The control of protein surface concentration on proteoliposomes

ColloidsandSurfaces,62 (1992) 177-184

177

Elsevier Science Publishers B.V.,Amsterdam

The control of protein surface concentration on proteoliposomes S h e i l a E. F r a n c i s a, F i o n a J. H u t c h i n s o n a, I a n G . L y l e b a n d M a l c o l m N . J o n e s a

aBiomolecular Organisation and Membrane Technology Group, Department of Biochemistry and Molecular Biology, University of Manchester, Manchester, UK bUnilever Research, Port Sunlight Laboratory, Bebington, Wirral, Merseyside, UK (Received 21 June 1991; accepted 20 August 1991)

Abstract Proteoliposomes having surface-bound succinylated concanavalin A (sConA) have been prepared by sonication of small unilamellar vesicles(SUV) and reverse-phaseevaporation (REV) from lipid mixtures of dipalmitoylphosphatidylcholine, phosphatidylinositol and the reactive lipid phosphatidylethanolamine derivatised with maleimidobenzoyl-N-hydroxysuccinimide. The liposomes were conjugated with protein by reaction with the N-succinimidyl-S-thioacetate(SATA) derivative of sConA and had weight-averagediameters (aVw)in the range 45-260 nm, as determined by photon correlation spectroscopy, and weight-averagenumbers of protein molecules per proteoliposome (/~w)up to approximately 2000. The factors controlling the extent of conjugation, including activation of the SATAderivative of sConA, the molar ratio of SATA to sConA and the reactive lipid content of the liposomes, have been studied. The surface concentration of sCoriA is directly controllable by manipulation of the reactive lipid content of the proteoliposomes. The results are compared with previous studies on wheatgerm agglutinin-conjugated proteoliposomes, and it is shown that for both types of proteoliposome the area per protein in the liposomal surfacesreaches limiting values of approximately 100 nm2 for REV and 40 nm2 for SUV. This observation is discussed in terms of the projected excluded area of protein on the liposomal surface.

Keywords:Proteoliposomes;succinylated concanavalin A; wheat germ agglutinin; liposomeconjugation; protein limiting area.

Introduction There is now considerable interest in the study of protein-conjugated liposomes (proteoliposomes) which has primarily arisen from their potential use as targeted delivery systems for drugs and other therapeutic agents [1-3]. The conjugation of antibodies is of particular importance, but plant lectins are also receiving attention as a means of targeting liposomes to specific carbohydrate receptors on biosurfaces [4-9]. Several methods are available for covalently attaching proteins to liposomes [10]; the most widely used procedures depend on derivatising phosphatidylethanolamine by reaction with succinimidyl reagents, e.g. N-succinimidyl-3(2-pyridylthiopropionate) (SPDP) or m-maleimido0166-6622/92/$05.00

benzoyl-N-hydroxysuccinimide ester (MBS) which, after incorporation into the liposome, is reacted with the dithiothreitol-reduced S P D P - p r o tein derivative [5,7] or the deacetylated succinimidyl-S-acetylthioacetate (SATA)-protein derivative respectively [6,8,9]. An alternative procedure involves the derivatisation of the protein with palmitic acid and incorporation of the palmitoylprotein into the liposome [4]. Whatever method is used, it is desirable to control the surface concentration of the protein and to be able to characterise the conjugated liposomes in terms of their size and the number of protein molecules per liposome in order to relate these parameters to the targeting efficiency of the system. As noted previously [8], values reported for the mass of conjugated protein

© 1992 - - Elsevier Science Publishers B.V. All rights reserved.

178

per mole of liposomal lipid extend over approximately four orders of magnitude and the numbers of protein molecules per liposome extend over approximately three orders of magnitude. While the latter parameter is dependent on liposome size and requires precise characterisation of the liposomes, the protein mass per mole of liposomal lipid partly reflects the different chemistries used for conjugation and the factors which determine the efficiency of reaction and packing of the proteins on the liposomal surface. In this study we have examined some of the factors controlling the conjugation of the succinylated lectin concanavalin A (sConA) to phospholipid liposomes prepared from mixtures of dipalmitoylphosphatidylcholine (DPPC), phosphatidylinositol (PI) and the reactive lipid derivative dipalmitoylphosphatidylethanolamine-maleimidobenzoyl-N-hydroxysuccinimide (DPPE-MBS). ConA is a site-directing molecule for cell surface mannosyl and glucosyl residues. The succinylated derivative was chosen in preference to conA because it has much less tendency to aggregate.

