Interactions between dermatan sulpahte chains. III Light-scattering and viscometry studies of self-association

179

Biochimica et Biophysica Acta,

586 (1979) 179--188 © Elsevier/North-Holland Biomedical Press

BBA 28958

INTERACTIONS BETWEEN DERMATAN SULPHATE CHAINS. III LIGHT-SCATTERING AND VISCOMETRY STUDIES OF SELF-ASSOCIATION *

L.A. FRANSSON

**, I.A. NIEDUSZYNSKI,

C.F. PHELPS and J.K. SHEEHAN

Department of Biological Sciences, University of Lancaster, Lancaster LA 1 4YQ (U.K.) (Received December 4th, 1978)

Key words: Dermatan sulfate; Light scattering; Viscometry ; Glycosaminoglycan

Summary

1. Two dermatan sulphate preparations from pig skin, DS-18 and DS-36 with L-iduronic acid and D-glucuronic acid ratios of 90 : 10 and 75 : 25, respectively, were studied by light scattering and viscometry. 2. In 0.15 M NaC1, both preparations yielded high particle weights (224 000 and 322 000, respectively) and viscometry at very low shear also yielded abnormally high intrinsic viscosities (108 and 128 ml/g, respectively). However, the two dermatan sulphate preparations afforded 'monomeric' weight-average molecular weights (30 000 and 25 000) and normal intrinsic viscosities (49 and 35 ml/g) in 0.15 M KCI. Gel chromatography experiments further confirmed that dermatan sulphate chains have a tendency to self-associate in the presence of Na ÷. 3. Dissociation of these 'superaggregates' in NaC1 solutions could be accomplished by: (i) increasing the ionic strength to 0.5 M NaC1; (ii) increasing the shear rate to 10--20 s-'. 4. When these 'superaggregates' were dissociated in 0.5 M NaCI, weight* The preceding papers of this series axe Refs. 3 and 4. ** Present address: D e p a r t m e n t of Physiological Chemistry 2, University of Lund, P.O. Box 750, S-220 07 Lurid, Sweden. Abbreviations: IdUA, L-iduronic acid; GIcUA, D-giucuronic acid; Glycans c o m p o s e d of uxonosylGalNAc-S04 repeats are generally referred to as galactosaminoglycans. A p o l y m e r c o m p o s e d exclusively of GIcUA-GalNAc-SO4 is b e t t e r k n o w n as c h o n d r o i t i n sulphate. In the c o n v e n t i o n a l nomenclature, d e r m a t a n sulphate denotes a P o l y m e r t h a t conta i ns solely IdUA-GalNAc-SO4 units. However, a~chetypal p o l y m e r s are rarely seen and chains c o m p o s e d of b o t h GIcUA-GalNAc-SO4 and IdUAGa]NAc-SO4 repeats may simply be designated as c o p o l y m e r i c galactosaminoglycans. In the present w o r k two prep arations of c o p o l y m e r i c galactosaminoglycans were used, DS-18 and DS-36, which were precip itated at 18% and 36% ethanol, respectively.

180

average particle weights of 32 000 and 41 000 were found for DS-18 and DS-36 respectively. It is suggested that a further level of self-association, probably dimerisation, occurs in 'copolymeric' dermatan sulphates that have a high glucuronic acid content, as previously proposed (Fransson, L.-.~. (1976) Biochim. Biophys. Acta 437,106--115).

Introduction

Dermatan sulphate is found in the extracellular matrix of fibrous connective tissue. In the native form several glycan chains are covalently bound to a protein core to form a proteoglycan [1]. By interaction with collagen, elastin and structural glycoproteins proteodermatan sulphates are considered essential for maintaining the structural integrity of the tissue [2]. However, the molecular mechanisms behind this interaction are poorly understood. Previous studies have shown that dermatan sulphate-chondroitin sulphate 'copolymers' may self-associate [ 3,4 ]. This was demonstrated both by affinity chromatography and by gel chromatography [3]. Self-association was most pronounced among 'copolymers' that contained similar proportions of IdUA and GlcUA. Hydrogen-bond breaking agents abolished the self-association. In a preceding report the 'copolymeric' structure of aggregating and non-aggregating galactosaminoglycans from various tissues was investigated [4]. By degradation of aggregating chains with testicular hyaluronidase, oligosaccharide fragments (decasaccharide or larger) which displayed affinity for a dermatan sulphate ligand were obtained. These oligosaccharides possessed alternating IdUAGalNAc-SO4 and GlcUA-GalNAc-SO4 sequences. Oligosaccharides derived from non-aggregating chains showed no binding and lacked the above-mentioned structural features. In the present study dermatan sulphate aggregates formed under various conditions have been investigated by light scattering and low-shear viscometry. Experimental

