Study of the structure of Shigella flexneri o antigen II. Physicochemical aspect

BIC~CHIMICA ET BIOPHYSICA ACTA

167

BBA 26273 STUDY OF T H E S T R U C T U R E OF S H I G E L L A

FLEXNERI

0 ANTIGEN

II. PHYSICOCHEMICAL ASPECT E. HANNECART-POKORNI, D. DEKEGEL, F. DEPUYDT Institut Pasteur du Brabant, Brussels (Belgium) (Received September Ilth, 1969)

AND J.

DIRKX

SUMMARY Molecules of the purified cell-wall antigen from Shigellaflexneri F6S serotype 5b, are long filaments of variable length and thickness, whereas the unit preparations with s°2o,w = lO.2 S are more homogeneous. The antigen filaments are probably polymerization artifacts of unit molecules which might be the normal biological entities present "in situ". The interpretation of physicochemical parameters leads to a 613-A long, 33-A thick ellipsoid of revolution for the IO.2-S unit. This long cylinder itself is made of io subunits having a molecular weight of 25000 and presenting the same geometry as the IO.2-S unit constituent. The steric arrangement of IO.2-S unit is different from that of the purified antigen which m a y be obtained by a skew association of the units along the axis of the filaments (skewness angle of 45-80°).

INTRODUCTION The somatic antigen of Shigella flexneri F6S serotype 5b is a lipopolysaccharide complex having a molecular weight of IO. lO6-45 • lO6. During the purification process the antigen molecules show a strong tendency to polymerize and their morphology as seen in the electron microscope varies considerably. This phenomenon has been studied b y DIRKX1 using the light scattering technique: the multiple associationdissociation equilibrium depends upon the concentration of the substance. In a previous paper 2 we found the antigen molecules to be highly heterodispersive as revealed b y their sedimentation constant and molecular weight. We suggested that the antigen is built by association of unit macromolecular structures having a sedimentation constant, s°2o,w, of lO.2 S which are themselves built of 1.44-S subunits. I t would be interesting to know the steric arrangement of the chemical groups such as sugars, f a t t y acids, subunits and unit molecules in the lipopolysaccharide complex. We therefore tried to determine different physicochemical parameters of these molecules. The present paper mainly concerns a study of the steric structure of the Io-S particles. Biochim. Biophys. Acta, 2Ol (197o) 167-178

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MATERIALS AND METHODS The extraction, purification and chemical degradation of antigen were made as described previously 2. Ph~vsicochemical measurements Hexosamines were determined b y the m e t h o d of ELSON AND MORGAN3, 4, hexoses b y the quantitative reaction of DISCHE 5 and heptoses by the cysteine-H2SO 4 reaction as modified b y OSBORN6. Citrate ions were detected b y gas c h r o m a t o g r a p h y with F and M research chrom a t o g r a p h model 81o using a Diatoport solid support and a silicon gum rubber S.E.3o at I °'o as liquid phase. Injection temperature was 33 o°, detector temperature, 325 °, column temperature, 12o-2oo ° (I °/rain), and helium flow, 4 ° ml/min. Spectrophotometrical determinations were done with a Beckman DB recording apparatus. Sephadex G-2oo filtration was used for the separation of glucidolipidic antigen molecules from degradation products adsorbed onto them. The Sephadex G-2oo column (2.5 cm × 5 ° cm) was equilibrated and eluted with o.I M NaC1 at a speed of 20 ml/h collecting 5-ml fractions. A Spinco model H electrophoresis apparatus equipped with Raleigh interferometer optics was used for diffusion gradient determinations. Infrared absorption spectra were obtained with a P e r k i n - E l m e r Infracord model 21o instrument after pelleting the antigen in K B r (I:IOO). The r o t a t o r y dispersion curves of glucidolipidic antigen and its degradation products were established with a Bullingham and Stanley-type Polarimetrix 62. The interpretation of the steric structure of all fractions was done according to equations established b y DRUDE ~ and MOFFIT8,10 and MOFFIT AND YOUNG9. The dimensions of molecules were determined from the relation of the axial ratio to the friction ratio by the formula of PERRIN 11. Electron microscope preparations were made by adsorption of glucidolipidic antigen and degradation products suspended in water on carbon-coated grids. After a few minutes the grids were sucked dry with a filter paper and either r o t a t o r y shadowed at small angle (6-1o °) with platinum iridium or negatively stained with / uranyl acetate or a I o/,/oa m m o n i u m m o l y b d a t e solution. a 2 OJo Molecular weight determinations (a) The Mechrolab model 2Ol A pressure osmometer was used for estimations of the small molecular weights according to WILSON et al. 12 using saccharose as a calibration standard with probe No. 5303 at 37 °. W a t e r was used throughout as a solvent. (b) The m e t h o d of Archibald, as described in detail by ELIAS13, was followed using a Kel-F cell of 12 m m 4 ° sector, with an F 43 b o t t o m and a rotation speed of 5563 rev./min. (c) Light-diffusion measurements were done with a Brice Phoenix light scattering photometer using a semi-octagonal I5-ml cell at 546 rap. The molecular weight of glucidolipidic antigen was calculated using the formula of NEUGEBAUER14 for long cylindrical particles. Biochim. Biophys. Acta, 2Ol (197o) i67-178

