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Chapter 12 Lipopolysaccharides
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Chapter 1 2 LIPOPOLYSACCHARIDES PAUL F. COLEMAN and OTHMAR GABRIEL L i p o p o l y s a c c h a r i d e s (LPS) a r e complex molecules f o u n d i n t h e o u t e r membrane o f gram n e g a t i v e b a c t e r i a . A r e p r e s e n t a t i v e s t r u c t u r e f o r t h i s t y p e o f m o l e c u l e i s shown i n F i g . 12.1.
L i p i d A, c o n t a i n i n g t h e e s t e r i f i e d f a t t y a c i d s shown i n t h e
f i g u r e i n v a r i o u s p r o p o r t i o n s , c o n f e r s n e t hydrophobic p r o p e r t i e s t o t h e m o l e c u l e which cause a g g r e g a t i o n i n aqueous media and make i t necessary t o conduct e l e c t r o p h o r e t i c f r a c t i o n a t i o n s i n t h e presence o f d e t e r g e n t . The e x a c t p o s i t i o n and
qp-EtNH2-
Gal ‘Glc-Hep-Hep-~I(DO--KOO GlcFAc
(*re
Man-Rha-Gal
L
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-o-antigen&. poLyrner
/
12-Gal
I HeD
KDO
H
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4
0
!
BHMA
P
BHMA
I
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Carbohydrate
F
I
E ~ N H ~
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--Lipid
moiety core
A moiety--
3- M y r i s t o x y m y r i s t a t e
F i g . 12.1. The g e n e r a l l y accepted chemical s t r u c t u r e o f SuZrnoneZZu typhimuriurn LT2 i s g i v e n above. The v a r i a b l e s u b s t i t u t i o n of f a t t y a c i d s on t h e L i p i d A m o i e t y i s i n d i c a t e d . BHMA = B - h y d r o x y m y r i s t i c a c i d , KDO = 2-keto-3-deoxyoctonate (3-deoxyD-mannooctulosonate), Hep = L-glycero-D-mannoheptose, Abe = Abequose, Rha = L-rhamnose, AraNH2 = 4-amino-4-deoxy-L-arabinose, E t N H 2 = ethanolamine.
d i s t r i b u t i o n of f a t t y a c i d residues e s t e r i f i e d t o t h e L i p i d A moiety are a t pres e n t unknown. N a t i v e LPS e x h i b i t s a s i n g l e s t a i n a b l e zone i n g e l e l e c t r o f o c u s i n g i n t h e presence o f n o n - i o n i c detergent’”.
S i m i l a r l y , g e l e l e c t r o p h o r e s i s o f LPS
i n n o n - i o n i c d e t e r g e n t y i e l d s a broad smear w h i c h does n o t appear u s e f u l f o r r e s o l v i n g LPS. Therefore, a l l f r a c t i o n a t i o n s o f LPS c o n s i d e r e d i n t h i s r e v i e w
282
F i g . 12.2. D i s c o n t i n u o u s SDS-PAGE o f (SaZrnoneZZa typhirnuriurn LT2) - LPS ( r e f . 1 2 ) . Gel c o n c e n t r a t i o n : 10% T , 2.5% C. The bands a r e v i s u a l i z e d by t h e p e r i o d a t e S c h i f f s t a i n (see r e f . 3 ) . L e f t : 0.2 mg LPS. Center: 0.5 mg LPS. R i g h t : 0 . 2 mg LPS. The e l e c t r o p h o r e t i c c o n d i t i o n s a r e t h e same as above b u t i n c l u d e 46 m~ HC1 i n t h e r e s o l v i n g g e l b u f f e r and 27 mM HC1 i n t h e anodic e l e c t r o l y t e . These cond i t i o n s r e s o l v e t h e t o p 20 0-Antigen c o n t a i n i n g bands as shown. The bands a r e numbered f r o m t h e t o p o f t h e p a t t e r n down, d i s r e g a r d i n g t h e uppermost 2 d i f f u s e bands. r e l a t e t o t h e sodium dodecyl s u l f a t e (SDS) d e r i v a t i v e o f LPS. Continuous SDS-PAGE f r a c t i o n a t i o n o f LPS d e r i v e d f r o m e i t h e r I?.
c o l i o r SaZmoneZla y i e l d s d i f f u s e
zones3, w h i l e d i s c o n t i n u o u s SDS-PAGE systems r e s o l v e LPS i n t o a m u l t i p l i c i t y o f w e l l d e f i n e d bands w i t h t h e o r d e r l y appearance o f an o l i g o m e r i c s e r i e s l Y 4 ( F i g .
