THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255. No. 3, Issue of February IO. pp 1221-1226, 1980 Printed m 1IS.A.

The Dispersion of Gram-negative Lipopolysaccharide by Deoxycholate SUBUNIT MOLECULAR WEIGHT*

(Received for publication, May 3, 1979)

Joseph W. Shands, Jr., and Paul W. Chun From the Departments of Medicine a n d Biochemistry, University of Florida College of Medicine, Gainesuille, Florida 32610

The lipopolysaccharide (LPS) was isolated from three strains of Salmonella typhimurium, a “smooth” strain, STM 7, the Ra mutant, TV 119, and the Re mutant, SL 1102. The effect of depletion of divalent cations on structure and the effect of deoxycholate on hydrody- namic behavior were studied. The results confirmed previous work by others that divalent cations and hy- drophobic forces are important factors influencing LPS size and morphology. The binding of deoxycholate to LPS was measured. When the weight average molecu- lar weights of the deoxycholate-dissociated LPS were determined by sedimentation equilibrium and cor- rected for bound deoxycholate, the values 5,555,10,607, and 15,592, respectively, for Re, Ra, and “smooth” LPS were in good agreement with calculated formula weights. Although others have suggested that the basic LPS subunit is a trimer, our results suggest that it exists in the dimeric form.

Lipopolysaccharides are found in the outer or L membrane of Gram-negative bacteria (1). They can be extracted, isolated, and resuspended in aqueous solvents to form colloidal solu- tions or unstable suspensions. LPS’ has self-aggregating prop- erties which result in large, polydisperse molecular forms. LPS isolated from “smooth” strains of bacteria has a molec- ular weight (M,) which ranges from 5 X lo5 to 20 X lo8 (2). The molecular weight of LPS from deep rough mutants is even greater.

LPS from smooth and from rough strains may be dispersed by detergents such as sodium dodecyl sulfate (3-5) and sur- factants such as Triton X-100 (6) or sodium deoxycholate (2, 7 , 8). Upon removal of excess surfactant by dialysis, a more homogeneous population of particles with a mean molecular weight of about 5 X lo5 to 1 X lo6 forms (2). Such observations suggest that hydrophobic interactions between subunits of LPS are important determinants of particle size.

Gram-negative lipopolysaccharides also have a high content of the cations, calcium and magnesium. Olins and Warner (4) and Galanos (9) have demonstrated that cations play an important role in the aggregation of LPS. The removal of the divalent cations (Ca2‘ or Mg”) reduces the size of LPS particles, and the readdition of divalent cations greatly en- larges particle size.

A number of attempts have been made to determine the

* This work was supported by Public Health Service Grant AI 07257 from the National Institute of Allergy and Infectious Diseases and in part by National Science Foundation PCM 76-04367. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: LPS, lipopolysaccharides; STM 7, a smooth strain of Salmonella typhimurium.

size of a monomer of LPS by modifying the hydrophobic and ionic interactions which join its subunits. A comparison of the results of these studies suffers from the disadvantage that the investigators studied LPS from different species and genera of bacteria by different techniques. Nevertheless, the discrep- ancies are of sufficient magnitude to raise doubts about some of the values found. Ribi et al. (2) dissociated LPS from Escherichia coli and Salmonella enteritidis with 2% deoxy- cholate and found a subunit with a molecular weight of approximately 20,000. Hannecart-Pokorni et al. ( 7 ) reported a similar subunit molecular weight for deoxycholate-dissoci- ated LPS extracted from Salmonella and from Shigella. On the other hand, Olins and Warner ( 4 ) dissociated the LPS from Azotobacter vinelandii with sodium dodecyl sulfate and EDTA and reported a particle weight of 65,000. McIntire et al. (8) dissociated E. coli LPS with 0.15% deoxycholate and found a subunit with a molecular weight of 118,000. The lipopolysaccharides of rough mutants have also been studied. Weiser and Rothfield (6) dissociated the LPS of an Rc mutant of Salmonella typhimurium in 0.1% Triton X-100 and found a subunit of 29,000 daltons. The same LPS when acetylated and dissolved in benzene had a subunit molecular weight of 10,300 (10). Molecular weight values ranging from 5,000 (11) to 10,000 ( 7 ) have been reported for LPS from Re mutants of Enterobacteriaceae.