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(d) Estimation of molecular weight according to the Svedberg procedure 15 was done referring to the following constants : (i) Sedimentation velocity of the endotoxins were performed in a Spinco model E ultracentrifuge. The observed sedimentation coefficients reported in Svedberg units (I S is I . lO -13 cm/sec per unit field) were corrected to values corresponding to solvent having the viscosity and density of water at 20 °. (ii) The diffusion coefficients were calculated by plotting the squares of the second moments against time TM and were corrected to correspond to a temperature of 20 ° in a solvent with the viscosity of water and reported to zero concentration of antigen. D a t a for diffusion coeffÉcients were obtained with a synthetic boundary Felled-Epon cell of 12 m m 3 ° sector in a Spinco ultracentrifuge run at 5000 rev./min. They are reported in units of I - l O -7 cm~/sec. (iii) Partial specific volumes were determined by the picnometer technique described by ELIAS17 at a temperature of 3 °° (~: o.oi °) with 5 vol. Values were reported to zero concentration of antigen. (e) The molecular weights are determined by Schlieren optics centrifugation in density gradients of CsC1 (starting density 1.4o89) for i8 h at 50740 rev./min using the double-sector capillary-type cell of 12 m m with an Epon centerpiece 2 ° sector TM. RESULTS

Purity and homogenicity of products Before applying physicochemical measurements, we tried to determine the degree of purity and homogenicity of glucidolipidic antigen and its degradation products. We stated previously that glucidolipidic antigen was seen under the electron microscope (Fig. I) as threads of different length having a molecular weight of several millions. The nonhomogeneous nature of glucidolipidic antigen was also observed by ultracentrifugation (Fig. 3) and chromatography on Sephadex G-200 and Sepharose 2B. The i0.2-S unit appears homogeneous and spherical under the electron microscope (Fig. 2).

Fig. I. Purified glucidolipidic antigen showing filaments of about 2o0 A thickness. Preparation conically shadowed with platinum-iridium. Magnification, 600oo ×. Fig. 2. Serum albumin-treated purified glucidolipidie antigen showing a complete degradation of the filaments into isolated particles of different shape having a mean diameter of about 2oo A. Preparation conically shadowed with platinum-iridium. Magnification, 6oooo ×.

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E. HANNECART-POKORNIet al.