- 1 . 2 ) . E l u t e d g e l f r a c t i o n s s u b j e c t e d t o r e - e l e c t r o p h o r e s i s produced t h e same bands w i t h i d e n t i c a l e l e c t r o p h o r e t i c m o b i l i t i e s . LPS from o t h e r organisms such as Neisseria appears t o y i e l d d i f f u s e bands i n a d i s c o n t i n u o u s PAGE system as
283 w e l l 5 . To e x p l a i n t h e band m u l t i p l i c i t y ( F i g . 12.2) i t has been assumed t h a t SDS binds e x c l u s i v e l y t o t h e L i p i d A p o r t i o n o f t h e
Based on t h a t assumption, LPS species d i f f e r i n e l e c t r o p h o r e t i c m o b i l i t y s o l e l y due t o s i z e f r a c -
t i o n a t i o n o f t h e c a r b o h y d r a t e moiety, as has been shown f o r c o r e mutants ( F i g . 6 i n ref.
3, F i g . 4 i n r e f . 6 ) . Making t h e same assumption, b u t u s i n g c i r c u i t o u s
reasoning, t h e numbers o f bands i n SDS-PAGE have been c o r r e l a t e d w i t h v a r i a b l e c h a i n l e n g t h o f t h e 0 - a n t i g e n polymer4 Double l a b e l i n g experiments, i n w h i c h t h e
.
0-Antigen and L i p i d A/core m o i e t i e s ( F i g . 12.1) were d i f f e r e n t i a l l y l a b e l l e d by r a d i o i s o t o p e p r e c u r s o r s , a l l o w e d one t o a r r i v e a t a r e l a t i o n between c h a i n l e n g t h of t h e c a r b o h y d r a t e m o i e t y and m o b i l i t y . C l e a r l y , t h e c o r r e l a t i o n h o l d s f o r t h e h i g h m o b i l i t y bands ( t h e l i n e a r p o r t i o n o f F i g . 68 i n r e f . 1 and F i g . 3C i n r e f . 6 ) I t does n o t seem t o h o l d f o r t h e l o w e r m o b i l i t y bands which c o n t a i n t h e m a j o r i t y
o f the 0-Antigen.
To f u r t h e r i n v e s t i g a t e t h e r e l a t i o n between m o b i l i t y , SDS c o n t e n t and c h a i n l e n g t h , Fergusonl p l o t s 7 were c o n s t r u c t e d f o r t h e LPS components d e p i c t e d i n F i g . 12.1.
There was no change i n t h e o r d e r o f bands a t t h e v a r i o u s g e l c o n c e n t r a -
t i o n s as determined by t r a n s v e r s e p o r e g r a d i e n t g e l a n a l y s i s 8 ”
(unpublished data).
C l e a r l y , t h e j o i n t 95% c o n f i d e n c e enveloDes o f KR ( m o l e c u l a r s i z e ) and Yo (molecu l a r n e t charge) ( F i g . 12.3) i n d i c a t e t h a t t h e SDS d e r i v a t i v e s o f LPS c o n s i s t s o f 2 p o p u l a t i o n s , one b e i n g v e r t i c a l l y d i s p l a c e d and t h e o t h e r h o r i z o n t a l l y d i s The b u l k o f t h e 0-Antigen c o n t a i n i n g LPS s p e c i e s f a l l s i n t o t h e f o r m e r c a t e g o r y . The v e r t i c a l l y d i s p l a c e d p o p u l a t i o n of e l e c t r o p h o r e t i c bands ( t h e t o p 18 bands o f F i g . 12.2) a l l have t h e same m o l e c u l a r s i z e (KR) w h i l e v a r y i n g i n t h e m o l e c u l a r n e t charge ( y o ) . A p o s s i b l e e x p l a n a t i o n f o r t h e o r i g i n
3-
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-
5.6
,
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F i g . 12.3. The Ferguson p l o t f o r (SuZmoneZZa typhimuriurn LT2) LPS i s shown on the. l e f t . The j o i n t 95% c o n f i d e n c e envelopes o f K~ and YO generated f r o m t h i s p l o t a r e shown a t t h e r i g h t . The p l o t r e p r e s e n t i n g t h e b e s t - f i t l i n e s and t h e c o n f i d e n c e envelopes were d e r i v e d by computer a n a l y s i s , u s i n g t h e PAGE-PACK p r o grams o f Rodbard and Chrambach7.
