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Comparative studies on cartilage aminopolysaccharide sulphates
496
BIOCHIMICA ET BIOPHYSICA ACTA
COMPARATIVE CARTILAGE I. P O L Y S A C C H A R I D E S
STUDIES
AMINOPOLYSACCHARIDE
ON SL'~L.~,~,~~
F R O M S H A R K , S K A T E , D O G F I S H A_.%-~-D~
~VftE~.EF.:
A. (5. LI.OYI), K. S. D()DGS()S AND R. G. PRICE
Department of Biochemistr.v, University of IVales. Cardiff (Great Britain) (Received June I8th, I 9 6 " ) SU~IMARY S t u d i e s on t h e p o l y s a c c h a r i d e c o n s t i t u e n t s of c a r t i l a g e f r o m a d u l t s p e c i ~ ~0ff5Ik~e s h a r k , s k a t e a n d dogfish r e v e a l t h e p r e s e n c e in e a c h of t h e s e prepar~.~i~m~,dfrm~e.fiat c o r r e s p o n d i n g t o c h o n d r o i t i n s u l p h a t e a n d k e r a t o s u l p h a t e . Howex, er. ,~m6h ,~t a m i n o p o l y s a c c h a r i d e s is c h a r a c t e r i s e d b y t h e p r e s e n c e of f r a c t i o n s ~¢ff~i~h~_" & ~ 5 ~ n g s u l p h a t e c o n t e n t . I n c o n t r a s t to t h e m a t e r i a l f r o m c a r t i l a g i n o u s ~ tt~e. ~.~tys a c c h a r i d e m i x t u r e from_ fin-whale c a r t i l a g e h a s c h o n d r o i t i n s u ~ ~ ~ ~ rn~jor constituent, both chondroitin sulphate C and keratosulphate being~r~ im.'~s t a n t i a l l y lower a m o u n t s . INTRODUCTION I t h a s n o w b e e n e s t a b l i s h e d t h a t m a m m a l i a n c a r t i l a g e s are chaxaa~emk~t 15~ r_he p r e s e n c e of t h r e e t y p e s of a r n i n o p o l y s a c c h a r i d e s u l p h a t e x. I n gemerat!, , d h 0 m ~ : ~ s u l p h a t e A ( r e p e a t i n g unit" 3-O-fl-D-glucuronido-N-acetyl-D-galactosamaineq4~d~tk~e) a n d c h o n d r o i t i n s u l p h a t e C ( r e p e a t i n g unit" 3 - O - f l - D - g l u c u r o n i d o - N - a c ~ ~ . ~ . ~ s a m i n e 6 - s u l p h a t e ) occur as m a i n c o n s t i t u e n t s , t o g e t h e r w i t h varbfiaag~rr~." , ~ 2 ~ r : ~ r n V ~ of k e r a t o s u l p h a t e ( r e p e a t i n g unit" 4-O-[3-D-galactosido-N-acety]-~nt~o~nrni_ne 6-sulphate). R e c e n t r e p o r t s 2-s reflect a s u b s t a n t i a l increase in i n t e r e s t i n ~Jae ~rrr/m~p)~Vs a c c h a r i d e c o n s t i t u e n t s of t h e c o n n e c t i v e tissues of c a r t i l a g i n o u s :fmh. ~nn r~ii~,~rrtText LLOYD et al. 9 r e p o r t e d t h e isolation a n d i n f r a r e d s p e c t r o s c o p i c exa,,m~rlii0m ,~fl poEy-s a c c h a r i d e s u l p h a t e s f r o m t h e c a r t i l a g e of t h e blue s h a r k (Carchart~,~s ~g~,aT~9),. t~e d o g f i s h (Scyllium canicula) a n d t h e s k a t e (Raja batis). T h e s e obserxr~tS~m~ lta~e, m~w b e e n e x t e n d e d t o i n c l u d e s t u d i e s on t h e c o m p o s i t i o n of t h e s e ma-~emia]~ :mx~i off ~ e s u l p h a t e d a m i n o p o l y s a c c h a r i d e s f r o m t h e c a r t i l a g e of t h e fin ~ 1 , ~ !g~h~n/hno~
physalus). MATERIAL~ AND .~IETHODS
A rninopolysacchari& sulphate preparat-ons P r e p a r a t i o n s of a m i n o p o l y s a c c h a r i d e ~ulphate m i x t u r e s were , 0 t ~ m ~ L p o t a s s i u m salts, f r o m a d u l t c a r t i l a g e s a m p l e s as d e s c r i b e d earlier.
a,s-v_tle
Biochim. Biophys. :4a;,. ,69 ((~sjt-5~4~-5o4!