Materials. Chondroitin 4-sulphate from beef nasal septum was prepared (by proteolysis) as described [5]. Pig skin dermatan sulphate was obtained after solubilization of the tissue by papain [6]. The galactosaminoglycans were further fractionated by ethanol (18 and 36% fractions were obtained) as calcium salts [6]. The materials were converted to the sodium salts by repeated precipitations with ethanolic sodium acetate. Sepharose and Sephadex gels were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Filters were obtained from Millipore S.A. 67120 Molsheim, France. Chemicals were of analytical grade. Analytical methods. Hexosamine was determined by the Elson and Morgan procedure as described by Antonopoulos et al. [7]. Separations of glucosamine, galactosamine and amino acids were achieved with the use of a Bio-Cal automatic amino acid analyser after hydrolysis (under N2) in 6 M HC1 at 100°C for 20 h. Total hexuronic acid was estimated by the carbazole [8] and orcinol [9] methods. Chromatographic separation of hexuronic acids (IdUA and GlcUA)

181

after acid hydrolysis was performed as described elsewhere [10]. Gel chromatography. Galactosaminoglycans (15 mg) were chromatographed on columns (11 X 1400 mm) of Sepharose CL6B with 0.5 M sodium acetate, pH 7.0, as eluting solvent (rate 10--15 ml/h). Fractions (1--2 ml) were collected and analysed for hexuronic acid [9]. Samples were also chromatographed on columns (12 X 1000 mm) of Sephadex G-100 with 0.20 M NaC1 or 0.20 KC1 as eluting solvents (rate 10 ml/h). Effluents were screened with an LKB Uvicord 3 using the 206 nm channel. Viscometry. Viscosity measurements were performed in a Couette viscometer similar to that described in [11] and modified to the cone and plate principle [12]. The stainless steel conical bob was suspended on a CuPd wire of 20 cm length and 0.0076 cm diameter. The sample volume required to fill the pot with the bob in position was 0.92 ml and the pot was maintained at 21°C with a water-filled brass jacket and a recirculating water bath. Measurements were made at six shear rates, 48.3, 19.3, 7.8, 3.1, 1.2 and 0.47 s -1, the rotation being generated by a kymograph motor. The deflection, 4, was measured using a long optical lever, the null position being obtained by turning a torsion head accurate to 1 min of arc. To reduce zero errors measurements were taken with the rotation of the pot reversed so that a measurement of 24 was obtained at each rotation rate. Readings were obtained at four concentrations between 4 and 20 mg/ml and a value of the intrinsic viscosity was obtained for each of the shear rates. The values obtained were then plotted against shear rate and an extrapolate to zero shear obtained. Light-scattering. Solvents for light-scattering were made up from distilled water with salts as detailed below and a trace of sodium azide and adjusted to pH 7.0 with 2 M NaOH (or KOH). All solvents were filtered through a Buchner funnel with 1.0 ~m pore size. Galactosaminoglycan (sodium salts) stock solutions (usually 20--25 mg/ml) were diluted to yield a series of solutions over the range 1--10 mg/ml which were individually dialysed with stirring against the solvent to achieve Donnan equilibrium. The solutions and solvent blanks were purified from dust by filtering through a Millipore membrane (pore size 0.8/~m; type AAWP} followed by centrifugation at 20 000 X g for 1 h before being pipetted into the lightscattering cells. All light-scattering cells and wide-bore pipettes used were extensively washed and then cleaned in refluxing acetone and housed in a dust-free cabinet in which the cells were loaded. The intensity of light scattered at angles between 30 ° and 150 ° was determined with a Sophica model 42 000 photo-goniodiffusometer (Societ~ Francaise d'Instruments de Controle et d'Analyses, 78 Le Mesuil-Saint-Denis, France) with a light-source of 436 nm wavelength at 23°C. Calibration of the instrument was performed with redestilled AnalaR benzene with a Rayleigh ratio of 4.56 X 106 cm -1, and additional calibration used the polymer solid standard provided with the instrument. The data were evaluated by the reflection correction m e t h o d [13]. Linear least-squares analysis yielded the extrapolates to c = 0 and 0 = 0, the latter values being obtained from the data at 30, 34 and 37.5 ° . The refractive index increments of galactosaminoglycan solutions versus the