We have re-examined the dissociation of LPS resulting from the removal of divalent cations and the addition of the surfac- tant, sodium deoxycholate. The degree of binding of deoxy- cholate to LPS has been measured, and the molecular weight of the monomer determined by sedimentation equilibrium.

MATERIALS AND METHODS

Bacteria-S. typhimurium strain 7 (STM 7) is a smooth, mouse- virulent strain originally obtained from Dr. M. Henberg, University of Hawaii. TV 119 and SL 1102 are Ra and Re mutants, respectively, of S. typhimurium LT2. Both strains were obtained from Dr. B. A. D. Stocker, Stanford University. All bacteria were maintained as lyoph- ilized stocks and were grown in batch cultures in M9 minimal medium (12) supplemented with 0.1% casamino acids (Difco, Detroit, MI) and 0.1% tryptophan. The bacteria were killed by the addition of formalin to a 1% concentration, harvested by centrifugation, and washed with distilled water.

Extraction of Endotoxin (LPq-Endotoxin was extracted from STM 7 by the phenol/water procedure (13). The LPS contained approximately 8% ribonucleic acid as determined by the orcinol method. This contaminant was removed by digestion with ribonucle- ase and dialysis followed by re-extraction with phenol/water. Small molecular weight contaminants were removed by chromatography on Sephadex G-200. Lipopolysaccharide was extracted from TV 119 and SL 1102 by the phenol/chloroform/petroleum ether method of Gal- anos et aL. (14). Analysis by UV spectrum revealed that all of the preparations of LPS were free of or contained less than 1% contami- nating RNA, and all were biologically active as measured by mouse lethality and B lymphocyte mitogen assays. Mouse LDm values were about 250 pg for STM 7 and TV 119 LPS and about 800 pg for SL 1102. The optimal concentrations in mitogen assays varied from 10 to

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1222 Dispersion of Lipopolysaccharide by Deoxycholate

FIG. 1. Chemical structure of LPS from S. typhimurium. GlcNAc, N-ac- etylglucosamine; P, phosphate; KDO, 2- keto-3-deoxyoctulosonic acid; P-ETN, phosphorylethanolamine; HEP, heptose; GLC, glucose; GAL, galactose; RHA, rhamnose; MAN, mannose; ABE, abe- quose; O-AC, 0-acetyl. Wavy lines sig- nify fatty acids (from Refs. 15 and 16).

b P

Mr -2ooo- *

-LIPID A- M

-Mr -9OO--. . e

t Re LPS (SL 1102) - Mr 2900

h4r -I800 CORE FOLYSACCHARIDE -

30 pg/ml. Fig. 1 illustrates the chemical structure of these lipopoly- saccharides.

All of the LPS preparations were extracted in the dry state with chloroform/methanol (2:l) to remove noncovalently linked phospho- lipids.

Removal of Divalent Cations-LPS was dissolved in 0.05 M Tris- (hydroxymethyl)aminomethane (Tris, Sigma, St. Louis, MO) buffer, pH 7.0, containing 10.’ M (ethylenedinitrilo)tetraacetic acid (EDTA, Mallinckrodt, St. Louis, MO). The pH was dropped to 4.0 by the addition of HCl and then neutralized with NaOH within 2 min. The LPS was then dialyzed against daily changes of lo-’ M EDTA in 0.05 M Tris, pH 7.0, for 3 days. The material was then dialyzed against distilled water and lyophilized. The calcium and magnesium contents of LPS were determined by the method of Savory et al. (17).