Materials and methods

Materials L-a-Dipalmitoylphosphatidylethanolamine (DPPE, product no. P-0890), L-a-dipalmitoylphosphatidylcholine (DPPC, product no. P-0763) and succinyl concanavalin A (sConA, product no. L-3885) were obtained from Sigma (Poole, UK). Phosphatidylinositol (PI, grade I, from wheatgerm, molecular weight 846g mo1-1 [11]) was from Lipid Products (South Nutfield, UK). Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was from Pierce and Warriner (Chester, UK) and Nsuccinimidyl-S-acetylthioacetate (SATA) was from Calbiochem (Cambridge, UK). [3H]-DPPC was from Amersham International (Amersham, UK). All other reagents were of analytical grade and aqueous solutions were made up with doubledistilled water.

S.E. Francis et al./ Colloids Surfaces 62 (1992) 177-184

Preparation of DPPE-MBS DPPE (40 mg) was dissolved in a mixture of dry chloroform (16 ml), dry methanol (2 ml) and dry triethylamine (20 mg). MBS (20 mg) was added, and the reaction mixture was stirred under nitrogen at room temperature for 24 h, after which the organic phase was washed three times with phosphate-buffered saline (PBS, pH 7.3) to remove unreacted MBS. The DPPE-MBS derivative was recovered from the organic phase by rotary evaporation and analysed by thin layer chromatography (TLC) using a silica plate and a solvent mixture containing chloroform, methanol and glacial acetic acid (65:25:13, v/v). TLC confirmed that there was negligible contamination of D P P E - M B S (Rf=0.78) with DPPE (Re=0.56). The D P P E MBS was stored in a chloroform-methanol (9:1, v/v) mixture at 4 ° C.

Preparation of SA TA derivative of sConA sConA was derivatised with SATA using modifications of the method described by Duncan et al. [12] using a range of mole ratios of SATA to sConA up to 35. Specifically 0-8.75 pl of a stock solution of 9.08 mg SATA in 50 I.tl of dimethylformamide were added to sConA (10 mg in 2.5 ml phosphate (50 mM)-EDTA (1 mM) buffer, pH 7.5) at room temperature. After the reaction (15 min) the derivatised sConA was separated from unreacted SATA by gel filtration on a Sephadex G-50 column (15 cm × 2 cm). Fractions (2 ml) were collected, and both derivatised sConA and unreacted SATA were detected spectrophotometrically at 280 nm. The protein was assayed using a Lowry microassay 1-13] with sConA as the standard. The dependence of hydroxylamine concentration on the deacetylation of the SATA derivative of sConA was determined up to a hydroxylamine concentration of approximately 0.1 M by incubation of derivatised sConA fractions containing 0.9-1.1 mg ml-1 protein for 1 h. This was done by addition of 200 lal of stock solutions of hydroxylamine (01.25 M) (made up in 2.5 mM EDTA with sufficient

S.E. Francis et al./ Colloids Surfaces 62 (1992) 177-184

solid Na2HPO4 added to bring the pH to 7.5) to 2 ml of protein solution. After deacetylation the sulphydryl content of the derivatised sConA was determined by the method of Ellman [14].

Preparation of proteoliposomes Liposomes were prepared by sonication of small unilamellar vesicles (SUV) and by reverse-phase evaporation (REV) from mixtures of DPPC and PI incorporating 0-14mo1% D P P E - M B S and tracer levels of [3H]-DPPC. For SUV the required lipid mixture (total mass 10 mg) plus 100 Ixl of [3H]-DPPC (4 laCi ml- 1) was dissolved in chloroform (20ml) and methanol (5 ml) in a roundbottomed flask. The mixture was rotary evaporated at 60°C to form a thin film which was then dispersed in 5 ml of nitrogen-saturated PBS at 60°C. After vigorous shaking the suspension was transferred to a glass test-tube, purged with nitrogen, sealed with a suba seal, and sonicated in a bath sonicator (Decon FS100) at 60 °C until visibly clear (approximately 1 h). For REV a modification of the method of Szoka and Papahadjopoulos rl 5] was used. A lipid film was prepared using 3 ml of chloroform-methanol (4:1, v/v) as above, in a 50 ml round-bottomed flask. The film was redispersed in 6 ml of chloroform-methanol (4:1 v/v), 3 ml of nitrogen-saturated, diluted (1 : 10) PBS were added at 60°C and the mixture gently shaken, followed by sonication for 3 min under nitrogen at 50°C. The resulting emulsion was rotary evaporated at 60°C. After the formation of a viscous gel phase and 'frothing', the resulting aqueous phase was purged with nitrogen for a further 15 min at 50°C to remove traces of organic solvent, and was kept at 50 °C for a further 15 min to allow annealing to occur. After preparation of the initial liposomal suspensions the preparations were applied to a gel filtration column (Sephadex G-200, 30cm x 2cm) previously equilibrated with PBS (pH 7.3) at a flow rate of 0.2ml min -1 and fractions (2 ml) were collected for subsequent conjugation. Location of the liposomes in the fractions was determined by