182

TABLE I THE REFRACTIVE

INDEX INCREMENTS AT 436 NM

Sample

Solvent *

/"n/Ac ( m l / g )

DS-18 DS-36

0 . 5 M s o d i u m a c e t a t e , p H 7.0 0 . 5 M s o d i u m c h l o r i d e , p H 7.0

0.117 0.119

* V a l u e s w e r e also r e c o r d e d in o t h e r a q u e o u s s o l v e n t s y s t e m s b u t o n l y s m a l l d i f f e r e n c e s in t h e t h i r d decimal place were noted.

solvent was determined with a Brice-Phoenix differential refractometer (Phoenix Division of VirTis, Gardiner, NY 12525, U.S.A.) and are shown in Table I. Results The characteristics of the two dermatan sulphate preparations (DS-18 and DS-36) with respect to uronic acid composition and number-average molecular weight are shown in Table II. DS-18 had Mn = 17 500 and 90% of the uronic acids were L-iduronic acid, whereas DS-36 had Mn = 14 500 and only 75% of the uronic acids were L-iduronic acid. Both preparations were chromatographed on Sepharose CL6B in 0.5 M sodium acetate, pH 7.0, to yield the elution profiles seen in Fig. 1. A control sample of chondroitin 4-sulphate (A), Mn -- 20 000, was eluted as a symmetrical peak which coincided with the major c o m p o n e n t o f DS-18 (B). The relatively GlcUA-rich DS-36 (C) was eluted in a more excluded position. The latter behaviour has been ascribed to self-aggregation o f 'copolymeric' dermatan sulphate chains [ 3,4 ]. A light-scattering study of the t w o dermatan sulphate preparations in both 0.15 M NaC1 and 0.15 M KC1 was conducted (Fig. 2). In 0.15 M NaC1, DS-18 yielded a weight-average particle weight o f 224 000, while the particle (presumably molecular) weight was reduced to 30 000 in 0.15 M KC1 (Fig. 2 and Table III). Similarly, DS-36 had a high particle weight, 322 000, in 0.15 M NaC1 and a low particle (presumably molecular) weight of 25 000 in 0.15 M KC1 (Fig. 2 and Table III). A viscometric study (Fig. 3) yielded intrinsic viscosities extrapolated to zero shear of 108 m u g for DS-18 and 128 ml/g for DS-36 in 0.15 M NaCl. These high values, compared with values of 49 ml/g and 35 ml/g in 0.15 M KC1,

T A B L E II ANALYSES OF DERMATAN

SULPHATE

Analyses

DS-18

DS-36

G a I N (%) L - I d U A (%) * D - G l c U A (%) * GalN/Ser M n (GaiN/Set ×500)

25.2 90 10 35 17 5 0 0

24.8 75 25 29 14 5 0 0

• E x p r e s s e d as the p e r c e n t a g e o f t o t a l u r o n i c acid.

183

-

A

03-

~

02

0.1 4!

~

,

,

,

-~l

l

I

'

I

06_

o 04,~, _

02-

1.2. 0804-

~/, 60

80

100

EFFLUENT VOLUME

(m[)

Fig. 1. Gel c h r o m a t o g r a p h y on Sepharose CL6B of (A) c h o n d t o i t i n sulphate, (B) DS-18 and (C) DS-36 in 0.5 M so dium acetate, pH 7.0, V 0 = 60 ml.

together with the marked shear dependence, confirm the presence of shear
TABLE III PARTICL E WEIGHT OF DERMATAN SULPHATE UNDER V A R IO U S CONDITIONS Sample

Co nditions (all at pH 7.0)

10 -3 X Particle weight

DS-18

0.15 0.30 0.50 1.00 0.15 0.50

M M M M M M

NaCI NaCI NaCI NaCI KCI NaOAc

224 90 32 33 30 37

DS-37

0.15 0.30 0.50 1.00 0.15 0.50

M M M M M M

NaCI NaCI NaCI NaC1 KCI NaOAc

322 43 41 41 25 25

184

300-

/

A

200-

200-

Bf

100-

100.y

!