As shown in Fig. 4, presenting the variation of sedimentation coefficients at different concentrations of some antigen preparations degraded to different extent, the evolution of the parent molecules to their subunits appears clearly. This degradation of the large molecules is shown by the slope of the curve which is approaching zero for the IO.2-S particles, whereas it is rather steep for the undegraded glucidolipidic antigen. The IO.2-S unit obtained by thermic degradation shows one symmetrical peak in the ultracentrifuge (Fig. 5), and has a Gaussian diffusion gradient (Fig. 6). It should be noted, however, that at high temperature some links of sugar molecules are ruptured. We therefore verified its purity by chromatography on Sephadex G-2oo (Fig. 7). This fractionation gave two peaks: one corresponding to the IO.2-S unit,

150

u

uJ 100

X so

_

0

5 CONCN. ( r a g / m l )

5,6

10

Fig. 3. S e d i m e n t a t i o n p a t t e r n of g l u c i d o l i p i d i c a n t i g e n : 5 m g / m l , K e l F I 2 - m m s t a n d a r d cell, 2 ° sector, 2oooo r e v . / m i n , 20 min. Fig. 4. V a r i a t i o n of s e d i m e n t a t i o n c o n s t a n t s w i t h c o n c e n t r a t i o n for a n t i g e n p r e p a r a t i o n s d e g r a d e d to different e x t e n t . Curves i, 2 And 3, curves for three different batches of purified g l u c i d o l i p i d i c a n t i g e n . C u r v e 4, curve for gluci~lolipidic a n t i g e n heated at 95 ° d u r i n g 3 ° mi n. C urve s 5 a n d 6, curves for glucidolipidic a n t i g e n h e a t e d at 12o ° d u r i n g 20 m i n a n d 3 ° m i n , respectively.

99 9C 7( <~

3C

s~ 6

5

4

3

2

1

0

1

2

3

~

5

6 mm

Fig. 5. S e d i m e n t a t i o n p a t t e r n of t h e IO.2-S u n i t : 5 m g / m l , E e l F I 2 - m m s t a n d a r d cell, 2 ° sector, 6 o o o o r e v . / m i n , 2o min. Fig. 6. Diffusion of t h e io.2-S unit: 2 m g / m l , 125 h diffusion at + i °, expressed in galton coordinates.

Biochim. Biophys. Acta, 2Ol (197 o) 167-178

Shigellaflexneri 0

z7z

ANTIGEN. II

300

8 200

10 o <

E,

0 5

0

30 FRACTION5

50

40

,

200

300

400

500

m~

Fig. 7. Filtration p a t t e r n of lO.2 S ( O - - © ) and 9 S ( O - - Q ) through Sephadex G-2oo, 5 ° mg/I ml of o.15 M NaCI. Fig. 8. R o t a t o r y dispersion curves of glucidolipidic antigen and degradation products in phosphate buffer, pH 7.3. 0 - - 0 , glucidolipidic antigen; O - - O , io.2-S unit; A - - A , hydroxylamine polysaccharide; A - - ~ k , acetic acid polysaecharide.

v-

so

200

300

~.00 '

s~ 0

600 '

mu

Fig. 9. R o t a t o r y dispersion curves of 9- and io.2-S fractions.

0--0,

Io.2-S unit; ~ - - ~ ) , 9 S.

Fig. IO. Crude glucidolipidic antigen showing long filaments of variable thickness (about 80-330 ~) and patches. The t h i n n e s t p a r t s of the filaments also show the greatest c o n t r a s t and seem to be the least permeable to the stain. U r a n y l acetate negatively c o n t r a s t e d preparation. Magnification 120 ooo x .

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the other to a mixture of free sugar molecules (molecular weight about 2oo) having a chemical composition identical to those found in glucidolipidic antigen or the IO.2-S unit. Together with the sugar molecules we found by gas chromatography citrate anions adsorbed onto the IO.2-S particles during its preparation. The total amount of free sugar isolated by chromatography m a y be as high as IO % of the antigen weight, indicating a rather severe damage. We studied the infrared spectra of different cleavage products obtained after heat treatment in order to determine any increase in carboxylic radicals, the presence of which was indicated by electrotitration. The low resolution of the apparatus used for these measurements did not permit any clear interpretation of the observed differences in infrared spectra.