284
o f t h i s p o p u l a t i o n would be t h a t t h e L i p i d A m o i e t y o f these m o l e c u l e s d i f f e r s i n f a t t y a c i d c o m p o s i t i o n and/or conformation, r e s u l t i n g i n d i f f e r e n t i a l b i n d i n g o f SDS which l e a d s t o charge h e t e r o g e n e i t y . The h o r i z o n t a l l y d i s p l a c e d p o p u l a t i o n ( t h e bands f r o m 18-23 i n F i g . 12.2) a l l have t h e same charge d e n s i t y ( Y o ) b u t v a r y i n m o l e c u l a r s i z e ( K ~ )(see F i g . 12.3). The d o u b l e - l a b e l r a t i o d a t a c i t e d above would i n d i c a t e t h a t t h e s e LPS s p e c i e s w i t h h i g h m o b i l i t y d e r i v e f r o m a v a r i a b l e degree o f 0-Antigen p o l y m e r i z a t i o n . However, a decrease i n t h e s i z e o f t h e carboh y d r a t e m o i e t y a t c o n s t a n t SDS c o n t e n t o f t h e L i p i d A m o i e t y would be exoected t o l e a d t o an i n c r e a s e i n m o l e c u l a r charge d e n s i t y and t h e r e f o r e Y o . No such i n c r e a s e i n Yo i s seen among s p e c i e s 18-24 on F i g . 12.3. T h e r e f o r e , t h e f i n d i n g s r e p o r t e d h e r e a p p a r e n t l y c o n t r a d i c t t h e e a r l i e r h y p ~ t h e s i s ~t h’ a~t SDS b i n d s e x c l u s i v e l y and t o t h e same degree i n each L i p i d A m o i e t y . An a l t e r n a t i v e explanat i o n f o r t h e h o r i z o n t a l d i s t r i b u t i o n o f t h e h i g h m o b i l i t y s p e c i e s ( F i g . 12.3, numbers 18-24) i s t h a t t h e i r common f r e e m o b i l i t y (charge d e n s i t y , Y o ) d e r i v e s from a compensatory mechanism, whereby p r o g r e s s i v e s h o r t e n i n g o f t h e c a r b o h y d r a t e m o i e t y would l e a d t o ( a ) an i n c r e a s e i n Y o , due t o an i n c r e a s e d charge/mass r a t i o , assuming SDS b i n d i n g i n t h e L i p i d A m o i e t y ~ n l y and ~ ’ (~b ) a decrease i n y o , due t o an i n c r e a s e d p a r t i a l s p e c i f i c volume, 7 , s i n c e t h e L i p i d A m o i e t y has a o f a p p r o x i m a t e l y 0.9, w h i l e t h e carbohydrate m o i e t y has a 0.6,
7 value
7 value
o f approximately
i n t h e absence o f SDS. I n i t s presence, t h e d i f f e r e n c e i n 7 v a l u e s i s exac-
erbated. An i n c r e a s e d
7 indicates
an i n c r e a s e d e f f e c t i v e , h y d r a t e d m o l e c u l a r
volume and t h e r e f o r e a decreased f r e e e l e c t r o p h o r e t i c m o b i l i t y ( Y o ) . These app a r e n t l y c o n t r a d i c t o r y ways t o i n t e r p r e t t h e p r e s e n t l y a v a i l a b l e d a t a make f u r t h e r s t u d i e s on t h e i n t e r a c t i o n between SDS and LPS necessary. ACKNOWLEDGEMENTS The a u t h o r s w i s h t o thank D r . L.M. H j e l m e l a n d f o r v a l u a b l e h e l p i n t h e i n t e r p r e t a t i o n o f t h e data, D r . D. Rodbard f o r s u g g e s t i n g t h e t r a n s v e r s e p o r e g r a d i e n t g e l a n a l y s i s o f LPS, and D r . A. Chrambach f o r d i s c u s s i o n s and e d i t o r i a l h e l p . The o r i g i n a l m a t e r i a l r e p o r t e d i n t h i s r e v i e w comprises work done i n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r t h e Ph. D. degree a t Georgetown U n i v e r s i t y f o r P. F. C. REFERENCES
1 2 3 4 5
R.C. Goldman and L. L e i v e , Eur. J . Biochem., 107 (1980) 145-153. P. Coleman, unpublished r e s u l t s . B. Jann, K. Reske and K. Jann, Eur. J . Biochem., 60 (1975) 239-246. E.T. Palva and P.H. Makela, Eur. J . Biochem., 107 (1980) 137-143. R.R. R u s s e l l and K.G. Johnson, Can. J . MicrobioZ., 2 1 (1975) 2013-2018.