l

i

i

i

05

10

15

20

2.5

sin 2(0/2) + 200c

sin2(8/2) + 200c

2oot° I 0 0 - ~

I

0.5

200-

D

100-

1!0 1.15 210 sin2(O/2] + 200c

01.5

110 115 2'0 sin2le/2) ÷ 200c

F i g . 2. Z i m m p l o t s o f t h e l i g h t - s c a t t e r i n g d a t a f r o m s o l u t i o n s o f : ( a ) D S - 1 8 a t c o n c e n t r a t i o n s o f 2, 4 , 6, 8 a n d 1 0 m g / m l i n 0 . 1 5 M NAG1, p H 7 . 0 , w i t h a t r a c e o f a z i d e . T h e o p t i c a l c o n s t a n t K = 2 . 2 3 • 1 0 - 7 m l 2 • g2 . c m - 4 . ( b ) D S - 3 6 a t c o n c e n t r a t i o n s o f 1 . 5 , 3, 5, 7 a n d 9 m g / m l i n 0 . 1 5 M N a C I , p H 7 . 0 , w i t h a t r a c e o f a z i d e . T h e o p t i c a l c o n s t a n t K = 2 . 3 5 • 1 0 7 m l 2 • g2 . c m - - 4 . ( c ) D S - 1 8 a t c o n c e n t r a t i o n s o f 1, 2, 3 a n d 5 mg/ml in 0.15 M KCI, pH 7.0, with a trace of azide. The optical constant K = 2.23 • 107 ml 2 • g2 . cm-4. (d) DS-36 at concentrations o f 1, 2, 3, 5 a n d 7 m g / m l i n 0 . 1 5 M K C I , p H 7 . 0 , w i t h a t r a c e o f a z i d e . T h e o p t i c a l c o n s t a n t K = 2 . 3 5 • 1 0 - 7 m l 2 • g2 . c m - 4 . I n a l l c a s e s ( a - - d ) : 4 e x t r a p o l a t e d t o z e r o a n g l e ; o a t 9 0 °"

low particle weights. To investigate the effect of Na ÷ and K ÷ on gel chromatographic behaviour the two dermatan sulphate preparations were analysed on Sephadex G-100 (Fig. 5). Both glycans were more excluded from the gel in the presence o f 0.20 M NaC1 (A and C) as compared with 0.20 M KC1 (B and D). The behaviour of the dermatan sulphates in solution was also studied as a function of ionic strength (see Table III). The particle weights for DS-18 decreased with increasing ionic strength and at I = 0.5 essentially the same value was reached as in 0.15 M KC1 (Table III). However, although the particle

185

', A 1oo-8o-6o~ ~o: 2o:

7~

oois-

-#

., j

B

120 \,

o

o

2 00-t :::

o Q:

8060 40~...e-- •

•

~o : ,~

~-

100-

! 201"1

I

I ~-

I

I 8

I

| 12

I

I

I

16

Sheer (sec -1)

I 20

I

1J "lI 2& L,8

i 05

i 1.0

i 1.5

200 C

Fig. 3. Plots o f intrinsic viscosity ( [ ~ ] ) versus shear rate (co) for ( A ) D S - 1 8 in 0 . 1 5 M NaCI ( o o ) and in 0 . 1 5 M KC1 ( ¢ e ) . ( B ) D S - 3 6 in 0 . 1 5 M NaC1 ( o o ) and in 0 . 1 5 M KCI ( e =). T h e [ ~ ] values at co ~ 0 w e r e D S - 1 8 in 0 . 1 5 M NaCI, 1 0 8 m l / g ; D S - 1 8 in 0 . 1 5 M KCI, 49 ml/g; D S - 3 6 in 0 . 1 5 M NaCI, 1 2 8 ml/g; D S - 3 6 in 0 . 1 5 M KC1, 35 m l / g .

Fig. 4. A c/Rgo p l o t of the light-scattering d a t a f r o m s o l u t i o n s o f D S - 3 6 at c o n c e n t r a t i o n s o f 1, 2, 3, 4 and 5 m g / m l in ( o ) 0 . 1 5 M KC1, (D) 0 . 1 3 5 M K C I / 0 . 0 1 5 M NaCI, (4) 0 . 0 7 5 M KC1/0.075 M NaCl and (u) 0 . 0 1 5 M KC1/0.135 M NaCl and at c o n c e n t r a t i o n s o f 1.5, 3, 5 and 7 m g / m l in (o) 0 . 1 5 M NaC1; all solut i o n s at pH 7 . 0 w i t h a trace o f azide. The optical c o n s t a n t K = 2 . 3 5 • 1 0 7 m l 2 • g 2 . c m - 4 .