Degradation products of the antigen The polymerization of the antigen observed during its purification is probably an artifact, the IO.2-S particle being the real constituent found in the bacterial cell wall. This io.2-S unit structure which is obtained through different degradation procedures is itself made up of smaller subunits linked together by chemical bonds of the same nature as those binding the unit particles of glucidolipidic antigen. The conformation of the degrading agent is important for the degradation process. The subunit seems to be devoid of biological properties characteristic of the IO.2-S unit. Up to the present time, we were unable to isolate and purify the 1.44-S fraction in the absence of sodium deoxycholate. Through an acetic acid hydrolysis of a IO.2-S fraction, we obtained a polysaccharide having a molecular weight of 9000. This value is smaller than the molecular weight of the subunit (24ooo), 15 % of which are lipid constituents, leaving a polysaccharide having at least a molecular weight of 2100o. The fraction obtained through hydroxylamine hydrolysis has a molecular weight of 90000 which again is quite different from the value obtained for the subunit fraction. This hydrolysis product is probably made up of several subunits which are consequently not completely devoid of lipids. We studied some of the physicochemical parameters of different degradation products (Table I) and came to the conclusion that the io.2-S unit structure is made of lO-12 subunits. Table I I shows the calculated dimensions of length, diameter and thickness of either a long or flat revolutionnary ellipsoid for several degradation products studied. Apparently both models seem to be possible for the unit particle and eventually only an electron microscopical study m a y decide which model will best fit the value obtained b y rotatory dispersion study. As for the subunit and the polysaccharide obtained b y acetic acid hydrolysis, quite evidently a flat molecule having a calculated thickness of 2 A has to be eliminated. The same conclusion holds true for the hydroxylamine polysaccharide showing a thickness of 5 A. Thus for the subunit acetic acid polysaccharide and hydroxylamine polysaccharide, only the elongated ellipsoid model seems to be the most probable.

Rotatory dispersion The rotatory dispersion of glucidolipidic antigen and its degradation products were also examined. Table I I I gives the dispersion parameters for different substances, expressed per mean weight of residual sugar molecules. This mode of expression is

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ANTIGEN.

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II

TABLE I PHYSICOCHEMICAL

PARAMETERS

Fraction

Methods

Unit

Sedimentation velocity lO.2 Diffusion Picnolnetry Svedberg Gradient density Light scattering

Subunit

OF

DIFFERENT

GLUCIDOLIPIDIC

sO20,w

S e d i m e n t a t i o n velocity Diffusion Picnometry Svedberg

ANTIGEN

DO20,w ~

fo/f

2.534

o.516 o.518

FRACTIONS

P*

p **

18.52

26.15

tool. wt.

o. 684 31 o ooo 25 ° ooo 252 ooo

1.44

o.381 0.388

4-375

37.88

66.51

o.68 25 4 °0

Hydroxylamine S e d i m e n t a t i o n velocity polysaccharide Archibald

3.8

Acetic Sedimentation velocity polysaccharide Diffusion Picnometry Svedberg Archibald

0.88

0.453

25.51

39.9 ° 84 ooo

0.428 0.430

6.895

28.74

47.2o

o.66 9 126 8 2oo

* P, axial ratio for cylindrical molecules. p, axial ratio for flat molecules.

* *

TABLE II CALCULATED DIMENSIONS

OF D I F F E R E N T

Fraction

GLUCIDOLIPIDIC

FRACTIONS

Long ellipsoid of revolution

Flat ellipsoid of revolution

Diameter

Length

Diameter

Length

33 ii 19 9

613 428 49o 251

26o 153 193 97

IO 2 5 2

(d)

Unit, lO.2 S S u b u n i t , 1. 5 S H y d r o x y l a m i n e polysaccharide, 3.8 S Acetic acid polysaccharide, I S

ANTIGEN

(d)

(A)

(A)

TABLE III ROTATORY DISPERSION OF GLUCIDOLIPIDIC ANTIGEN AND ITS DEGRADATION PRODUCTS Since the s u b u n i t could not be kept as such in the absence of s o d i u m deoxycholate, which has a high r o t a t o r y dispersion b y itself, no valuable m e a s u r e m e n t s were o b t a i n e d in this case.

