Faraday Discuss. Chem. SOC., 1986, 81, 107-116 Physicochemical Studies of Vesicles and Biomembranes Spectroscopic Studies and Phospholipid Polymers Dennis Chapman,* David C. Lee and James A. Hayward Department of Biochemistry and Chemistry, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF Recent biophysical studies using a range of physical and spectroscopic techniques of both biomembranes and various lipid-water (vesicle) systems are described. The nature of lipid-protein interactions in some natural and model biomembranes has been examined with regard to the extent of lipid perturbation and protein conformational changes. The application of second- derivative and fourth-derivative infrared spectroscopy to these systems indi- cates clearly the bands associated with the secondary structure and also the weak bands associated with the minor amide components of the membrane proteins.The technique also provides evidence for the effects of intrinsic proteins on the lipid carbonyl groups when the lipid is below its T, transition temperature. Studies of phospholipid polymers in vesicle form and in Langmuir- Blodgett films are discussed. The introduction of new biomembrane-mimetic surfaces are also described. The latter are used to study protein and cell adsorption to particular phospholipid polar groups. In recent years we have been examining lipid vesicles and biomembranes using a variety of physical techniques. We have been particularly interested in examining lipid-protein interactions, i.e. how the lipid molecules are perturbed by the presence of intrinsic or integral proteins and also seeking methods to provide information about membrane protein structures.We have also set out to create new polymers, i.e. phospholipid polymers, so as to produce polymeric vesicles, thereby increasing their stability for certain applications. We have also developed derivatisation methods for treating the surfaces of glass and metals producing in some cases surfaces which mimic (in a simple form) the outer-surface character of the lipid portion of red blood cells. We describe some of our recent studies in this paper. I.R. Studies of Protein-Lipid Interactions and Protein Structure in Natural and Reconstituted Membranes Fourier transform infrared (f.t.i.r.) spectroscopy has been applied extensively in recent years to probe the structure and dynamics of biological membranes. Variations in band frequency, linewidth and intensity are sensitive to structural transitions of both lipid and protein components.The vibrations of individual groups provide structural informa- tion on highly localised regions of the lipid bilayer. Thus, the C-H stretching absorp- tions of the lipid acyl chains are readily distinguished from the carbonyl stretchings of the interfacial region and the phosphate stretchings of the polar headgroup. Of particular importance in the study of lipid-protein interactions is the non-perturbing nature of the technique. The addition of an external probe molecule is not required and the absorp- tions of the lipid and protein groupings reflect their genuine environments.The principal impediment to the study of aqueous membranes and their models, i.e. the strong and 107108 Spect roscopic Studies and Phospholipid Polymers broad i.r. absorptions of water, has been removed with the use of microcomputers for digital subtraction of the background. 192 Studies of the i.r. spectra of phospholipids have provided much information on the structure of the acyl chain, interfacial and headgroup regions of aqueous bilayer~.~ The main endothermic phase transition of aqueous phospholipid bilayers results in pro- nounced alterations in the methylene band parameters. The band maximum frequencies of the CH, asymmetric and symmetric stretching bands are sensitive to the static order of the acyl chains.The introduction of an increased proportion of gauche conformers above the phase transition causes a shift of these bands to higher freq~encies.