179

scintillation counting of 100 Ixl aliquots. The liposomes were conjugated by mixing and incubation with the deacetylated SATA derivative of sConA in appropriate proportions at room temperature for 2 h or at 4 ° C overnight. In general the liposome fraction (2 ml) containing approximately 2 mg of lipid is incubated with 2 ml of SATA-derivatised sConA containing approximately 2 mg of protein. After conjugation the reaction mixture was applied to a Sepharose 4B column to separate the proteoliposomes from unreacted protein. The lipid and protein content of the proteoliposome fractions were determined from the [3H1-DPPC counts and Lowry assay [13] respectively.

Proteoliposome size characterisation The liposomes and proteoliposomes were characterised in terms of size distribution at several stages during their preparation by photon correlation spectroscopy using a Malvern autosizer model RR146. The scattering data were fitted to an equivalent normal weight distribution W(di) to give the weight-average diameter (dw) and the standard deviation (aw) of the distribution. This information was used together with the protein to lipid molar ratio in the proteoliposomes to compute the weight-average number of protein molecules per proteoliposome (Pw) defined from the relations

where Pi and wi are the number of proteins per proteoliposome and weight of proteoliposomes of species i respectively as previously described I-8]. Results

A typical elution profile from a Sepharose 4B gel filtration column of a proteoliposome preparation after conjugation is shown in Fig. 1. The coincidence of the protein and liposome peaks confirms the attachment of the sConA to the liposomes which elute at a much lower elution volume than the unreacted sConA. The slight

S.E. Francis et al./Colloids Surfaces 62 (1992) 177-184

180 i .500

15C'0

VO+ V.

o

1

,,0.

1ooo

o

too

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°

/ soo

-

• \

r

60

Jt


•

5

10

15

20

Fraction number

25

50

35

40

0000 0.00

(2ml)

Fig. 1. Elution profile of sConA-conjugated REV (composition of DPPC/PI, 9 : 1 by weight plus 10.7mol% DPPE-MBS) from a Sepharose 4B column. The peak fraction of proteoliposomes had a weight-averagediameter (dw)of 145 nm and a Pw= 1400. The void (V0) and inner volume (Vo+ V/,.)of the column are indicated. shoulder at low elution volumes on the unreacted protein peak occurred in numerous preparations and probably arises from a small amount of aggregation of the unreacted sConA. It should be noted that succinylation of ConA gives a derivative which is a dimer at p H 7 (molecular weight 52 000) having two succinyl groups per subunit (molecular weight 26 000) [16]. The amount of unconjugated protein shows that the efficiency of conjugation is relatively low. In the preparation of proteoliposomes incorporating up to 13.8 mol% D P P E - M B S it was found that the percentage by weight of sConA in the reaction mixture which conjugates with the liposomes was 2.47 _+ 0.32% for each mol% D P P E MBS in the liposomes. We examined the effect of hydroxylamine concentration on the deacetylation of the SATA derivative of sConA prepared at a constant molar ratio of SATA to sConA of 10:1 (Fig. 2). The extent of deacetylation increases with hydroxylamine concentration reaching a limiting value at approximately 0.1 M. This concentration was used routinely to deacetylate the SATA-sConA derivative. Figure 3 shows the dependence of the number of sulphydryl groups per sConA (dimer) on the molar ratio of SATA to sConA in the reaction mixture. The maximum number of sulphydryl groups introduced is 1.76 + 0.1 at a molar ratio of 25:1 or above. Routinely a molar ratio of 10:1

0.02

0.06

O.04

Hydroxylumine

0.08

0.10

0.12

moles/I

Fig. 2. Dependence of deacetylation of the SATA derivative of sConA on hydroxylamine concentration. The concentration of the sConA derivative was approximately constant in the range 0.989 to 1.08mg ml- 1.