Binding of Deoxycholate to LPS-The binding of [carboxyl- ‘Cldeoxycholate (specific activity 7.4 mCi/mmol, Mallinckrodt, St. Louis, MO) to LPS was determined by equilibrium dialysis. [“C]Deoxycholate was diluted with nonradioactive deoxycholate in 0.1 M Tris buffer, pH 8.0. LPS (3 mg/ml) was dissolved in and dialyzed against deoxycholate in equilibrium dialysis cells. The concentrations of deoxycholate ranged from 0.15% to 2.4%. Samples were taken from both sides of the cell at 24 h, 48 h, and 72 h and were counted in a scintillation spectrometer. Intrinsically ‘C-labeled STM 7 LPS was dissolved in and dialyzed against 0.3% deoxycholate t,o determine whether dissociated subunits crossed the dialysis membrane. They did not. Similarly, after equilibrium dialysis of SL 1102 LPS in 0.3% deoxycholate, samples from both sides of the membrane were ana- lyzed for 2-keto-3-deoxyoctulosonic acid (18). 2-Keto-3-deoxyoctulo- sonic acid could not be reliably detected in that side of the chamber to which no LPS was added. The results showed that probably no LPS crossed the membrane. At most, it is possible that 5% of the LPS crossed the membrane.

The Partial Specific Volume of LPS-The va.,p of LPS was cal- culated as follows. LPS was dried over phosphorus pentoxide and the dried product was weighed and dissolved in 0.3% deoxycholate in 0.1 M Tris buffer, pH 8.0. Solvent was added to give a concentration of LPS of 0.3% (3 mg/ml). The va,,,, was determined by weight of the solvent and of the solute plus solvent in a 5-ml pyknometer. A minimum of five individual weights varying by no more than 0.004% was used for the calculations of v. The data from the deoxycholate- LPS binding studies were then used to calculate +‘, the partial specific volume of anhydrous LPS plus bound deoxycholate. This was done by the formula

1 (d - d,‘) wt LPS + wt deoxycholate bound (a$‘)

where d,,’ is the density of the solvent minus the density of that amount of deoxycholate bound to the LPS, and d is the density of the solution.

The rationale for this approach to the determination of p stems from the potential errors that may be made in multicomponent systems with interacting solute and solvent. Casassa and Eisenberg (19) (discussed by Woods et al., 20) have shown that this error can be rendered negligible by dialysis of the anhydrous solute against the solvent and determination of p using the density of the retentate and dialysate and concentration determined by optical density. We were

n=6 -Mr -4000 - - 0 SIDE CHAIN -

Ra LPSfTV 119) - Mr 4700

Complete LPS(STM 7)- Mr 0700

unable to do this since we could not redetermine the concentration of LPS in the solvent. Therefore, we have calculated +’ of the complex of LPS and deoxycholate by entering into the calculation the amount of deoxycholate bound to LPS. The validity of this procedure rests on the assumption that the p of deoxycholate does not change after binding to LPS. This assumption is probably correct since the P of deoxycholate is the same at concentrations below and above the critical micellar concentration (21). To ensure the accuracy of our measurements, p was determined simultaneously on bovine serum albumin. Relative viscosities were determined by an Ostwald capillary viscometer.

Sedimentation Equilibrium Measurements-Sedimentation equi- librium experiments were performed in a Spinco/Beckman model E analytical ultracentrifuge equipped with RTIC unit. Runs to deter- mine the concentration dependence of the molecular weight were made at 25’C, using a Yphantis six-channel centerpiece made from carbon-filled epoxy resin, at an initial concentration of approximately 0.1 g/dl, running three samples simultaneously. The total sample volume in the cell was 0.11 ml, giving a column height of 3 mm.

