FORCES BETWEEN NOVEL SURFACES.
3.1 Materials and Method.
GM1 w as ob tain ed from Sigm a C hem ical C om pany and u se d w ith o u t fu rth e r purification. P re ssu re -a re a iso th erm s an d tra n sfe r ratio s were recorded on a Langm uir-B lodgett a p p a ra tu s w hich h a s been previously described by M arra^.
T ra n sfer ra tio s ( th e ratio of th e h ea d g ro u p a re a of th e m olecules a t th e air-w ater interface to th e h ead g ro u p a re a on th e m ica surface) w ere determ ined as follows: A large sh e e t of freshly cleaved m ica (2cm x 4cm) w as firstly hydrophobed w ith e ith e r d ip a lm ito y lp h o s p h a tid y le th a n o la m in e (DPPE) or dim ethyldioctadecyl am m onium brom ide (DDOABr) . The second la y er w as d e p o site d a n d th e tr a n s f e r ra tio d e te rm in e d by dividing th e a re a th e b a rrie r h a d moved by th e a re a of th e m ica sh e et.
B ilay ers w ere d ep o sited u s in g th e L an g m u ir-B lo d g ett d ep o sitio n te c h n iq u e d escrib ed by M a rra ^ . F irstly, th e m ica su rfa c e s w ere h y d ro p h o b ed by d ep o sitio n of a m onolayer of DDOABr or DPPE. The second, G M 1-containing layer w as th en
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d e p o site d a n d th e s u rfa c e s tra n s fe rre d from th e L an g m u ir tro u g h to th e a p p a ra tu s in sm all b e a k e rs. The su rfa c e s were m ain tain ed u n d e r w ater du rin g th e tra n sfe r an d m ounting in the apparatus.
3.2 Results.
3.2.1 T ransfer Ratios.
The p r e s s u r e /a r e a iso th erm m e asu red for a m onlayer of p u r e GM1 (Fig. 2) w a s in c lo se a g re e m e n t w ith th e m e a su re m e n ts of Maggio. * 1 The T ran sfer ratio s m e asu red for th e v ario u s lipids are show n in Table 1. Force m e a su re m e n ts w ere p erform ed u s in g b o th DDOABr a n d DPPE to form th e hydrophobic m onolayer an d no difference in th e m easu red force w as reco rd ed . P ure GM1 d ep o sited onto DDOABr show s th e low est tra n sfe r ratio. W ith its large h ead group a re a an d wedge sh a p e th e m olecule req u ires a considerable a m o u n t of ch ain tilt to p a c k o n to a solid s u rfa c e . W hen GM1 is m ixed w ith dioleoylphosphatidylcholine(DO PC) tra n sfe r ratio s of 0.80, 0.85 for 10% a n d 20% GM1 are m e a su re d . GM1 m ixed w ith DPPC (which is in th e gel sta te a t room tem p eratu re) show s a m u ch higher tra n sfe r ratio of 0.92.
Table 1. T ransfer Ratios(for abbreviations see text).
Layer D eposited S u b strate IT (mN/m) T ran sfer Ratio
DDOABr Mica 2 5 1.14
GM1 DDOABr+Mica 3 0 0 .7 5
20% GM1/DOPC DDOABr+Mica 3 0 0 .8 5
10% GM1/DOPC DDOABr+Mica 3 0 0 .8 0
30
0l5
CO CO 0 i— CL 0o
0 t : C/D0 0.2 0.4 0.6 0.8
1
1.2 1.4 1.6 1.8
A/molecule (nm2)
Figure(2). The surface pressure/ area isotherm for GM1 spread on a water sub-phase.
6 0
3.2.2 Force M easurem ents.
The force betw een two deposited bilayers of p u re GM1 in w ate r (re su lts n o t shown) is repulsive a t all se p a ra tio n s. The force co n sists of a long-range double-layer force (Debye length of ~ 100 nm), a n d a steeply risin g rep u lsio n a t se p a ra tio n s less th a n 6 nm . W hen sodium chloride is added to th e a p p a ra tu s to give a final c o n c e n tra tio n of ImM , th e m e a su re d in te ra c tio n ap p e ars as show n in Fig.3. The d istan ces are m e asu red relative to h y d ro p h o b ic c o n ta c t in a ir for th is a n d all o th e r m e a su re m e n ts. The m e a su re m e n ts are highly reproducible an d the re su lts for two se p arate force ru n s are show n. The surfaces ca n n o t be p u sh e d to a se p aratio n closer th a n 5.7 nm (at a load exceeding l O m N m ' l ) . At d ista n c e s g reater th a n 10 nm th e force is fitted quite well by DLVO-theory (solid black line) u sin g th e n o n -lin ea r Poisson-B oltzm an eq u atio n w ith c o n s ta n t charge b o u n d a ry conditions. The plane of origin for th e charge is se t a t a se p a ra tio n of 6 nm . At se p a ra tio n s less th a n 9 n m th e force becom es slightly m ore repulsive th a n theory predicts.