weight for DS-36 decreased from 322 000 to 41 000 it did not attain the value of 25 000 found in 0.15 M KC1. Furthermore, the relatively high particle weights obtained for DS-36 in 0.15 M NaC1 were not appreciably altered by raising the temperature to 60°C. Since the chromatographic behaviour of dermatan sulphate in 0.5 M sodium acetate (Fig. 1) indicated that this might be an associative condition for the DS-36 preparation and that some of the DS-18 also exhibited association, lightscattering data were obtained in this solvent (Fig. 6). The shape of the line formed by the extrapolates to zero angle at finite concentrations seems to be indicative o f a dissociating system. At concentrations between 4 and 10 mg/ml a relatively high particle weight could be extrapolated. However, in the range 1--4 mg/ml the extrapolated particle weight was 37 000 (DS-18) and 25 000 (DS-36), respectively.

186

4020-

40-

B

20B

~- 40-

E 300"

-300 o

20. 200-

ooo 40-

o

o

-200

o

- 100

100-

20.

30

40

50

60

70

EFFLUENT VOL.(ml)

8'o

1 2

i 3

1

2

i 3

sin 2 (8/2) + 200c

Fig. 5. G e l c h r o m a t o g r a p h y o n S e p h a d e x G - 1 0 0 o f D S - 1 8 (A a n d B) a n d DS-36 (C a n d D ) in 0 . 2 0 M NaC1, p H 5.5 (A a n d C) a n d 0 . 2 0 M KCI, p H 5.5 (B a n d D). V 0 = 3 9 . 5 m l ; Vt = 123 m l . Fig. 6. Partial Z i m m p l o t s (c/Ro, A; c/R90, o) o f t h e l i g h t - s c a t t e r i n g d a t a f r o m s o l u t i o n s o f (A) D S - 1 8 a n d ( B ) D S - 3 6 at c o n c e n t r a t i o n s o f 1 - - 1 0 m g / m l in 0 . 5 M s o d i u m a c e t a t e , p H 7.0, w i t h a trace o f a z i d e .

Discussion The relationship between molecular weight of the closely related chondroitin sulphate and its elution behaviour upon gel chromatography has been determined by Wasteson [ 14]. In an unfractionated chondroitin sulphate preparation an Mn (osmometry) of 20 800 and an M w (sedimentation velocity, diffusion and sedimentation equilibrium studies) of 25 300 were obtained. The most extensive determination of the molecular weights of dermatan sulphate was made by Tanford et ai. [15]. Sedimentation equilibrium studies in 0.5 M NaC1 yielded Mn, Mw and Mz of 23 000, 27 000 and 41 000, respectively. In the present study, Mn values (end group analysis) of 17 500 and 14 500 (DS-18 and -36, respectively) were obtained. In 0.15 M KC1, the corresponding Mw values (light-scattering) were 30 000 and 25 000. In contrast, light-scattering measurements in 0.15 M NaC1 yielded very high particle weights for the two dermatan sulphates (224 000 and 322 000, respectively). Dissociation of these 'superaggregates' occurred either when the ionic strength was increased from 0.15 to 1.0 M NaC1 or in 0.15 M NaC1 by the application of mild shear rates of 10--20 s -~. In 1.0 M NaC1, the particle size of DS-18 was the same as in 0.15 M KC1. However, DS-36 was still almost twice as large as in 0.15 M KC1 (41 000 vs. 25 000). Clearly, light-scattering and viscometric measurements made at zero or low shear have detected a level of 'superaggregation' which is not readily observed in gel or affinity chromatography [3,4]. However, the DS-36 preparation un-