Fraction

Dispersion formula

~o

ao

bo

Glucidolipidic antigen, 17o S Unit, lO.2 S Fraction, 9 S H y d r o x y l a m i n e polysaccharide, 3.8 S Acetic acid polysaccharide, i S

MOFFIT--YOUNG9 MOFFIT--YOUNG9 MOFFIT--YOUNG9 DRUDE 7 DRUDE 7

152 152 152 152 152

+619 + 109 + 527 +583 + 77 °

--7OO + 791 -- 624 O o

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E. HANNECART-POKORNI ct at.

quite arbitrary, since no account is taken of the rotatory dispersion introduced by certain fatty acids such as fl-hydroxymyristic acid. To us, however, this seems to be the only means of comparison between the different subunits, the lipid fraction containing optically active compounds amounting to about 15 % (w/w) in the intact glucidolipidic antigen molecule. Although the data of Table I I I do not permit an)" definite conclusions about the steric structure of glucidolipidic antigen, we observed the following: (i) The polysaccharides of glucidolipidic antigen follow the simple DRUDE~ equation, whether they are obtained through acetic acid hydrolysis or by a more gentle hydroxylamine hydrolysis. This would indicate that they have no appreciable secondary structure, which agrees with the fact that these molecules are already highly degraded and rid of their lipid components. (ii) The other fractions, glucidolipidic antigen or the compounds obtained by thermic degradation, follow the equation of MOFFIT--YouNG9, with an important b0 value. This finding would point to a rather elaborated secondary structure for these substances. Rather surprising and more difficult to explain is the change of sign in b0 when passing from glucidolipidic antigen to the io.2-S unit and the new change for the 9-S fraction. Whatever the explanation of these changes is, it remains certain that the 9-S and the IO.2-S compounds, obtained under nonbiological circumstances are rather artificial. The secondary structure seems to be established spontaneously during the preparation of the fractions but remains inapparent in the polysaccharides derived from them. The lipids intimately linked to the polysaccharides m a y therefore be responsible for the secondary structure of the lipopolysaccharide molecules. (iii) The parameters a 0 and b0 are of the same order as those found for proteins, but the value of ~.0 is far lower than the value of 212 m/, usually admitted for protein molecules. This in itself is not astonishing since the electronic transitions between chemical radicals responsible for the rotatory dispersion of both types of substances are quite different. The value of 152 m# obtained for different glucidolipidic antigen preparations best fits the dispersion equation on the coordinates suitably modified according to the type of equation used. No definite conclusions can be drawn about the secondary structure of the lipopolysaccharide fractions from the fragmentary results of rotatory dispersion determinations. These measurements only reflect the existence of a secondary structure which is absent in the polysaccharides. The reality of secondary structure is also demonstrated by tile fact that the rotatory dispersion of glucidolipidic antigen and IO.2-S preparations varies reversibly when heated to 87 °, whereas polysaccharides do not show any change in these circumstances.

Electron microscopy Direct observations in the electron microscope of different fractions were made on conically shadowed or negatively stained preparations. The degrading influence of serum albumin on purified glucidolipidic antigen is clearly demonstrated in Figs. I and 3 obtained after conical shadowing. The IO.2-S units thus obtained appear essentially as globular particles with a mean diameter of 250 A which is comparable to the mean diameter of glucidolipidic antigen filaments. Glucidolipidic antigen submitted to detergents giving also IO.2-S particles leads to pictures quite identical to Fig. 3. :

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Quite obviously the shadowing technique is not adequate to detect fine differences in ultrastructure of I0.2-S units. We therefore applied negative staining. After purification of glucidolipidic antigen, a more homogeneous fraction mostly made of long filaments (Figs. I I and 12) is obtained. Differences in thickness along the filaments are observed. The thinnest parts seem to be less permeable to the contrasting agent. This may, however, be an artifact due to the greater dispersion of electrons b y the uranyl acetate stain accumulated around the filaments, resulting in a local increase in contrast observable at high resolution.