~ The width of these i.r. absorptions is determined by rotational, translational or collisional effects. Thus, the CH, bandwidths are sensitive to the degree of motional freedom of the CH, groups. Most i.r. studies of protein-lipid interactions have concentrated on measurement of the C-H bands. In an early study the effects of the incorporation of the intrinsic proteins Ca*+-ATPase and bacteriorhodopsin and the intrinsic polypeptide gramicidin A on the acyl chain order of saturated phosphatidylcholine bilayers were r e p ~ r t e d . ~ It was found that below the phase-transition temperature, these molecules behaved in a similar manner to cholesterol, causing a disordering of the chains.Above the phase transition, the perturbations of the acyl chains differed from the progressive ordering effect observed with cholesterol. At high lipid protein molar ratios, an ordering of the acyl chains was observed. However, when the concentration of the intrinsic protein was increased this effect was removed and the static order of the chains was essentially the same as in the pure lipid system. More recently, we have repeated this type of study using dimyris- toylphosphatidylcholine in which the acyl chain hydrogen atoms have been completely replaced by deuterium atoms.6 This allows examination of the acyl chain C-'H stretching modes without overlap from the C-H stretching modes of the intrinsic protein.Our study indicated that bacteriorhodopsin, at low concentration, has no effect on the static order of the lipid racy1 chains above the phase-transition temperature (fig. 1). At high protein concentration there was some disordering of the lipid acyl chains, indicated by an increase in band frequency compared to the pure lipid. 1.r. spectroscopy may be used to probe the interfacial region of lipid bilayers via examination of the C=O stretching bands between 1750 and 1700 cm-'. Two absorption bands in this region are associated with the two ester groupings in diacyl lipids. The sn-1 carbonyl absorbs near 1740 cm-' while a band near 1725 cm-' is due to the carbonyl at the sn-2 position3 (see fig. 2). After the subtraction of the aqueous background the i.r.spectrum normally reveals only a single broad C=O band envelope which shifts by 4 cm-' to lower frequencies at the phase transition. This shift arises from alterations in intensity rather then frequency of the sn-1 and sn-2 components, as revealed by second- derivative (fig. 2) or deconvolution calculations. On passing through the phase transition, we observed a decrease in the intensity of the 1740cm-' component relative to the 1726-1728 cm-' component. Values for the intensity ratio of the two components of the carbonyl stretching band are given in table 1. Intensity ratios were calculated by measuring the negative absorbance from zero at 1726 and 1740cm-' in the second- derivative spectra. For DMPC, conversion from the gel to liquid-crystalline state produced a decrease in the relative intensity at 1740 cm-'. Incorporation of bacterio- rhodopsin or Ca2+-ATPase into the bilayers caused a large reduction in 11726/11740 below T, (23 "C).Above T,, a slight increase in was observed for the bacteriorhodop- sin-containing liposomes and a slight decrease for those containing Ca2+-ATPase. However, these differences were small compared to those observed below T'. We conclude that the presence of the integral proteins in the bilayer reduces the conforma- tional inequivalence below the phase transition so that a number of the sn-2 chains are now constrained in a conformation similar to that of the sn-1 chain.D. Chapman, D. C. Lee and J. A. Hayward 109 2095 2094 2093 7 2092 2- 2091 E 2090 2089 2088 2087 Q J. I I I I I I I I 10 15 20 25 30 35 40 45 temperature/"C Fig.1. Variation of C2H2 symmetric stretching frequency with temperature for 0, dmpc A, dmpc [2H]54/bacteriorhodopsin (102 : 1) and V, dmpc-[2H],2/bacteriorhodopsin (22: 1) molar ratios. 1.r. spectroscopy is an established technique for the study of the structures of polypeptides and proteins. The i.r.