T /

/I z T/ ./I

iooo

// / / // //

/ 5

10

15

mole

ratio

20

25

30

35

40

SATA/S,CONA

Fig. 3. Dependence of the sulphydryl content of sConA (dimer) (i.e. number of -SH groups per dimer) on the SATA to sConA molar ratio. A concentration of 0.1M hydroxylamine was used for deacetylation. was used for derivatisation which introduced 1.21 _+ 0.05 - S H groups per sConA. These figures are comparable with those for SATA-horseradish peroxidase (molecular weight 44 000) derivatives found by Duncan et al. [12]. The dependence of the extent of conjugation on the D P P E - M B S content of the proteoliposomes is shown in Fig. 4. The data relating to the REV size (dw) range 152-252 nm and six SUV preparations (dw, 67-97 nm) containing 7.4 mol% D P P E MBS are represented by a single point. The extent of conjugation increases linearly with D P P E - M B S mol percent but has a non-zero intercept due to a small amount of physical adsorption of sConA

S.E. Francis et al./ Colloids Surfaces 62 (1992) 177-184

181

T G.

150

T ~

1 ] o_"

100

iooo

._c 0 ~ o_

50

2

4-

6

8

10

12

0

14

5

6

9

12

15

mole % PEMBS

mole % PEMBS

Fig. 4. Dependence of the extent of conjugation of sConA to DPPC/PI (9 : 1 by weight) liposomes as a function of D P P E MBS incorporation. ©, REV; O, SUV.

found when liposomes containing no D P P E - M B S are incubated with sConA. From the extent of conjugation and the measured size distributions of the proteoliposomes the weight-average number of sConA molecules per proteoliposome (/~w) were obtained. Figure 5 shows Pw as a function of D P P E - M B S mol percent for sConA-conjugated proteoliposomes together with corresponding data for wheatgerm agglutinin (WGA)-conjugated proteoliposomes taken from our previous work [8]. It should be noted that since Pw increases with proteoliposome size it is only possible to obtain a relationship between Pw and D P P E - M B S mol percent for proteoliposomes of approximately constant size. Table 1 shows characterisation data for typical sConA-conjugated proteoliposomes obtained dur-

Fig. 5. Weight-average number of proteins per liposome (/~w) (DPPC/PI composition 9 : 1 by weight) as a function of D P P E MBS incorporation. O, sConA=REV (dw = 228 _+23 nm); 0 , sConA-SUV (dw=gl_+llnm); ram, WGA-REV (dw = 163_+6nm); O, WGA-SUV (aTw=83_+4nm). The data for WGA-conjugated REV and SUV were taken from Ref. [8].

ing the course of their preparation. The weightaverage diameters change little on conjugation, although on subsequent fractionation by gel filtration the proteoliposomes in the peak fractions were sometimes larger than the values in the unfractionated samples; a small degree of aggregation may be partly the cause of this observation. The long-term stability of sConA-conjugated proteoliposomes was studied over a six month period. These data are shown in Table 2 together with data for wheatgerm agglutinin (WGA)-conjugated proteoliposomes which were prepared in connection with previous studies [8,9]. The sConAconjugated proteoliposomes showed a small increase in size (on average 12.9%) over six months,

TABLE 1 Characterisation of liposomes (DPPC/PI, weight ratio 9:1) at stages during their preparation and co~ugation with succinyl concanavalin A

~.

//wb

~o

(Mol%) D P P E - M B S

Type

(nm)

awa

(nm)

awb

(nm)

awc

/~w

7.4 7.4 10.7 13.8

SUV REV REV REV

67 203 177 252

43 126 103 155

64 201 171 230

41 118 102 143

97 202 252 256

61 112 152 148

341 1084 1635 1807

aParameters of peak fraction after gel filtration (Sephadex G-200). bParameters after conjugation with sConA. cParameters of peak fraction after gel filtration (Sepharose 4B).

