y 1969

SURFACE ACTIVITY OF UREA TABLE SURFACE

I

CONCENTRAr['ION OF UREA AREAS FOR STEARIC ACID

Area of stearic acid

AT

FIX~D

I X 10l°

(A n per molecule)

x

(moles/cm2)

25 25 25 30 30 30 32 32 32 45 45

0.018 0.051 0.083 0.018 0. 051 0.083 0.018 0. 051 0.083 0.051 0. 083

1.18 3.16 5.00 1.40 3.73 5.91 1.27 3.40 5.38 2.65 4.16

defined by the expression I = (1 -- x)I~2 - xPl. At the mole fractions of urea used here (x < 0.083) I is approximately the surface concentration of urea. The results of this calculation are shown in Table I. A comparison of the surface and bulk concentrations of urea is possible if an assumption is made concerning the effective thickness of the interfacial region. Choosing 10 A 2 (or 10.7 cm) as the order of magnitude of the thickness, for 1, 3, and 5 M bulk concentrations, the number of gram moles of urea in a bulk volume 1 em X 1 em X 10_7 em would be 1, 3, and 5 X 10-1°, respectively As shown in Table I these are approximately the concentrations of urea at the interface in the volume available around the stearie acid molecules. DISCUSSION

Inclusion complexes do not form between urea and the hydrocarbon chains when the f a t t y acid molecules are arranged in a monolayer at an air-water interface. The evidence for this is the fact that f a t t y acid monolayers compress to approximately the same limiting areas per molecule independent of the urea concentration in the substrate. Interaction with earboxyl groups of the f a t t y acids through dipole-dipole interaction or hydrogen bonding is indicated f r o m the surface potential measurements for nil areas per stearic acid molecule. Davies and Rideal (3) consider the surface potential of a f a t t y acid

293

monolayer to be the sum of potentials due to hydrocarbon chains, polar groups, and oriented water molecules. The present state of the theory does not allow quantitative interpretation of dipole-dipole interactions between the fatty acid molecule and urea. However, measurements of the potential per fatty acid molecule, as shown in the figures, reveal that increasing the concentration of urea progressively makes the potential per molecule more negative at areas per f a t t y acid molecule where urea is forced out of positions coplanar with the carboxyl groups. Furthermore the lower the temperature the more negative the potential change produced by urea. These facts are consistent with a dipole-dipole interaction between the urea and the carboxyl groups: increasing concentrations of urea increase the probability of finding a urea molecule in association with the earboxyl group; increasing the temperature tends to oppose orientation of urea with the earboxyl groups through increase in molecular thermal energy or molecular agitation. Since the potential per molecule remains negative during compression to a solid monolayer, at high compression, urea molecules are most probably beneath the monolayer. Increase in collapse pressure at 15°C due to urea in the substrate shows that the presence of oriented urea beneath the monolayer makes disruption of the monolayer from the substrate more difficult. Calculations show that the surface eoncentration of urea required to provide energy for the observed expansions of the f a t t y acid monolayers is of the same order of magnitude as the bulk concentration of urea. There was no surface excess concentration of urea; that is, urea is either desorbed from the interfacial region or enters the interface along its diffusion or concentration gradient. But there is no excess accumulation of urea as occurs with surface-active molecules. The interfaeial region expands to accommodate the urea molecules which penetrate. Similar expansions occur when surface layers of lung extract are exposed to xenon (10) and when pellicles of paramecia are exposed to sufficient pressures of xenon (11). Thus molecules which can penetrate into the interracial region increase the free energy of the interface

Journal of Colloid and Interface Science, Voh 29, No. 2, February I969

294

SEARS

in the same manner as adding more gas molecules to a piston and cylinder assembly would increase the energy of this three-dimensional system. That urea is able to produce expansion of surface-oriented molecules is important in speculating about how urea may alter cell membrane permeability. This permeability alteration is discussed below. Urea did not affect the surface pressure or surface potential of soap monolayers. There was no effective penetration of urea into this interfacial region. According to the picture presented by Sears and Schulman (4), the structure of sodium and potassium stearate monolayers is determined by the presence of hydrated cations associated with the carboxyl groups. Dipole association between the urea and the carboxyl groups was precluded by the cation and the reduction of the surface potential due to the ion-carboxyl association; there was not as great a charge attraction to orient the urea dipole at the soap monolayers and the water associated with the cations is assumed to be highly oriented. There is nothing unique about the desorption of urea from certain interfaces and penetration of others. Urea differs from other non-surface-active molecules only by degree. Sodium hydroxide is strongly desorbed from the surface of its water solution. The energy required to place the highly polar NaOH in the surface is much greater than the energy required for a water molecule to occupy the surface position. Therefore water molecules are in the surface region and the NaOH molecules remain in the homogeneous bulk of the solution. If insoluble polar molecules such as stearic acid are placed at the surface, the coulombic attraction between the carboxyl groups and the Na ion supplies the energy required to displace some of the surface water and bring Na ions into the interfacial region. This reaction results in changes in surface pressure and surface potential. Urea behaves in a similar but weaker fashion. Effect of Temperature. Both urea and temperature cause expansion of the fatty acid monolayers. Change in surface pressure with temperature can be measured at constant areas per molecule and these changes are an Journal of Colloid and Interface Science,

Vol.