The calculation of the molecular weight distribution was based on the Yphantis (22) and Richards and Schachman (23) manipulation using computer programs which can be modified for use with an Amdahl470/0-2 unit (modified IBM 360/1800, University of Florida’s NERDC computing facilities), plotting In J or In C as a function of X’. The weight average molecular weight of the LPS. deoxycholate complex was calculated from the slope of a plot of In C versus XV, based on the expression:

zfr = 2RT dln C

(1 - va’p,w’ d(X’)

where (1 - vo) is the buoyancy factor and w is the angular velocity, using the partial specific volumes previously determined by pykno- metric measurements. The In C versus X’ data were fitted to a least square polynomial and the values of dln C/d(X’) were calculated by a modification of the sliding five-point least quadratic treatment of Yphantis (22). The molecular weights of the anhydrous LPS were derived by correcting for the amount of deoxycholate bound to LPS.

Electron Microscopy-Samples of LPS were stained positively with uranyl acetate or negatively with vanatadomolybdate as de- scribed previously (24). The grids were examined and photographed in a Siemens 1A electron microscope.

RESULTS

Removal of Divalent Cations-The levels of Ca2+ and Mg” found in the LPS preparations before and after EDTA treat- ment are shown in Table I. Removal of divalent cations grossly affected the interaction of the LPS with water. The complete LPS became quite water-soluble and the Re LPS made finer dispersions. The morphological consequences of EDTA treatment are shown in Fig. 2. The ribbon-like and the trilaminar structures of STM 7 LPS seen in Fig. 2A were reduced to much smaller particles after EDTA treatment (Fig. 2B). The vesicular structures of SL 1102 LPS shown nega- tively stained with vanatadomolybdate (Fig. 2C) and posi-

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Dispersion of Lipopolysaccharide by Deoxycholate 1223

tively stained with uranyl acetate (Fig. 2 0 ) were transformed hydrodynamic behavior of LPS in the ultracentrifuge. AS by EDTA to the ribbon-like or trilaminar structures seen in shown in Table 11, the sedimentation coefficient of the deox- Fig. 2E and 2F. The LPS of TV 119 (not shown) was ribbon- ycholate-dissociated STM 7 LPS was slightly reduced, and a like before treatment. These ribbons were made shorter by fast and a slow peak formed by the LPS of TV 119 were EDTA treatment, but there was no qualitative change in LPS resolved into one slow peak after the removal of Ca2+ and morphology. The removal of divalent cations did affect the Mg".

TABLE I Calcium and magnesium concentrations in LPS TABLE I1

Sedimentation coefficients of LPS in deoxycholate before and after Caz+ and Mg2* depletion Per cent hy weight

Ca" Mg"' Un- EDTA- Un- EDTA-

treated treated treated treated

LPS s:*,.. in 0.3% deoxycholate LPS (4 mg/ml)

Ilntreated EDTA-treated

STM 7 0.69 0.01 0.21 0.008 STM 7 4.2 3.2 TV 119 1.56 0.02 0.38 0.004 TV 119 10.4 SL 1102 0.50 0.03 0.62 0.000 2.1 2.4

4

FIG. 2. Electron micrographs of LPS before and after treatment with EDTA. A, STM 7 LPS stained positively with uranyl acetate: X 98,000. B, STM 7 LPS treated with EDTA and stained with uranyl acetate: X 98,000. C, SL 1102 LPS stained negatively with vanatadomolybdate: X 98,000. D, SL 1102 LPS stained positively with uranyl acetate: X 98,000. E, SL 1102 LPS treated with EDTA stained negatively with vanatadomolybdate: x 98,000. F, SL 1102 LPS treated with EDTA stained positively with uranyl acetate: X 98,000.

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1224 Dispersion of Lipopolysaccharide by Deoxycholate

FIG. 3. Schlieren patterns of EDTA-treated STM 7 LPS (3 mg/ml) in different concentrations of deoxycholate in 0.1 M Tris, pH 8.0. A (upper), 0% deoxycholate; (lower), 0.075% deoxycho- late; B (upper), 0.15% deoxycholate; (lower), 0.3% deoxycholate.