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6 R.S. Munford, C . L . Hall and P. Rick, J . Bacteriol., 144 (1980) 630-640. 7 D. Rodbard a n d A. Chrambach, Anal. Biochem., 40 (1971) 95-134. 8 G. Kapadia, A. Chrambach and D. Rodbard, Electrophoresis and I s o e Z e c t r i c Focusing on polyacrglamide Gel, W. de Gruyter, Berlin, 1974, p . 115. 9 J . Margolis and K.G. Kenrick, Nature Ilondonl, 214 (1967) 1334-1336. 10 0. Rodbard a n d A. Chrambach, EZectrophoresis and I s o e l e c t r i c Focusing on Polyacrylamide Gel, W. de Gruyter, Berlin, 1974, p. 28. 11 P . F . Coleman and 0. Gabriel, Fed. Proe., Fed. Amer. Soc. E q . BioZ., 40 (1981) 1846. 12 U . K . Laemmli and M. Favre, J. Mol. BioZ., 80 (1973) 575-599.
Chapter 1 2 LIPOPOLYSACCHARIDES PAUL F. COLEMAN and OTHMAR GABRIEL L i p o p o l y s a c c h a r i d e s (LPS) a r e complex molecules f o u n d i n t h e o u t e r membrane o f gram n e g a t i v e b a c t e r i a . A r e p r e s e n t a t i v e s t r u c t u r e f o r t h i s t y p e o f m o l e c u l e i s shown i n F i g . 12.1.
L i p i d A, c o n t a i n i n g t h e e s t e r i f i e d f a t t y a c i d s shown i n t h e
f i g u r e i n v a r i o u s p r o p o r t i o n s , c o n f e r s n e t hydrophobic p r o p e r t i e s t o t h e m o l e c u l e which cause a g g r e g a t i o n i n aqueous media and make i t necessary t o conduct e l e c t r o p h o r e t i c f r a c t i o n a t i o n s i n t h e presence o f d e t e r g e n t . The e x a c t p o s i t i o n and
qp-EtNH2-
Gal ‘Glc-Hep-Hep-~I(DO--KOO GlcFAc
(*re
Man-Rha-Gal
L
f
-o-antigen&. poLyrner
/
12-Gal
I HeD
KDO
H
R
4
0
!
BHMA
P
BHMA
I
A”
Carbohydrate
F
I
E ~ N H ~
-
--Lipid
moiety core
A moiety--
3- M y r i s t o x y m y r i s t a t e
F i g . 12.1. The g e n e r a l l y accepted chemical s t r u c t u r e o f SuZrnoneZZu typhimuriurn LT2 i s g i v e n above. The v a r i a b l e s u b s t i t u t i o n of f a t t y a c i d s on t h e L i p i d A m o i e t y i s i n d i c a t e d . BHMA = B - h y d r o x y m y r i s t i c a c i d , KDO = 2-keto-3-deoxyoctonate (3-deoxyD-mannooctulosonate), Hep = L-glycero-D-mannoheptose, Abe = Abequose, Rha = L-rhamnose, AraNH2 = 4-amino-4-deoxy-L-arabinose, E t N H 2 = ethanolamine.
d i s t r i b u t i o n of f a t t y a c i d residues e s t e r i f i e d t o t h e L i p i d A moiety are a t pres e n t unknown. N a t i v e LPS e x h i b i t s a s i n g l e s t a i n a b l e zone i n g e l e l e c t r o f o c u s i n g i n t h e presence o f n o n - i o n i c detergent’”.