182

S.E. Francis et al./ Colloids Surfaces 62 (1992) 177-184

TABLE 2 Long-term stability of conjugated D P P C - P I - D P P E - M B S proteoliposomes. Weight average diameter dw as a function of time dw (nm)

Time (months) Conjugate

Lipid wt. ratio

/~w

0

sConA sConA sConA sConA

6:4:2 8:2:1 9:1:1 9:1:2

54 610 1200 2868

117 180 244 253

WGA

9:1:1

531

193

which most probably arises from aggregation. The size of the WGA-conjugated proteoliposomes remained almost constant over the entire period of study. Discussion The overall objective of the study was to investigate some of the factors relating to the conjugation of protein to liposomal surfaces and the control of the surface concentration of protein in proteoliposomes. It is clear that the extent of conjugation of sConA can be manipulated by controlling the mol percent of reactive lipid (DPPE-MBS) in the liposomes, as was previously found for WGA [8]. Similar control of conjugation was found for WGA and phaseolus vulgaris agglutinin (PHA) by Liautard et al. [5] using the SPDP (N-succinimidyl-3-(2-pyridylthiopropionate)) reagent to conjugate through DPPE. It is significant, however, that the extent of conjugation at a given mol percent of reactive lipid varies with the lectin for liposomes of approximately constant size (Fig. 5), being greater for sConA than for WGA. Liautard et al. [5] found that conjugation was more efficient for WGA than PHA. Regression analysis on the data in Fig. 4 gives a gradient of 10.9_ 2.9 (g protein per mole lipid) per mol% DPPE-MBS for sConA conjugation which compares with values 4.45 and 4.87 for WGA-conjugated REV and SUV respectively [8].

4

6

8

12

18

24

192

190

191

194

152 186 259 284 202

-

Figures 2 and 3 demonstrate that the conditions of deacetylation (hydroxylamine concentration) of the SATA derivative of the protein is a controlling factor together with the SATA to sConA molar ratio. The maximum number of sulphydryl groups which can be introduced into sConA is relatively small although comparable to the values found for other proteins of similar size [6,8,12]. At the routinely used molar ratio of SATA to sConA of l0 : 1 under optimum (0.1 M hydroxylamine) deacetylation conditions, the introduction of a mean value of 1.21 +0.05 -SH groups per sConA molecule (dimer) means that assuming a Poisson distribution of thioalkylated molecules, 29.8% of the sConA molecules will not be thioalkylated, 36.1% will have one thioalkyl group, 21.8% two groups and 12.3% more than two. From the sequence of ConA [17] there are 24 lysine residues per dimer, four of which are succinylated in sConA [16], leaving a possible 20 for thioalkylation so that the SATA reaction has a relatively low yield under the conditions described. Similar conditions introduce two -SH groups into WGA [6] which has a possible 11 lysine residues for thioalkylation [18]. The packing of protein on the liposomal surface would be expected to reach a limiting value provided that the amount of reactive lipid is not limiting. This expectation is confirmed for both REV and SUV by plotting the area per protein molecule against Pw as shown in Figs 6 and 7. The data are for sConA-conjugated proteoliposomes

S.E. Francis et al./ Colloids Surfaces 62 (1992) 177-184

" •

183

15oo,

10oo,

E

t 0

o o

50o,

o

°° ~"°° ~ ~ ' - e I 500

-

I 1000

•

-

I 1500

__e__ 2000

P

w

Fig. 6. Surface area of protein (area per moelcule) on proteoliposomes (REV) conjugated with sConA ( e ) and WGA ((3) as a function of weight-average number of proteins per proteoliposome (Pw).

8oo-.!

E

°-/

vc

D

400-

20o-- •

0

I 20o

~ m

gl_

.

-II

I 4oo P

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w

Fig. 7. Surfacearea of protein (area per molecule) on proteoliposomes (SUV) conjugated with sConA (I) and WGA ([]) as a functionof weight averagenumber of proteins per proteoliposome (Pw). from this study and for WGA-conjugated proteoliposomes taken from previous work [8,9]. For both REV and SUV the data points fall on the same curve within the experimental uncertainty and show that at high values of Pw the area per protein molecule approaches a limiting value which for REV is of the order of 100 nm 2 per molecule and for SUV 40 nm 2 per molecule. At the pH of these studies (7.4), from the amino acid sequences of the proteins [17,18] the net charge on the molecules will be of the order of - 1 8 for sConA (dimer) and near zero for WGA; however at physiological ionic strength in PBS, charge repulsion due to double-layer overlap will be largely screened so