4

3 3

$ cn 2

I

I I .o 20 3.0 4.0 5.0

CONCENTRATION OF LPS IN mg / ml

LPS in deoxycholate from STM 7 (M), TV 119 (M), FIG. 4. Plot of s~ , , ,~ versus concentration of EDTA-treated

and SL 1102 H.

V# FIG. 5. Reciprocal plot of binding of deoxycholate to LPS

when r = micromolar concentration of deoxycholate bound per mg of LPS and c = micromolar concentration unbound. M, STM 7; m, TV 119 H, SL 1102.

Sedimentation Coefficients of LPS in Deoxycholate-Fig. 3 shows the effect of increasing concentrations of deoxycholate on the sedimentation coefficient of LPS from STM 7 in 0.1 M Tris buffer, pH 8.0. Each increase in deoxycholate concen- tration from 0 to 0.3% resulted in a decrease in the S ~ O . ~ ~ , , ~ .

1792 - A

1318 -

0844 - 0

5 0 371 -

-0861 4 2 W 42461 42654 42S47 43.04043253 431)s 43619 43812 4 4 m 44d98

X 2

2.140

1.633

1.126

0

z 0610

0111

3

.i -0.396 1 /

l/

FIG. 6A-C.

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Dispersion of Lipopolysaccharide by Deoxycholate 1225

FIG. 6. Molecular weight distribution of LPS as a function of radial distance. A, In C uersus X' plot of STM 7 LPS in 0.3% deoxycholate in 0.1 M Tris, pH 8.0. Concentration of LPS, 0.75 mg/ mi; speed, 24,630 rpm. Open circle is experimental point coincident with theoretical point based on a computer plot. Solid circles are experimentally determined points. B, In C versus X' plot of TV 119 LPS in 0.3% deoxycholate in 0.1 M Tris, pH 8.0. Concentration of LPS, 0.75 mg/ml; speed, 24,630 rpm. Open circles are experimental points coincident with theoretical points based on a computer plot. Solid circles are experimentally determined points. C, In C versus X2 plot of SL 1102 LPS in 0.3% deoxycholate in 0.1 M Tris, pH 8.0. Concentration of LPS, 0.75 mg/ml; speed, 35,600 rpm. Open circles are experimental points coincident with theoretical points based on a computer plot. Solid circles are experimentally determined points. D, schlieren pattern of SL 1102 LPS in 0.3% deoxycholate in 0.1 M Tris, pH 8.0. Concentration, 4 mg/ml.

TABLE 111 Subunit molecular weights and partial specific volumes of LPS

Molecular weiaht v a ~ ~ ! '#''&dg Weight 0.3% and de- average Weight

ml/g In calcu-

LPS deoxy- oxycho- MrLPS average Weight dimer lated

cholate late and de- M. LPS average M . uncor- cor- Oxycho- (whole 25'; from rected rected cell) for-

mula cell)

STM 7 0.683 0.704 32,214" 24,965" 15,592" 17,400 TV 119 0.697 0.710 23,515" 16,698" 10,607" 9,400 SL 1102 0.731 0.740 11,476" 8,044" 5,555" 5,800

9,350" 6,545"

Equilibrium sedimentation at high speed and low concentration. Equilibrium sedimentation at low speed and high concentration.

The maximal concentration of deoxycholate that could be used was 0.3% since a 0.6% solution of deoxycholate exceeded the critical micellar concentration.

The effect of LPS concentration on the rate of sedimenta- tion in deoxycholate is shown in Fig. 4. In this experiment, the LPS of strain STM 7 was dissolved in 0.15% deoxycholate, which in retrospect probably failed to cause complete disso- ciation (see Fig. 3). It is also clear that there was little or no concentration-dependent change in the value of the ~m.,,.~,,, , . The LPS of both T V 119 and SL 1102 were dissolved in 0.3% deoxycholate and a decrease in the Sm,u,.pp with decreasing concentration of LPS was observed. When extrapolated to zero concentration, the Sn.,,, value for SL 1102 was about 0.75 and that for TV 119 was about 0.9.