187 d o u b t e d l y shows a residual self-association when the superaggregation is disrupted. It is always hazardous to speculate on the size of the self-associates formed from data acquired b y extrapolation to zero concentration (as in lightscattering) since this very dilution may perturb the position of the multimerm o n o m e r equilibrium or equilibria. In principle, the association may be examined by t w o extremes o f behaviour. Either nearly all of the molecules may be involved in a small association, e.g. a dimerization, or a small proportion of the molecules may be involved in a large associate. Gel chromatography on Sephadex G-100 (Fig. 5) shows no evidence of large aggregates b u t the whole distribution is more excluded in 0 . 2 M NaCI than in 0.2 M KC1. Furthermore, the viscometric data (Fig. 3B) on DS-36 at high shear rates where 'superaggregation' must be disrupted, shows a similar ratio of intrinsic viscosities between 0.15 M NaC1 and 0.15 M KC1, to the ratio of the light-scattering molecular weights for DS-36 in 1 . 0 M NaC1 (41 000), where 'superaggregation' is disrupted (Table III) compared with 0.15 M KC1 (25 000). Since light-scattering particle sizes are weight averages b u t viscosity sizes are intermediate between number- and weight averages, large aggregates would influence the light-scattering data to a greater extent than the viscosity data. Thus, the results of gel chromatography, viscometry and light-scattering indicate a small associate in which nearly all of the molecules participate. The light-scattering data from DS-36 in 0.5 M sodium acetate has the appearance of a dissociating system with high concentration/low concentration particle weight extrapolate ratio of 2 : 1. If this is indeed a dissociating system, then the dissociation constant KD, calculable from the data in Fig. 6, is 10 -4 M. The self-association of 'copolymeric' dermatan sulphate chains noted by gel and affinity chromatography [3,4] may, therefore, tentatively be assigned to dimerization. However, the mechanism of the 'superaggregation' p r o m o t e d in 0 . 1 5 M NaC1 b u t inhibited b y 0.15 M KC1 is not understood. The present findings have important implications for the structure and function of proteodermatan sulphate macromolecules. These molecules are capable of forming supramolecular aggregates (CSster, Fransson, Nieduszynski, Sheehan and Phelps, unpublished observations) which are sensitive to shear. The self-associating properties of the sidechains of such proteoglycans enable them to form large networks. The finding that n e t w o r k formation could be enhanced by Na ÷ and inhibited b y K÷pro vides a mechanism by which proteoglycans remain monomeric intracellularly, while exposure to the extracellular environment induces multimerization.

Acknowledgements Grants from: The Wellcome Trust (Visiting Scientist Fellowship to L.-/~.F.), the Arthritis and Rheumatism Council, The Nuffield Foundation, The Swedish Medical Research Council (B75-13X-00139), Gustaf V :s 80-£rsfond and 'Greta och Johan Kocks Stiftelser'. We thank Mrs. Birgitta Havsmark for excellent technical assistance and the Department of Chemistry, Lancaster University, for use of a differential refractometer.

188

References 1 Fransson, L.-A. (1970) in Chemistry and Molecular Biology of the Intercellular Matrix (Balasz, E.A., ed.), pp. 823--842, Academic Press, New York 2 Lindahl, U. and H66k, M. (1978) Annu. Rev. Biochem. 4 7 , 3 8 5 - - 4 1 7 3 Fransson, L.-A. (1976) Biochim. Biophys. Acta 4 3 7 , 1 0 6 - - 1 1 5 4 Fransson, L.-A. and C~ister, L. (1979) Biochim. Biophys. Acta 5 6 2 , 1 3 2 - - 1 4 4 5 Fransson, L.-A. and Malmstr6m, A. (1971) Eur. J. Biochem. 18,422---430 6 Fransson, L.-A. (1968) Biochim. Biophys. Acta 1 5 6 , 3 1 1 - - 3 1 6 7 A n t o n o p o u l o s , C.A., Gardell, S., Szirmai, J.A. and de Tyssonsk, E.R. (1961) Biochim. Biophys. Acta 83, 1--15 8 Dische, Z. (1949) J. Biol. Chem. 1 6 7 , 1 8 9 - - 1 9 8 9 Brown, A.H. (1948) Arch. Biochem. 1 1 , 2 6 9 - - 2 7 5 10 Fransson, L.-A., R o d i n , L. and Spach, M.L. (1968) Anal. Biochem. 23, 317--330 11 Ogston, A.G. and Stanier, J.E. (1953) J. Physiol. 1 1 9 , 2 4 4 - - 2 4 6 12 Mooney, M, and Ewaxt, R.H. (1934) Physics 5, 350--354 13 T o m i m a t s u , Y., Vitello, L. and Fong, K. (1968) J. Colloid Interface Sci. 27,573---580 14 Wasteson, A. (1969) Biochlm. Biophys. Acta 1 1 7 , 1 5 2 - - 1 5 4 15 Tanford, C., Marler, E., Jury, E. and Davidson, E.A. (1964) J. Biol. Chem. 239, 4 0 3 4 - - 4 0 4 0