that packing may not be limited by protein net charge but more by effective size. From the threedimensional structure of the two proteins the sConA dimer has dimensions of the order of 8nmx4nmx5nm [19] and WGA 4 n m × 4 n m x 6 nm [19]. It follows that the projected areas of these molecules will be in the ranges 13-50 nm 2 (sConA) and 13-28 nm 2 (WGA). However, some excluded volume effect would be expected. For rigid spherical molecules the excluded projected area would be four times the physical projected area, giving values in the ranges 5 2 - 2 0 0 n m 2 (sConA) and 5 2 - 1 1 2 n m 2 (WGA) depending on the orientation of the protein at the surface. The observed limiting area for REV falls in these ranges, whereas for SUV the limiting area is less than four times the excluded projected area but greater than the physical projected areas. While it is possible that the surface concentration of protein is not uniform over the liposomal surface and some surface aggregation may occur, the observed limiting areas are not inconsistent with a uniform distribution. The smaller value of the limiting area for SUV is consistent with a higher surface curvature facilitating tighter packing and hence a greater surface density. It should be noted that for the proteoliposomes with higher values of Pw, the surface area per molecule of reactive lipid (assuming an area per lipid molecule of 0.50 nm 2 [20]) is 4 nm 2 per reactive lipid, which is greatly in excess of the protein surface area per molecule, and hence the reactive lipid is not limiting the extent of conjugation. A further interesting aspect of the properties of these proteoliposomes is their stability over relatively long time periods (Table2). While the sConA-conjugated proteoliposomes show a degree of aggregation in 6 months, the WGA-conjugated proteoliposomes were very stable, a property which is of potential value should proteoliposomes of this type find application in drug delivery.

Acknowledgements We thank SERC for financial support for F.J.H. and for a CASE studentship for S.E.F.

184

References 1 P.A.M. Peeters, G. Storm and D.J.A. Crommelin, Adv. Drug Delivery Rev, 1 (1987) 249. 2 L. Leserman and P. Machy, in M.J. Ostro (Ed.), Liposomes from Biophysics to Therapeutics, Marcel Dekker, New York, 1987, Chapter 5, p. 157. 3 S. Wright and L. Huang, Adv. Drug Delivery Rev., 3 (1989) 343. 4 S. Carpenter-Green and L. Huang, Anal. Biochem., 135 (1983) 151. 5 J.P. Liautard, M. Vidal and J.R. Philippot, Cell Biology Int. Rep., 9 (1985) 1123. 6 FJ. Hutchinson and M.N. Jones, FEBS Lett., 234 (1988) 493. 7 L.B. Margolis, A.A. Bogdanov, Jr., L.V. Gordeeva and V.P. Torchilin, in G. Gregoriadis (Ed.), Liposomes as Drug Carriers, John Wiley, 1988, Chapter 52, p. 727. 8 F.J. Hutchinson, S.E. Francis, I.G. Lyle and M.N. Jones, Biochim. Biophys. Acta, 978 (1989) 17. 9 F.J. Hutchinson, S.E. Francis, I.G. Lyle and M.N. Jones, Biochim. Biophys. Acta, 978 (1989) 212.

S.E. Francis et al./Colloids Surfaces 62 (1992) 177-184

10 F.J. Martin, T.D. Heath and R.R.C. New, in R.R.C. New (Ed.), Liposomes a Practical Approach, I.R.L. Press, Oxford, 1990, Chapter 4, p. 163. 11 L. Ter-Minassian-Saraga and G. Madelmont, J. Colloid Interface Sci., 85 (1982) 373. 12 R.J.S. Duncan, P.D. Weston and R. Wrigglesworth, Anal. Biochem., 132 (1983) 68. 13 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (195l) 265. 14 G.L. Ellman, Arch. Biochem. Biophys., 82 (1959) 70. 15 F. Szoka and D. Papahadjopoulos, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 4194. 16 G.R. Gunther, J.L. Wang, I. Yahara, B.A. Cunningham and G.M. Edelman, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 10r2. 17 B.A. Cunningham, J.L. Wang, M.J. Waxdal and G.M. Edelman, J. Biol. Chem., 250 (1975) 1503. 18 C.S. Wright, J. Mol. Biol., 194 (1987) 501. 19 Brookhaven Protein Data Bank Entries 3CNA (K. Hardman) 3WGA (C, Wright). 20 M.J. Janiak, D.M. Small and G.G. Shipley, J. Biol. Chem., 254 (1979) 6068.