-active amide bands are associated with the CONH grouping which these molecules have in common. Initial qualitative studies related the frequencies of the relatively strong amide I and amide 11 bands to the presence of specific types of secondary structure in various soluble polypeptides and proteins. In principle, a globular protein containing several types of substructure will give several amide I maxima. However, the large half-widths of these components prevents their resolution.A method for assessing the number and position of component peaks is derivative spectroscopy. Second-derivative i.r. spectra of water-soluble proteins have been obtained7 and peaks associated with a-helical, @-sheet and @-turn conformations together with the vibrations of some amino acid side-chains were resolved. We have recently obtained the first second-derivative i.r. spectra of membrane protein^.'^' First we concentrate on our studies of the sarcoplasmic reticulum (SR), a membrane system which is involved in the regulation of the contraction-relaxation cycle of striated muscle. The majority of the protein present in the membrane is the Ca2+ activated ATPase which causes the active accumulation of Ca2+ into the SR during relaxation of the muscle.We have studied the secondary structure of this protein in three environments: the isolated SR, vesicles of purified Ca2'-ATPase in SR lipids and reconstituted into bilayers of dimyristoylphosphatidylcholine.' In fig. 2 we present difference, second- and fourth-derivative f.t.i.r. spectra of SR membrane after the subtraction of the aqueous buffer background. Negative bands in the second-derivative correspond to positive bands in the fourth-derivative, which, in turn, correspond to positive absorption bands in the difference spectrum. Three main absorption bands are110 Spectroscopic Studies and Phospholipid Polymers 1 1 1 1 1 1 i 1 1 1 1 1 1 1 1 1 1 1 1 t I 1 I I I I I I l - l l I I I I I I I 1 19 00 1800 1700 1600 1500 1 1 wavenumber/ cm- ' I 30 Fig.2. (a) Difference, (b) second-derivative and ( c ) fourth-derivative f.t.i.r. spectra of sarcoplasmic reticulum at pH 7.4 and 20 "C after subtraction of the aqueous background absorption. seen in the difference spectrum, the amide I and amide I1 bands from the protein at 1655 and 1547 cm-', respectively, and a C=O stretching band from the lipid at 1737 cm-'. The frequency of the amide I maximum of 1655 cm-' may be assigned to the presence of a predominantly a-helical protein in the membrane. However, analysis of the second- derivative reveals the presence of p-sheet structure, with absorptions at 1632 cm-' and 1680-1690 cm-', which has been predicted from the primary sequence." Further analysis using the fourth-derivative reveals components in the amide I region which may be assigned to a-helical structure (1657 and 1643 cm-') P-sheet structure (1681 and 1630 cm-') and p-turns (1690 ~ m - ' ) .~ , ~ The band at 1531 cm-' in the amide I1 region may also be due to P-structure.' The fourth-derivative spectrum also reveals bands which may be assigned to amino acid side-chains," which were previously lost beneath the broad amide I and I1 band envelopes. These are tyrosine (1516 and 1613 cm-'), glutamate (1568 cm-') and arginine or aspartate (1581 cm-'). These bands may provide future probes for structural alter- ations in enzyme active sites in a wide range of systems. The bands at 1742 and 1727cm-' in the second-derivative are C=O stretching absorptions, assigned above, from the lipid present in the membrane.The fourth- derivative reveals a third component at 1709 cm-' which has also been reported by other workers using spectral deconv~lution.