S i m i l a r l y , g e l e l e c t r o p h o r e s i s o f LPS
i n n o n - i o n i c d e t e r g e n t y i e l d s a broad smear w h i c h does n o t appear u s e f u l f o r r e s o l v i n g LPS. Therefore, a l l f r a c t i o n a t i o n s o f LPS c o n s i d e r e d i n t h i s r e v i e w
282
F i g . 12.2. D i s c o n t i n u o u s SDS-PAGE o f (SaZrnoneZZa typhirnuriurn LT2) - LPS ( r e f . 1 2 ) . Gel c o n c e n t r a t i o n : 10% T , 2.5% C. The bands a r e v i s u a l i z e d by t h e p e r i o d a t e S c h i f f s t a i n (see r e f . 3 ) . L e f t : 0.2 mg LPS. Center: 0.5 mg LPS. R i g h t : 0 . 2 mg LPS. The e l e c t r o p h o r e t i c c o n d i t i o n s a r e t h e same as above b u t i n c l u d e 46 m~ HC1 i n t h e r e s o l v i n g g e l b u f f e r and 27 mM HC1 i n t h e anodic e l e c t r o l y t e . These cond i t i o n s r e s o l v e t h e t o p 20 0-Antigen c o n t a i n i n g bands as shown. The bands a r e numbered f r o m t h e t o p o f t h e p a t t e r n down, d i s r e g a r d i n g t h e uppermost 2 d i f f u s e bands. r e l a t e t o t h e sodium dodecyl s u l f a t e (SDS) d e r i v a t i v e o f LPS. Continuous SDS-PAGE f r a c t i o n a t i o n o f LPS d e r i v e d f r o m e i t h e r I?.
c o l i o r SaZmoneZla y i e l d s d i f f u s e
zones3, w h i l e d i s c o n t i n u o u s SDS-PAGE systems r e s o l v e LPS i n t o a m u l t i p l i c i t y o f w e l l d e f i n e d bands w i t h t h e o r d e r l y appearance o f an o l i g o m e r i c s e r i e s l Y 4 ( F i g .
- 1 . 2 ) . E l u t e d g e l f r a c t i o n s s u b j e c t e d t o r e - e l e c t r o p h o r e s i s produced t h e same bands w i t h i d e n t i c a l e l e c t r o p h o r e t i c m o b i l i t i e s . LPS from o t h e r organisms such as Neisseria appears t o y i e l d d i f f u s e bands i n a d i s c o n t i n u o u s PAGE system as
283 w e l l 5 . To e x p l a i n t h e band m u l t i p l i c i t y ( F i g . 12.2) i t has been assumed t h a t SDS binds e x c l u s i v e l y t o t h e L i p i d A p o r t i o n o f t h e
Based on t h a t assumption, LPS species d i f f e r i n e l e c t r o p h o r e t i c m o b i l i t y s o l e l y due t o s i z e f r a c -
t i o n a t i o n o f t h e c a r b o h y d r a t e moiety, as has been shown f o r c o r e mutants ( F i g . 6 i n ref.
3, F i g . 4 i n r e f . 6 ) . Making t h e same assumption, b u t u s i n g c i r c u i t o u s
reasoning, t h e numbers o f bands i n SDS-PAGE have been c o r r e l a t e d w i t h v a r i a b l e c h a i n l e n g t h o f t h e 0 - a n t i g e n polymer4 Double l a b e l i n g experiments, i n w h i c h t h e
.