Fig. I I. Purified glucidolipidic antigen showing essentially long filaments of variable length and thickness (about 5o-12o A). Again the thinnest parts (arrows) of the filaments seem to be the most contrasting. Uranyl acetate negatively contrasted preparation. Magnification, 12o0o0 ×. Fig. 12. Purified glucidolipidic antigen in the presence of 0.04% poxesmol showing about 8o-& thick, i5o-6oo-]k long rod-like structures. This preparation corresponds to 20 S. Uranyl acetate negatively contrasted preparation. Magnification, i2o ooo ×.

The effect of the detergent polyoxyethylenesorbitmonolaurate at a concentration of 0.04 % giving a fraction of 20 S is clearly demonstrated in Fig. 12 which shows a homogeneous preparation of rod-like structures. RIBI et al. 19 also obtained such structures after treatment of Salmonella enteritidis endotoxin with sodium deoxycholate and dialysis. A sodium dodecylsulphate-(o. 4 %)-treated purified glucidolipidic antigen giving a IO.2-S fraction shows a network made of very thin (4° A) long filaments together with globular particles of different shapes which m a y correspond to rolled up thin filaments. Due to the greater resolution obtainable by the negative contrast technique the very thin rod-like structures and filaments now become clearly Biochim. Biophys. Acta, 2Ol (197 o) 167-178

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visible. The observed dimensions correspond rather well with the calculated values indicated in Table II. DISCUSSION AND CONCLUSION

Purified glucidolipidic antigen molecules are rather heterogeneous long filaments having variable thickness whereas io-S unit structure preparations tend to be more homogeneous as shown by ultracentrifugation, molecular diffusion, Sephadex chromatography and electron microscopy. It is important to know what is the morphology of the IO.2-S units and how, through a process of polymerization, they may eventually give rise to the long glucidolipidic antigen filaments, seen in the electron microscope. The IO.2-S constituent has a molecular weight of 25oooo-3ooooo compatible only with a certain degree of asymmetry. The axial ratio resulting from this molecular asymmetry depends upon the chosen model, either a long (cylindrical) ellipsoid of revolution (613 A long by 33/k diameter) or a flat disk-like ellipsoid (26o A diameter by IO/k thickness). It is difficult to determine clearly which of both models should be retained but quite evidently the polymerization of Io.2-S units must fulfill certain requirements: (i) For a degree of polymerization of IOO, giving a molecular weight of about 25" lO8 the filament must show a high axial ratio (3o-2oo). (ii) The polymerization model must account for the differences in thickness along the glucidolipidic antigen filament as observed in the electron microscope (diameter values of 5o-3oo )~). It is difficult to imagine how flat disk-like units might associate closely so as to fulfill these requirements. Long ellipsoid of revolution molecules, however, may easily line up to form long filaments by a skew association along the filament axis (Fig. 14). The length of

!

I

C C C C

\ Fig. 13. Purified glucidolipidic a n t i g e n in t h e presence of 0.4% s o d i u m d o d e c y l s u l p h a t e s h o w i n g a f u r t h e r d e g r a d a t i o n r e s u l t i n g in t h i n (4 ° ~) f i l a m e n t s a n d particles of no definite s h a p e (arrows) p o s s i b l y c o r r e s p o n d i n g to p a r t i a l l y rolled u p filaments. U r a n y l a c e t a t e n e g a t i v e l y c o n t r a s t e d p r e p a r a t i o n . Magnification, 120000 × . Fig. 14 . Skew a s s o c i a t i o n of e l o n g a t e d ellipsoids of r e v o l u t i o n a l o n g t h e axis of t h e filament. a, half of t h e g r e a t axis of t h e ellipsoid; b, half of t h e s m a l l axis of t h e ellipsoid; 6, angle of t h e s m a l l axis of t h e ellipsoid a n d t h e axis of t h e filament.