Binding of Deoxycholate to LPS-Fig. 5 shows a double reciprocal plot of the binding of deoxycholate to LPS. More deoxycholate bound to the rough lipopolysaccharides per unit weight than to the wild type, and there was a rough correlation between the amount of deoxycholate bound and the degree of incompleteness of the LPS. The binding curve behaved as expected up to a concentration of 0.6% deoxycholate. At concentrations of deoxycholate higher than 0.6%, there was

an anomalous decrease in binding. At a deoxycholate concen- tration of 0.3% the per cent weight of the LPS. deoxycholate complex contributed by the deoxycholate was 22.5% for STM 7,28% for TV 119, and 30% for SL 1102.

Molecular Weight Determinations-Fig. 6, A to C shows plots of In C displacement versus radius squared during equi- librium sedimentation at high speed and low concentration. The LPS from STM 7 and that from T V 119 always showed slight deviation from the ideal, suggesting heterogeneity or an associating system. The LPS from SL 1102 showed near homogeneity during sedimentation equilibrium and during velocity sedimentation, as illustrated by the schlieren pattern formed in a synthetic boundary cell (Fig. 6D). The vapp, $', the experimental molecular weights, and the calculated mo- lecular weights are listed in Table 111.

DISCUSSION

These data support those reported by others which indicate that divalent cations are important determinants of the sue and shape of LPS particles. The removal of divalent cations resulted in dispersion of the STM 7 LPS, changing ribbon-like lamellar structures into small lamellae and discs. Paradoxi- cally, the removal of divalent cations produced lamellar struc- tures from vesicles of lipid-rich SL 1102 LPS. It has been reported that divalent cations are required for the reassembly of dissociated outer membrane components into vesicles (25, 26) and for the incorporation of exogenous LPS into intact cells (27). The morphological transformatjon of SL 1102 LPS from vesicle to lamella upon removal 'of divalent cations suggests that these cations are essential for the maintenance of the vesicular structure in addition to its formation.

How Ca2+ and Mg" interact with LPS at the molecular level is unknown. One assumes that they act as counterions for the phosphate groups present in lipid A and the phosphate and the carboxyl groups in the core polysaccharide. Neutral- ization of the negative charge of these groups by cations would reduce repulsive charges between LPS subunits and allow LPS particles to aggregate. Conversely, removal of the diva- lent cations would increase the net negative charge, resulting in dispersion of the LPS by charge-charge repulsion. This is analogous to the effect of succinylation of LPS, a process which also results in dispersion (3). Such reasoning is sup- ported by the observable effect of EDTA on the size and morphology of the LPS from STM 7. It also explains ason- ably the necessity for divalent cations for the recons ution or reassembly of the Gram-negative outer membrant . The failure of SL 1102 LPS to disperse like that of STM 7 after Ca" and M 2 + depletion and the persistence of a ribbo!l-like structure could be due to lack of the negatively charged phosphate groups normally found in core polysaccharide and a lack of hydrophilic 0 sugars. The potentially lesser negative charge might be insufficient to disrupt the hydrophobic inter- actions of the lipid A subunits.

The studies of binding of sodium deoxycholate to LPS revealed both expected and unexpected phenomena. The rough LPS bound more deoxycholate per unit weight than the smooth LPS, as one might expect. With all of the LPS preparations, an increase in deoxycholate concentration up to 0.3 to 0.6% resulted in an increase in deoxycholate bound to LPS. At higher concentrations, binding behaved in an anom- alous fashion. Further increases in deoxycholate concentration resulted in decreases in the amount bound to LPS. Such a phenomenon might be explained by two events. First, 0.6% deoxycholate exceeds the critical micellar concentration. At this point, deoxycholate binds to itself, perhaps in preference to LPS. Second, LPS seems to be maximally disaggregated in 0.3 to 0.6% deoxycholate, and potentially, the disaggregated

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1226 Dispersion of Lipopolysaccharide by Deoxycholate

“monomer” could traverse the dialysis membrane. This pos- sibility was tested and disproved.