0-Antigen and L i p i d A/core m o i e t i e s ( F i g . 12.1) were d i f f e r e n t i a l l y l a b e l l e d by r a d i o i s o t o p e p r e c u r s o r s , a l l o w e d one t o a r r i v e a t a r e l a t i o n between c h a i n l e n g t h of t h e c a r b o h y d r a t e m o i e t y and m o b i l i t y . C l e a r l y , t h e c o r r e l a t i o n h o l d s f o r t h e h i g h m o b i l i t y bands ( t h e l i n e a r p o r t i o n o f F i g . 68 i n r e f . 1 and F i g . 3C i n r e f . 6 ) I t does n o t seem t o h o l d f o r t h e l o w e r m o b i l i t y bands which c o n t a i n t h e m a j o r i t y
o f the 0-Antigen.
To f u r t h e r i n v e s t i g a t e t h e r e l a t i o n between m o b i l i t y , SDS c o n t e n t and c h a i n l e n g t h , Fergusonl p l o t s 7 were c o n s t r u c t e d f o r t h e LPS components d e p i c t e d i n F i g . 12.1.
There was no change i n t h e o r d e r o f bands a t t h e v a r i o u s g e l c o n c e n t r a -
t i o n s as determined by t r a n s v e r s e p o r e g r a d i e n t g e l a n a l y s i s 8 ”
(unpublished data).
C l e a r l y , t h e j o i n t 95% c o n f i d e n c e enveloDes o f KR ( m o l e c u l a r s i z e ) and Yo (molecu l a r n e t charge) ( F i g . 12.3) i n d i c a t e t h a t t h e SDS d e r i v a t i v e s o f LPS c o n s i s t s o f 2 p o p u l a t i o n s , one b e i n g v e r t i c a l l y d i s p l a c e d and t h e o t h e r h o r i z o n t a l l y d i s The b u l k o f t h e 0-Antigen c o n t a i n i n g LPS s p e c i e s f a l l s i n t o t h e f o r m e r c a t e g o r y . The v e r t i c a l l y d i s p l a c e d p o p u l a t i o n of e l e c t r o p h o r e t i c bands ( t h e t o p 18 bands o f F i g . 12.2) a l l have t h e same m o l e c u l a r s i z e (KR) w h i l e v a r y i n g i n t h e m o l e c u l a r n e t charge ( y o ) . A p o s s i b l e e x p l a n a t i o n f o r t h e o r i g i n
3-
1.8 -
i 6
0
2
4
6
8 %T
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1.21.04.2
’
’
4.4
4.6
4.8
,
5.0
,/I,
5.2 5.4 KRxlOZ
-
5.6
,
5.8
,
6.0
F i g . 12.3. The Ferguson p l o t f o r (SuZmoneZZa typhimuriurn LT2) LPS i s shown on the. l e f t . The j o i n t 95% c o n f i d e n c e envelopes o f K~ and YO generated f r o m t h i s p l o t a r e shown a t t h e r i g h t . The p l o t r e p r e s e n t i n g t h e b e s t - f i t l i n e s and t h e c o n f i d e n c e envelopes were d e r i v e d by computer a n a l y s i s , u s i n g t h e PAGE-PACK p r o grams o f Rodbard and Chrambach7.