W hen th e so d iu m chloride c o n c en tratio n is in creased to 100 mM th e m easu red in teractio n ap p e ars a s show n in Fig.4. A w eak attra ctiv e force is m e a su re d a t s e p a ra tio n s g re a te r th a n 15nm . The force is repulsive a t closer se p a ra tio n s a n d h a s a n ex p o n e n tia l d ecay len g th of 1.2nm . The su rfa c e s c a n n o t be p u sh e d closer th a n 6 nm .
The forces m e a su re d betw een GM1 m ixed w ith DOPC are show n in Figs. 5 an d 6. At 10 mole% GM1 th e m e asu red in te ra c tio n in p u re w a te r is show n in Fig. 5. The long-range d o u b le-lay er force (diam onds) is q u ite well fitted by th e n o n lin e a r P o issio n -B o z ltm a n n e q u a tio n w ith c o n s ta n t ch a rg e
<3)= 9.8 m C m k’ ^= 9nm f% n V = 8 3 mV 0 5 1 0 1 5 20 2 5 30 3 5 4 0 4 5 D (nm)
Figure(3). Measured forces ( scaled as F/R where R is the radius of curvature of the surfaces) between hydrophobed mica surfaces coated with a monolayer of GM1. The distance is measured relative to hydrophobic contact in air. Results are shown for two separate force runs. The solid line is the theoretical DLVO force calculated and includes a non-retarded van der Waals interaction with A \2 i= 1 xlO ^T. The curve is calculated assuming a constant charge interaction with a Debye length k = 9nm and a charge of 9.8 mCm4 with
F
/R
(
fi
N
n
r
1)
F
/R
(
|L
iN
m
a Q =15.6 mCm k"^ = 1.3nm <)/^=26mV 4— . —1 10 12 14 16 18D (nm)
300 -D (nm)
Figure(4). Measured forces between surfaces coated by GM1 at a sodium chloride concentration of 100 mM. The results for three separate force runs are shown plotted on a logarithmic scale (top) and linear scale (below). The solid line is the theoretical DLVO fit with a Debye length k* = 1.3 nm and a charge
of 15.6 mCm'z with the potential of the surfaces at infinite separation = 26
F
/R
(|
iN
rr
i
a 0 = 26 mCrri'k"^ = 42nm V 21 mV40
80
120
160
200
D (nm)
Figure(5). The force at 10% Gml in DOPC. The double layer force is well fitted at large separations by the non-linear Poisson-Boltzmann equation.
F
/R
(
|i
N
m
<T0 = 1 mCm k ^ = 68nm V T = 72m V■t— I— i— I— f 1— |— i— i— i— i— |— f
0
50
100
150
200
D (nm)
Figure(6). Forces between bilayers containing GM1 at a concentration of 2.5%. The force is much lower in magnitude than for the higher concentration case in figure(4).
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b o u n d a ry conditions. A hard-w all rep u lsio n is m e asu red a t 4.5 nm an d the force rem ains repulsive a t all se p aratio n s. The forces betw een bilayers containing 2.5 mol% GM1 in w ater w ith o u t any added electrolyte are show n in Fig. 6. Again th e m easu red force re m a in s rep u lsiv e a t all s e p a ra tio n s a n d is m u c h low er in m a g n itu d e th a n th e force m e a s u r e d a t th e h ig h e r GM1 co n cen tratio n . The force is well fitted by double layer-theory a t large separations.
The forces m easu red betw een bilayers of 10% GM1 mixed w ith DPPC w ere sim ilar to th o se o b tain ed w hen m ixed w ith DOPC, except th a t th e h a rd wall in th e force occurred betw een 8.3 an d 9.3 nm .