The ratios of the moles of deoxycholate bound per mol of LPS were calculated using the weight average molecular weight as concentration approaches zero (C + 0). The values were: STM 7, 11.1; TV 119, 10.2; and SL 1102, 6.1. The similarity of the binding values for STM 7 and TV 119 suggests that deoxycholate does not bind to the 0-side chains. The lesser binding to SL 1102 when compared to TV 119 suggests that some binding occurs in the core region (shown in Fig. 1). The probable binding sites include the fatty acids of lipid A, the phosphorylethanolamine groups of the deep core region, and perhaps the amino group of N-acetylglucosamine of the core region. One could propose a model in which 1 deoxycho- late molecule binds to the group of fatty acids linked to each glucosamine of the backbone, 1 to each phosphorylethanola- mine, and 1 to the amino sugar of the core. In such a model, the number of molecules of deoxycholate bound to the Re, Ra, and complete LPS shown in Fig. 1 would be 3, 5, and 5, respectively. If we assume that the monomeric unit of LPS is a dimer of the structure shown in Fig. 1, the values are very close to those found experimentally. Unfortunately, we cannot determine whether, under the conditions of equilibrium di- alysis, all of the binding sites were occupied and whether the LPS was completely dissociated.

Sedimentation equilibrium revealed some polydispersity of the LPS from STM 7 and from TV 119. It is impossible to determine whether this phenomenon is caused by true heter- ogeneity of “monomers,” or whether it is caused by association of monomers, or whether it is due to the Donnan effect. The Re mutant LPS, on the other hand, appeared to be monodis- perse and approached the ideal in sedimentation equilibrium.

The weight average molecular weight that we determined for the smooth LPS subunit was compatible with values reported for LPS of other Enterobacteriaceae by Ribi et al. (2) and by Hannecart-Pokorni et al. (7). When our data were extrapolated to infinite dilution, however, the value was smaller. The molecular weight found for the Ra mutant LPS was similar to the value obtained for an Rc mutant of Sal- monella by Romeo et al. (lo), and the value for the Re mutant LPS was compatible with the values reported by Kasai et al. (11). Although one might expect the LPS of different bacteria to have different molecular weights because of different chem- ical structure, it appears that the size of the LPS monomer may be relatively uniform among members of the Enterobac- teriaceae.

Are these data sufficiently reliable to allow construction of a theoretical model of LPS? There are several potential sources of error. First, there is no absolute assurance that in such self-associating systems true monomers are resolved. Second, as previously discussed, in a multicomponent system with interacting solute and solvent, there may be substantial errors in the calculation of the partial specific volume.

It is of interest that our uncorrected partial specific volumes (Table 111) are very similar to values reported by others. McIntire et al. (8) reported a t of 0.66 ml/g for Escherichia coli LPS in 0.15% deoxycholate. Hannecart-Pokorni et al. (7) reported a P of 0.68 ml/g for a complete Salmonella LPS and 0.80 ml/g for Re LPS. Presumably, both of these were meas- ured in 2 to 3% deoxycholate. We have reason to believe, therefore, that our values for P are accurate, and since we have corrected for the binding of deoxycholate and for changes

in solvent density, we assume that our molecular weight values are more accurate than others which were determined by centrifugation of multicomponent systems.

The data we have obtained suggest that a subunit of Sal- monella LPS consists of a dimer of the structure shown in Fig. 1. This interpretation is consistent with the calculated molecular weights of the three LPS preparations tested. Oth- ers have proposed that the subunit of LPS is a trimer (10,26). The reasons for this difference in interpretation include small differences in the determined molecular weights and a possible underestimation of formula weights in previous studies.