~ Bands in the 1468-1421 cm-' region are CH2D. Chapman, D. C. Lee and J. A. Hayward Table 1. The effects of Ca2+-ATPase and bacteriorhodopsin on the intensity ratio of the carbonyl stretching bands of DMPC" 111 DMPC/Ca2--ATPase DMPC/bacteriorhodopsin T/"C DMPC 245 : 1 135: 1 10 0.52 0.3 1 20 0.45 0.43 25 0.50 0.55 35 0.79 0.70 45 0.80 0.8 1 0.32 - - 0.83 - Data were obtained from the second-derivative spectra presented, in part, in ref. (8). The negative absorbance intensities at 1726 and 1740 cm-' were measured at each temperature. deformation modes from the lipid acyl chains. The region 1900-1800 cm-' is free from absorption bands and gives an indication of the low noise in the spectra.Fig. 3 presents difference, second-derivative and fourth-derivative spectra of purple membrane, a light-energy transducing membrane which forms part of the plasma membrane of Halobacterium halobium and other extreme halophiles. These patches contain a single protein, bacteriorhodopsin, which uses light energy to translocate protons across the membrane, thereby setting up an electrochemical gradient. This gradient is used to synthesize ATP and subsequently, to provide energy for the metabolism of the cell. The amide band maximum at 1660cm-' may be assigned to the presence of a,,-helices in the protein. A predominantly a-helical structure is also indicated by the amide I1 band frequency of 1545 cm-', in agreement with the electron diffraction study of Henderson and Unwin.12 However, second- and fourth-derivative analysis of the broad amide bands reveals components at 1684, 1635 and 1530cm-', which may be assigned to @-structure.' This @-structure has also been revealed by circular dichroism and was proposed to be transmembrane antiparallel P-sheet.13 Further studies are in progress in our laboratory using selective modification of bacteriorhodopsin by enzyme cleavage to determine the location of the structures giving rise to these i.r. absorptions.Other absorptions in the fourth-derivative f.t.i.r. spectrum of purple membrane may be assigned to amino acid side-chains in bacteriorhodopsin' as for the Ca2+-ATPase. Phospholipid Polymers In order to probe the molecular structure and motional dynamics of biological mem- branes and their models, l4 photosensitive moieties have been incorporated into lipid structures.The presence of a small photolabile group permits the generation of reactive species with minimal perturbation of the structure of the membrane in which they reside. In contrast to their use as photoaffinity labels, our approach toward photosensitive phospholipids has been for the preparation of stable polymers. We have used polymeric phospholipids as models of biological membranes and in biomedical applications. A wide variety of polymerizable functional moieties has been incorporated into an assortment of amphipathic compounds to produce stable surfactant assemblies.' Many of these compounds have contained a photolabile diacetylene group and were based upon the initial observations of Wegner, who showed that these triple-bonded structures formed crystalline polymers upon irradiation with U.V.light. l6 We have synthesised phospholipids that contain diacetylene groups in one or both of their acyl chains [fig. 4( a)]."," The physical properties of the monomeric, diacetylenic112 Spectroscopic Studies and Phospholipid Polymers 1900 1800 1700 1600 1500 wavenumberj cm- ' 10 Fig. 3. (a) Difference, (b) second-derivative and ( c ) fourth-derivative f.t.i.r. spectra of purple membrane at pH 7.4 and 20 "C after subtraction of the aqueous backgound absorption. phospholipid were studied by optical and magnetic resonance spectro~copy~~-~* and by calorimetric and monolayer techniques22923 and were found to resemble the properties of naturally occurring lipids.N.m.r. relaxation times both for the head groups and for the resolved resonances in the acyl chains were very similar in diacetylenic and non-poly- merizable phospholipids. Calorimetric studies showed that the temperature of the thermotropic phase transition, T,, is lower for the diacetylenic phospholipids than for the corresponding saturated phosphatidylcholines, but is higher than that of the cis- unsaturated homologue~.