3.3 Discussion.
DLVO th eo ry p red ic ts th a t th e in te ra c tio n betw een an y two su rfa ce s will be attractiv e a t s h o rt se p a ra tio n s du e to th e V an der W aals interaction. The forces betw een GM1 bilayers are repulsive a t sh o rt se p aratio n s a n d the ab sen ce of a m e asu rab le V an d er W aals force m u s t be d u e to th e p rese n ce of eith er a h y d ratio n or a steric repulsion. G angliosides are know n to bind a b o u t 60 w a te r m olecules for each h e a d g ro u p 13 t h en ce a significant h y d ratio n rep u lsio n could be expected. F u rth erm o re, th e term in al galactose residue of GM1 is likely to be exposed on th e su rfa c e a n d M arra h a s sh o w n th a t th e force betw een galactolipids is repulsive a t sh o rt se p aratio n s due to hydration of th e h ead g ro u p ^. As a re su lt it is reasonable to expect th a t GM1 w ould show a sim ilar hydration force.
6 2
The p resen ce of h y d ra tio n /s te ric rep u lsio n betw een th e h e a d g ro u p s com plicates the in te rp re ta tio n of th e m e a su re d force curves w ith DLVO theory. It is im possible to determ ine the position of th e sialic acid residue relative to th e m ica surface and as a re s u lt th e position of the plane of origin of charge is also u nknow n. The force applied did n o t exceed 10 m N m 'l d u rin g th e force m easu rem en ts, and at th is force it is im possible to fully d eh y d rate phospholipid head groups, a s a re s u lt a larger th a n ex p ected b ila y er th ic k n e s s is o b ta in e d . To overcom e th is p ro b le m M a rra c o m p a re d h is m e a s u r e m e n ts w ith th e d eh y d rated layer th ic k n e ss. This q u an tity , how ever, c a n n o t be estim a ted for GM1 b e c a u se the conform ation of th e head g ro u p in th ese bilayers is unknow n.
A h a r d w all re p u ls io n o c c u rs a t 5 .7 n m w h ic h is co n sid erab ly less th a n th e th ic k n e ss expected for a bilayer of GM1 w ith th e h ead groups a t m axim um extension. D ata from X- ra y diffraction s tu d ie s ^ and estim a tes from m o lecu lar m odels p u t th e expected b ilay er th ic k n e ss a t 9.5 to lO nm . The large h ead g ro u p a re a re su ltin g from th e low tra n s fe r ratio d u rin g the deposition indicates th a t there is a considerable am o u n t of chain tilt. The a re a p e r h e a d group a t th e p re s s u re of deposition is equal to 0.71 nm ^ a n d w ith a tra n sfe r ratio of .75 the deposited h e a d g ro u p a re a is ln m ^ . Since th e m o le c u la r volum e m u s t rem ain c o n s ta n t th e layer thickness calculated sh o u ld be ab o u t 6.9 nm . B ecause of th e large h eadgroup of GM1 it is likely th a t th e th ic k n e ss m e a su re d a t 10 m N m 'l in force is considerably th in n e r th a n th e th ic k n ess of the bilayer w ith o u t a n applied load. The m easu red in te ra ctio n begins to deviate from th a t calculated from th e n o n -lin ear Poisson-B oltzm an eq u atio n a t se p aratio n s
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less th a n 8nm . This additional repulsion m ay be due to the steric in te ra c tio n or h y d ra tio n re p u ls io n a n d in d ic a te s th a t th e h e a d g ro u p s m ay extend fu rth e r th a n th e 5 .7 n m force b a rrie r into the aqueous phase.
W hen GM1 is mixed w ith DOPC the h a rd wall in the force law o c c u rs a t 4 .5 n m . T h is s e p a r a tio n c o rre s p o n d s to th e th ic k n ess expected for DOPC bilayers an d th e ir hyd ratio n layers. The d eh y d rated th ic k n ess o b tain ed from X -ray m e a su re m e n t is 3.5 nm . At th e higher tran sfer ratio th e GM1 head group would be expected to be m ore extended th a n in p u re bilayers formed from GM1 alone. A s h o rt ra n g e ste ric re p u lsio n w ould be ex p ected a t a m u c h la rg e r s u rfa c e s e p a ra tio n d u e to th e in te ra c tio n of th e GM1 head g ro u p s b u t th is is n o t observed.