29,

No.

2,

February

AATF - vsA

A T Stearic acid

4o

0.0[ N HCI ~, 1.0 M Urea •

o 5.0 M Urea [] 5.0 M Urea

-~ 35 30

~ 2~ ~~ 20. ~ 15 ~

Io. 5. 0

i

21

i

J

i

23

Fro. 9. AAII/AT

1

25

i

,

,

I

,

i

,

i

i

27 29 31 33 Areo (A Z/molecule)

vs.

area

per

molecule

,

55

~

i

37 curves

for stearie acid on 0.01 N tIC1, 1.0 M urea, 3.0 M urea, and 5.0 M urea. Temperature range 25° to 37°C. alteration in entropy. That is, change in pressure at constant area for the monolayer is similar to change in pressure for constant volume for a three-dimensionai gas; no work is done by either system, the change being in the entropy of the system. In either of the systems, two- or three-dimensional, the change in pressure can be accomplished by increasing the temperature, by adding more molecules, or both. Plotting A (AII)/(AT) against the area per fatty acid molecule shows that temperature has a greater effect the greater the area per molecule. This is shown in Fig. 9. The slopes are not very different over the range of urea concentrations. Thus urea does not produce any significantly greater disordering of the interface than that produced by the expansion of the monolayer in the absence of urea.

Speculations Concerning Interactions of Urea with Cell Membranes. When the interfacial tension between cells and their exterhal medium has been measured, the values obtained have been less than 1 dyne/cm. Therefore the surface pressure is high. This means that the cell would tend to expand if not sufficiently restrained, in the same manner as a gas under high pressure will expand if released. The restraining forces to hold the membrane molecules in position 1969

SURFACE ACTIVITY OF UREA arise from (a) cohesion between the hydrocarbon chains in the hydrophobic portion of the membrane, (b) association of the phospholipid molecules with calcium which acts to diminish the repulsive forces between like charges of the polar groups, (c) orientation of water to form "soft ice," and (d), protein networks adsorbed to the surface of the cell. Penetration of urea into this region will provide additional forces leading to expansion. In this study the effect of urea to expand monolayers was demonstrated; however, high concentrations of urea were required before any measurable expansion occurred. In the case of the cell membrane, however, subtle expansions are expected to produce more marked effects. Shah and Schulman (12) have shown that the number of double bonds of the hydrocarbon chains of phospholipids or the inclusion of spacer molecules such as cholesterol in phospholipid monolayers is sufficient to alter the association of calcium with the phospholipid molecules. If urea penetrates the interfacial region of the cell membrane in sufficient concentration to release calcium, marked changes in permeability to ions and water would result. This hypothesis can be tested by examining whether urea causes release of isotopically labeled calcium from cell membranes.

295

ACKNOWLEDGMENTS This work was supported by grant GM-07072 from the National Institutes of Health. The many helpful discussions with the late Professor Jack H. Schulman are gratefully acknowledged. REFERENCES 1. GOOD, W., Biochim. Biophys. Acta, 52, 545 (1961). 2. MITCHISON, J. M., AND SWANN, M. ~., J. Exptl. Biol. 31,443 (1954). 3. DAVIES, J. T., AND ]~IDEAL, E. K., "Inter-

facial Phenomena." Academic Press, New York, 1961. 4. SEARS, D. F., AND SCHULMAN,J. I-I., J . Phys. Chem. 68, 3529 (1964). 5. DREHER, K. D., AND SEARS, D. F., T r a n s . Faraday Soc. 62,741 (1966).

6. ADAMSON, A. W., "Physical Chemistry of Surfaces." Interscieace Publishers, New York, 1960. 7. ADAM,N. K., "The Physics and Chemistry of Surfaces," 3rd ed. Oxford University Press, London, 1941. 8. LANGMUIR, I., J. Chem. Phys. 1,756 (1933). 9. GUGGENIIEIM, E. A., "Thermodynamics." North-Holland Publishing Company, Amsterdam, 1957. 10. CLE~ENTS, J. A., AND WILSON, K. M., Proc. Natl. Acad. Sci. U.S. 48, 1008 (1962). 11. SEARS, D. F., AND GITTELSON, S. M:., J . Protozool. 11,538 (1964). 12. StaSH, D. O., ANDSCH~'L~AN,J. tI., J. Am. Oil Chem. 2, 52 (1967).

Journal of Colloid and Interface Science, Vol. 29, No. 2, February 1969