~~ In mixed-chain phosphatidylcholines, replacement of the long diacetylene-containing acyl chain with a saturated acyl chain disrupts the packing of the lipid and further decreases T,. This disruption in the packing of the acyl chains is also evident in the inability of mixed-chain diacetylenic phosphatidylcholines to form a stable monolayer at the air-water interface, even at low temperatures." These phospholipid molecules containing diacetylene groups form polymers upon irradiation with U.V.light. The incorporation of polymeric phospholipids within the bilayers of liposomes increases their resistance to precipitation and leakage, and permits the preparation of stable surfaces that mimic the surfaces of biomembranes. Phospholipid Polymerisation in Whole Cells The physicochemical similarities between diacetylenic phospholipid monomers and conventional lipids suggested that phospholipid polymers might be found in situ follow-D, Chapman, D. C. Lee and J. A. Hayward $=o $=O F=O y=o $=O :=o- ./’ where PC is ‘.CHz-C/H -$H2 r--9: - - - -1 I o=p-0 I 113 A/nm Fig. 4. (a) Formation of the polyconjugated phospholipid polymer from the diacetylenic monomer; n may be varied to produce monomers of different lengths and different phase-transition tem- peratures. The hydrocarbon chains may be esterified to different polar head groups. ( 6 ) Visible spectra of 86 layers of diacetylenic phosphatidylcholine after various irradiation times. The sample consists of 43 layers of lipid on each side of a quartz slide. The upper surface is hydrophilic and formed by the polar head groups. ing incorporation of the monomeric lipid into the membranes of living cells. The rigid structures thus formed would permit the study of ( a ) isothermal ‘freezing’ of cellular membranes; ( 6) cellular ‘capsules’ containing entrapped cytoplasmic and intrinsic membrane proteins; (c) permanently asymmetric lipid bilayers; and ( d ) lateral discon- tinuities in membrane fluidity that would accompany localized polymer formation.The biosynthetic incorporation of diacetylenic fatty acids into membrane phos- pholipids and glycolipids was accomplished with a fatty acid auxotroph of the bacterium Acholeplasma laidlawii. When A. laidlawii cells were grown in the presence of diacety- lenic fatty acids, up to 90% of the membrane acyl chains were derived from the medium.24 Shorter-chain diacetylenic fatty acids were most suitable as substrates for the growth of these cells, and resulted in the most extensive uptake of the lipid. The distribution of the C20-diacetylenic fatty acid was very similar to that obtained for cells grown on a monounsaturated fatty acid, oleic acid.Similarities in uptake, in capacity to support growth, and in distribution among lipid classes suggest that the diacetylenic lipids are arranged in the bilayer in a manner similar to the oleate-containing lipids.114 Spec? roscop ic Studies and Phospholipid Polymers Polymerization of the membrane lipid in A. laidlawii was accompanied by a loss in the activity of an intrinsic membrane protein, NADH oxidase. In contrast, the activity of an extrinsic membrane protein, ribonuclease, was unaffected by p~lymerization.~~ Blodgett and Langmuir first demonstrated that a multilayered coating of lipid could be deposited onto a solid support by successively dipping the support through a m ~ n o l a y e r .~ ~ As with most models of biological membranes, instability is the primary limitation of multilayered films. Diacetylenic phospholipids pose a significant advantage for the utilization of multilayered films; polymerization inhibits the rearrangement and decay evident with multilayers of non-polymerizable lipids. We have developed procedures for the preparation of Langmuir-Blodgett-type multi- layers of diacetylene-containing Multilayers of diacetylenic phos- pholipids consistently presented a hydrophobic surface if they were polymerized after withdrawal from the subphase. In situations involving biocompatible surfaces, however, it is sometimes important that the prosthetic surface be polar. A polar surface could be stabilized, however, by irradiating the multilayer under water.Alternatively, the diacetylenic film was replaced with stearic acid before the last upstroke. After irradiation the layer of stearic acid was washed away, exposing the underlying polar surface. Varied materials (glass, quartz, Perspex, Teflon and mica) have been coated with ordered layers of diacetylenic phosphatidylcholines in which the phosphocholine moiety formed the outer coated surface. Fig. 4(b) shows the visible spectrum of a multilayer at various irradiation times. An increase in absorption in both the visible and U.V. regions of the spectrum accompanied polymerization. The layers after polymerization were quite stable in aggressive media and, with some precautions, could be handled without damage. An alternative method for the preparation of coated surfaces has been described by Regen et al.27y28 This method involves the polymerization of vesicles composed of diacetylenic or methacryloylic phosphatidylcholine in the presence of an insoluble support.The polymerized vesicles were assumed to associate with the phase bondary between the aqueous medium and the solid support. The capacity to modify the surface properties of existing materials by deposition of polymerizable multilayers may find important biomedical applications. The mechanical and topological properties of the support can be retained while the interfacial properties are changed to mimic those of cellular surfaces. Biomimetic Surfaces Asymmetries in the distribution of phospholipid head groups have been found in a variety of cells.29 The accumulated evidence suggests strongly that in the case of blood cells, the observed lipid asymmetry serves a biological purpose in the maintenance of the delicate balance between haemostasis and thrombosis.The extracellular surfaces of the plasma membranes of blood cells are thromboresistant; in strong contrast, their cytoplasmic surfaces are highly thrombogenic. The simplest common feature of the blood-compatible surfaces is the presence of large quantities (up to ca. 90% of the total surface lipids) of phosphorylcholine-containing phospholipids. We have previously investigated the haemostatic potential of polymeric phos- phorylcholine surfaces in dispersi~n.~~*'' Polymerized liposomes of diacetylenic phos- phatidylcholines did not alter the recalcification clotting times of citrated, pooled normal plasma regardless of lipid concentration.This non-thrombogenic nature of polymeric phosphatidylcholine was not altered after incubation in human plasma in vitro for up to 1 week at 37 "C. We have now developed32 a panel of reactive species which should result in the covalent deposition of phosphorylcholine on a variety of solid substrates. The resultant monomolecular layer provides a self-assembling surface which, in theory, should resemble the lipid interface presented by blood cells. The hydrophilic, membrane-D. Chapman, D. C'Lee and J. A. Hayward 115 ( a ) parent compound ( b ) natural phosphatidylcholines X,=O--CH, 0 I II CH-0-C- [CH21n - CH, I CH,-0- C- [CH,l,-CH, II 0 ( c ) hydroxyl-reactive phosphorylcholines (I) x,= c1 CH3 I I CH3 X,= 0-CH2-CH2-O- Si-Cl x, = 0- x,= C I x,= 0- ( d ) reaction products I " m 0 CHI h + I1 1 I k* (IIP HO-Si k \ + (CH,),N-CH,-CH,-O-P-O-CH2-CH~O-Si-O-Si~ t& O CH, Fig.5. Structures of phosphorylcholine ( a ) and its natural ( b ) and synthetic ( c ) and ( d ) derivatives. In the phosphatidylcholines found in biological membranes, the phosphorylcholine head group is esterified to a diacylglycerol. Two hydroxy-reactive phosphorylcholines have been prepared ( c ) , choline dichlorophosphate (I) and phosphatidylcholine ethylene glycol dimkthylsilylchloride (11). The immobilised species formed by reaction with silicon hydroxide are shown ( d ) . mimetic character of this surface provides opportunities for the generation of new, hybrid biomaterials.The modified surfaces retain the chemical, physical and topological properties of the substrates, with the intention of mimicking the characteristics of biomembranes (extreme thinness, low antigenic potential and increased potential for haemo- and bio-compatibility) (fig. 5 ) . Evidence for the covalent deposition of a self-assembing phosphorylcholine layer has been provided for the hydroxylated surfaces of glass and silica.33 Structural