Acknowledgments-We would like to thank Mrs. J. McGraw and Mr. E. McClain for their technical assistance. The first author (J. W. S.) is thankful for the many helpful discussions with Dr. Parker Small and appreciates his valiant efforts to teach him physical chemistry.

REFERENCES 1. Frank, H., and Dekegel, D. (1967) Folia Microbiol. 12, 227-233 2. Ribi, E., Anacker, R., Brown, W., Haskins, W., Malmgren, B.,

Milner, K., and Rudbach, J. (1966) J. Bucteriol. 92, 1493-1509 3. McIntire, F. C., Sievert, H., Barlow, G., Finley, R., and Lee, A.

(1967) Biochemistry 6,2363-2372 4. Olins, A. L., and Warner, R. (1967) J. Biol. Chem. 242,4994-5001 5. Oroszlan, S. I., and Mora, P. (1963) Biochem. Biophys. Res.

6. Weiser, M. M., and Rothfield, L. (1968) J. Biol. Chem. 243, 1320-

7. Hannecart-Pokorni, E., Dekegel, D., and Depuydt, F. (1973) Eur.

8. McIntire, F. C., Barlow, G., Sievert, H., Finley, R., and Yoo, A.

9. Galanos, C. (1975) 2. Zmmunitaetsforsch. 149,214-229

Commun. 12,345-349

1328

J. Biochem. 38,6-13

(1969) Biochemistry 8,4063-4067

10. Romeo, D., Girard, A,, and Rothfield, L. (1970) J. Mol. Biol. 53,

11. Kasai, N., Hayashi, Y., and Komatsu, N. (1970) Jpn. J. Med. Sci.

12. Anderson, E. H. (1946) Proc. Natl. Acad. Sci. U. S. A. 32, 120-

13. Westphal, O., Luderitz, O., and Bister, F. (1952) 2. Naturforsch.

14. Galanos, C., Luderitz, O., and Westphal, 0. (1969) Eur. J. Bio- chem. 9,245-249

15. Hammerling, G., Luderitz, O., and Westphal, 0. (1970) Eur. J. Biochem. 15,48-56

16. Luderitz, O., Galanos, C., Lehmann, V., Nurminen, M., Rietschel, E. T., Rosenfelder, G., Simon, M., and Westphal, 0. (1973) J. Infect. Dis. 1285, SI7429

17. Savory, J., Wiggins, J., and Heintges, M. (1969) Am. J. Clin. Pathol. 51, 720-727

18. Karkhanis, Y. D., Zeltner, J. Y., Jackson, J. J., and Carlo, D. J. (1978) Anal. Biochem. 85,595-601

19. Casassa, E. F., and Eisenberg, H. (1964) Adu. Protein Chem. 19, 287-395

20. Woods, E. F., Himelfarb, S., and Harrington, W. F. (1963) J. Biol. Chem. 238,2374-2385

21. Tanford, C., Nozaki, Y., Reynolds, J., and Makino, S. (1974) Biochemistry 13,2369-2376

22. Yphantis, D. A. (1964) Biochemistry 3,297-317 23. Richards, E. G., and Schachman, H. (1959) J. Phys. Chem. 63,

24. Shands, J. W., Jr., Graham, J., and Nath, K. (1967) J. Mol. Biol.

25. DePamphilis, M. L. (1971) J. Bacteriol. 105, 1184-1199 26. Nakamura. K.. and Mizushima. S. (1975) Biochim. Biophys. Acta

475-490

Biol. 23,361-364

128

7b, 148-155

1578-1591

25, 15-21

413,3711393 . .

27. Jones, N. C., and Osborn, M. J. (1977) J. Biol. Chem. 252, 7398- 7404

Eur. J. Biochem. 7,239-246 28. Malchow, D., Luderitz, O., Kickhofen, B., and Westphal, 0. (1969)

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J W Shands, Jr and P W Chunmolecular weight.

The dispersion of gram-negative lipopolysaccharide by deoxycholate. Subunit

1980, 255:1221-1226.J. Biol. Chem. 

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