284
o f t h i s p o p u l a t i o n would be t h a t t h e L i p i d A m o i e t y o f these m o l e c u l e s d i f f e r s i n f a t t y a c i d c o m p o s i t i o n and/or conformation, r e s u l t i n g i n d i f f e r e n t i a l b i n d i n g o f SDS which l e a d s t o charge h e t e r o g e n e i t y . The h o r i z o n t a l l y d i s p l a c e d p o p u l a t i o n ( t h e bands f r o m 18-23 i n F i g . 12.2) a l l have t h e same charge d e n s i t y ( Y o ) b u t v a r y i n m o l e c u l a r s i z e ( K ~ )(see F i g . 12.3). The d o u b l e - l a b e l r a t i o d a t a c i t e d above would i n d i c a t e t h a t t h e s e LPS s p e c i e s w i t h h i g h m o b i l i t y d e r i v e f r o m a v a r i a b l e degree o f 0-Antigen p o l y m e r i z a t i o n . However, a decrease i n t h e s i z e o f t h e carboh y d r a t e m o i e t y a t c o n s t a n t SDS c o n t e n t o f t h e L i p i d A m o i e t y would be exoected t o l e a d t o an i n c r e a s e i n m o l e c u l a r charge d e n s i t y and t h e r e f o r e Y o . No such i n c r e a s e i n Yo i s seen among s p e c i e s 18-24 on F i g . 12.3. T h e r e f o r e , t h e f i n d i n g s r e p o r t e d h e r e a p p a r e n t l y c o n t r a d i c t t h e e a r l i e r h y p ~ t h e s i s ~t h’ a~t SDS b i n d s e x c l u s i v e l y and t o t h e same degree i n each L i p i d A m o i e t y . An a l t e r n a t i v e explanat i o n f o r t h e h o r i z o n t a l d i s t r i b u t i o n o f t h e h i g h m o b i l i t y s p e c i e s ( F i g . 12.3, numbers 18-24) i s t h a t t h e i r common f r e e m o b i l i t y (charge d e n s i t y , Y o ) d e r i v e s from a compensatory mechanism, whereby p r o g r e s s i v e s h o r t e n i n g o f t h e c a r b o h y d r a t e m o i e t y would l e a d t o ( a ) an i n c r e a s e i n Y o , due t o an i n c r e a s e d charge/mass r a t i o , assuming SDS b i n d i n g i n t h e L i p i d A m o i e t y ~ n l y and ~ ’ (~b ) a decrease i n y o , due t o an i n c r e a s e d p a r t i a l s p e c i f i c volume, 7 , s i n c e t h e L i p i d A m o i e t y has a o f a p p r o x i m a t e l y 0.9, w h i l e t h e carbohydrate m o i e t y has a 0.6,
7 value
7 value
o f approximately
i n t h e absence o f SDS. I n i t s presence, t h e d i f f e r e n c e i n 7 v a l u e s i s exac-
erbated. An i n c r e a s e d
7 indicates
an i n c r e a s e d e f f e c t i v e , h y d r a t e d m o l e c u l a r
volume and t h e r e f o r e a decreased f r e e e l e c t r o p h o r e t i c m o b i l i t y ( Y o ) . These app a r e n t l y c o n t r a d i c t o r y ways t o i n t e r p r e t t h e p r e s e n t l y a v a i l a b l e d a t a make f u r t h e r s t u d i e s on t h e i n t e r a c t i o n between SDS and LPS necessary. ACKNOWLEDGEMENTS The a u t h o r s w i s h t o thank D r . L.M. H j e l m e l a n d f o r v a l u a b l e h e l p i n t h e i n t e r p r e t a t i o n o f t h e data, D r . D. Rodbard f o r s u g g e s t i n g t h e t r a n s v e r s e p o r e g r a d i e n t g e l a n a l y s i s o f LPS, and D r . A. Chrambach f o r d i s c u s s i o n s and e d i t o r i a l h e l p . The o r i g i n a l m a t e r i a l r e p o r t e d i n t h i s r e v i e w comprises work done i n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r t h e Ph. D. degree a t Georgetown U n i v e r s i t y f o r P. F. C. REFERENCES
1 2 3 4 5
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6 R.S. Munford, C . L . Hall and P. Rick, J . Bacteriol., 144 (1980) 630-640. 7 D. Rodbard a n d A. Chrambach, Anal. Biochem., 40 (1971) 95-134. 8 G. Kapadia, A. Chrambach and D. Rodbard, Electrophoresis and I s o e Z e c t r i c Focusing on polyacrglamide Gel, W. de Gruyter, Berlin, 1974, p . 115. 9 J . Margolis and K.G. Kenrick, Nature Ilondonl, 214 (1967) 1334-1336. 10 0. Rodbard a n d A. Chrambach, EZectrophoresis and I s o e l e c t r i c Focusing on Polyacrylamide Gel, W. de Gruyter, Berlin, 1974, p. 28. 11 P . F . Coleman and 0. Gabriel, Fed. Proe., Fed. Amer. Soc. E q . BioZ., 40 (1981) 1846. 12 U . K . Laemmli and M. Favre, J. Mol. BioZ., 80 (1973) 575-599.