integrity of the deposited group is supported by the equimolar association of phosphorus and116 choline with the reacted surfaces. Covalent modification of the treated surfaces is demonstrated by i.r. spectroscopy. The modified surfaces are thermostable at tem- peratures up to 375 "C for extended Surfaces modified by the deposition of phosphorylcholine should exhibit characteris- tics highly desirable for biomaterials.The teleological arguments for possible haemo- compatibility have already been presented and are based upon the resemblance of phosphorycholine surfaces to the thromboresistant lipid surfaces of human blood cells. Additionally, the zwitterionic character of phosphorylcholine, attributed to the presence of both a strongly basic quaternary ammonium ion and an acidic phosphate ion of approximately equal strength, may impart useful physicochemical properties to the surface. This ionic balance may alter the adhesion of proteins and cells to modified surfaces. Further studies of these treated and other biologically relevant properties are in progress.Spectroscopic Studies and Phospholipid Polymers We thank the Wellcome Trust, The Commission of the European Communities and the Humane Research Trust for financial support. References 1 D. G. Cameron, H. L. Casal and H. H. Mantsch, J. Biochem. Biophys. Methods, 1979, 1, 21. 2 D. Chapman, J-C. Gomez-Fernandez, F. M. Goni and M. Barnard, J. Biochem. Biophys. Methods, 1980, 3 H. L. Casal and H. H. Mantsch, Biochim. Biophys. Acta, 1984, 779, 381. 4 M. Cortijo and D. Chapman, FEBS Lett., 1981, 131, 245, 5 M. Cortijo, A. Alonso, J-C. Gomez-Fernandez and D. Chapman, J. Mol. Biol., 1982, 157, 597. 6 D. C. Lee, A. A. Durrani and D. Chapman, Biochim. Biophys. Acta, 1984, 769, 49. 7 H. Susi and D. M. Byler, Biochem. Biophys.Res. Commun., 1983, 115, 391. 8 D. C. Lee, J. A. Hayward, C. J. Restall and D. Chapman, Biochemistry, 1985, 24, 4364. 9 D. C. Lee, D. A. Elliot, S. A. Baldwin and D. Chapman, Biochem. SOC. Trans., 1985, 13, 684. 2, 315. 10 D. H. MacLennan, C. J. Brandl, B. Korczak and N. M. Green, Nature (London), 1985, 316, 696. 1 1 Yu. N. Chirgadze, 0. V. Fedorov and N. P. Trushina, Biopolymers, 1975, 14, 679. 12 R. Henderson and P. N. T. Unwin, Nature (London), 1975, 257, 28. 13 B. K. Jap, M. F. Maestre, S. B. Hayward and R. M. Glaeser, Biophys. J., 1983, 43, 81. 14 P. Chakrabarti and H. G. Khorana, Biochemistry, 1975, 14, 5021. 15 J. H. Fendler, Science, 1984,, 223, 888. 16 G. Wegner, Makromol. Chem., Makromol. Chem., 1972, 154, 35. 17 D. S. Johnston, S. Sanghera, M. Pons and D. Chapman, Biochim. Biophys. Acta, 1980, 602, 57. 18 D. S. Johnston, L. R. McLean, M. A. Whittam, A. D. Clark and D. Chapman, Biochemistry, 1983, 22, 19 M. Pons, D. S. Johnston and D. Chapman, Biochim. Biophys. Acta, 1982, 693, 461. 20 M. Pons, D. S. Johnston and D. Chapman, J. Polyrn. Sci., Polyrn. Lett. Ed., 1982, 20, 513. 21 M. Pons, C. Villaverde and D. Chapman, Biochim. Biophys. Acta, 1983, 730, 306. 22 0. Albrecht, D. S. Johnson, C. Villaverde and D. Chapman, Biochim. Biophys. Acta, 1982, 687, 165. 23 J. Leaver, A. Alonso, A. A. Durrani and D. Chapman, Biochim. Biophys. Acta, 1983,732,210. 24 J. Leaver, A. Alonso, A. A. Durrani and D. Chapman, Biochim. Biophys. Acta, 1983, 727, 327. 25 K. B. Blodgett and I. Langmuir, Phys. Rev., 1937, 51, 964. 26 L. R. McLean, A. A. Durrani, M. A. Whittam, D. S. Johnston and D. Chapman, Thin Solid Films, 27 S. L. Regen, P. Kirszensztejn and A. Saingh, Macromolecules, 1980, 16, 335. 28 S. L. Regen, BV. Czech and A. Singh, J. Am. Chem. SOC., 1980, 102, 6638. 29 R. F. A. Zwaal and H. C. Hemker, Huemostasis, 1982, 11, 12. 30 J. A. Hayward and D. Chapman, in Biocompatibility of Tissue Analogs, ed. D. F. Williams (CRC Press, 31 J. A. Hayward and D. Chapman, Biomaterials, 1984, 5, 135. 32 A. A. Durrani, J. A. Hayward and D. Chapman, Biomaterials, 7, 121. 33 J. A. Hayward, A. A. Durrani, C. J. Shelton, D. C. Lee and D. Chapman, Biomaterials, 1986, 7, 126. 3192. 1983, 99, 127. Boca Raton, Florida, 1985). Received 13th December, 1985