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T A B L E IV CALCULATED D I M E N S I O N S OF A STACK OF IOO u n i t

IO.2-S

Angle of skewness (degrees)

Apparent diameter Apparent length (~t) (•)

45 5° 55 60 65 7° 75 8o 85

311 283 252 22o 186 15o 114 76 38

4 5 6 7 8 IO 13 20 4°

95 ° 445 lO2 ooo 280 23 ° 5 °0 15o 260

M O L E CU L E S

Axial ratio (A) 16 19 24 31 44 68 119 264 lO57

the filament and its local thickness will only depend upon the local degree of inclination of the unit molecule axis and the filament axis. Unit molecules having a length 2a and a diameter 2b, forming an angle a with the filaments axis, the apparent filament thickness will be 1 = 2a cosa and the total filament length L = 2bn/cosa, n being the degree of polymerization. Table IV shows the values of l and L and the axial ratios of the polymer for different angles of skewness. The values agree rather well for inclination angles of 45-80 ° with what is found by electron microscopy and moreover a possible local variation of the inclination m a y well account for the differences in thickness observed. This polymerization model is only adequate for unit molecular structures having a long cylinder morphology and does not hold for flat molecules for which it is also difficult to imagine a polymerization process giving continuous thickness variations of 5o-3oo ft. Moreover the smallest dimension (12 A) of the flat disk equals about the dimension of the constituent molecules (e.g. 8 A for glucose 19) but is therefore incompatible with a secondary steric arrangement as shown b y rotatory dispersion. It should be emphasized that this polymerization model presupposes that most of the polymerization links are more or less mobile and present in the middle part of the unit molecule. ACKNOWLEDGEMENTS

We thank Professor J. Beumer for his advice and encouragement during this work and Mrs. De Vuyst-Vanheule for her technical skill in electron microscopy. REFERENCES I J. DIRKX, Contribution ~ l't2tude de la Fixation des Bactdriophages sur les Bactdries Sensibles, A c t a Medica Belgica, 1963, p. 116. 2 E. HANNECART-POKORNI, D. DEKEGEL, F. DEPUYDT AND J. DIRKX, Biochim. Biophys. Acta, 2Ol (197 ° ) 155. 3 L. A. ELSON AND W. T. M O R G A N , Biochem, J., 27 (1933) 1824. 4 L. A. ELSON AND W. T. MORGAN, Biochem, J., 28 (1934) 988. 5 Z. DlSCHE, Microchemie, 7 (1929) 37. 6 M. J. OSBORN, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 49.

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P. DRUDE, Lehrbuch der Optik, Hirzel, Leipzig, 19oo. W. MOFFIT, J . Chem. Phys., 25 (1956) 467 . W. MOFFIT AND J. T. YOUNG, Proc. Natl. Acad. Sci. U.S., 42 (1956) 596. W. MOFFIT, Proc. Natl. Acad. Sci. U.S., 42 (1956) I. F. J. PERRIN, J. Phys. Radium, 7 (1936) i. A. WILSON, L. BINI AND I~. HOFSTAOER, Anal. Chem., 33 (1961) 135. H. G. ELIAS, Mdthodes d'Ultracentrifugation Analytique, 3rd ed., B e c k m a n I n s t r u m e n t s I n t e r national S.A., Gen~ve, 1969, p. 16o. T. NEUGEBAUER, Ann. Phys., 42 (1943) 509 • TH. SVEDBERG AND K. O. PEDERSON, The Ultracentrifuge, Oxford Univ. Press, New York, 194o, p. 4 o. H. K. SCHACHMAN,Methods in Enzymology, Vol. 4, Academic Press, New York, 1957, p. 32. H. G. ELIAS, Theory and Application of Ultracentrifugal Techniques, B e c k m a n I n s t r u m e n t s GMBH, Munich, 1964, p. 89. J. DIRKX, Analytical Density Gradient Ultracentrifugation, B e c k m a n I n s t r u m e n t s GMBH, Munich, 1964, p. 19. E. RIBI, R. L. ANACKER, R. BROWN, W. T. HASKINS, B. MALGREN, N. C. MILNER AND J. A. RUDBACH, J. Bacteriol., 92 (1966) 1493.

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