AMINOPOLYSACCHARIDE
SULPHATES
497
31onosaccharide constituettts of polysaccharide preparations Portions (Ioo-2oo mg) of t h e polysaccharides dissolved in IO ml of I N H~SO4 were h e a t e d in sealed t u b e s for 4-5 h at IiO °. After cooling, t h e h y d r o l y s a t e s were n d-n.:l tre,,L,u with Ba(OH)o to p H 8.0 a n d t h e n c e n t r i f u g e d to remove BaSO4. Excess B a ~+ in t h e s u p e r n a t a n t s was t h e n precipiti~ted b y gassing with CO2 a n d t h e BaCO3 precipitates r e m o v e d b y centr!fuging. The p r o d u c t s were t h e n c o n c e n t r a t e d to d r y n e s s b y r o t a r y e v a p o r a t i o n u n d e r reduced pressure a t 35 ° and the residues redissolved in o.5-ml portions of water. T h e c o n c e n t r a t e d h y d r o l y s a t e s were c h r o m a t o g r a p h e d on W h a t m a n No. I or 3MM p a p e r s using d o w n w a r d irrigation w i t h p h e n o l - a m m o n i a - w a t e r t° or e t h y l a c e t a t e - p y r i d i n e - w a t e r (I2O'5O:4o, v/v) as solvents. The results of u n i - d i m e n s i o n a l s e p a r a t i o n s were checked b y t w o d i m e n s i o n a l c h r o m a t o g r a p h 3, using c o m b i n a t i o n s of p h e n o l - w a t e r - a m m o n i a 1°, e t h y l a c e t a t e - p y r i d i n e - w a t e r or b u t a n . . I - o l - a c e t i c a c i d - w a t e r (5o" I 2 : 2 5 , v/v) as developing solvents. S e p a r a t e d c o m p o n e n t s were d e t e c t e d w i t h aniline h y d r o g e n p h t h a l a t e n or p - a n i s i d i n O 2 sprays for r e d u c i n g s u b s t a n c e s a n d w i t h E l s o n - M o r g a n r e a g e n t s Is for hexosamines. T h e n a t u r e of t h e h e x o s a m i n e c o m p o n e n t s was also a s c e r t a i n e d by conversion to t h e c o r r e s p o n d i n g p e n t o s e d e r i v a t i v e s ~4, ~5 Chromatography on Ecleola-cdlttlose Commercial g r a d e Ecteola-ceilulose (Brown Co., Berlin, N.Y.) was s u s p e n d e d in o.5 M NaCI and, after p a c k i n g in a glass column, t r e a t e d successively w i t h o.5 M N a O H , 3.o M NaC1 a n d o.I M N a C l - o . I M HCI ( I ' I , v/v), according to RlXGEI~TZ AND
RE1CHARD
16.
For a n a l y t i c a l s e p a r a t i o n s t h e modified cellulose was p a c k e d u n d e r g r a v i t y flow in a glass t u b e (15 cm i cm) fitted at t h e lower end w i t h a f r i t t e d glass disc. T h e column was t h e n w a s h e d w i t h 5 h o l d - u p v o l u m e s of t h e o.I M N a C I - o . I M HC1 (i :I, vlv ) m i x t u r e . F o r t h e s e p a r a t i o n t h e p o l y s a c c h a r i d e ( I 5 - 2 o rag), dissolved in 2-4 ml of w a t e r , was c o n v e r t e d t.'~ t h e free acid b y passing t h r o u g h a Dowex-5o c o l u m n (H + 2orm, 5o-xoo mesh) a n d t h e acid e l u a t e a n d w a s h i n g s c o n c e n t r a t e d to I ml by freeze-drying. T h e sample was t h e n a d d e d to t h e c o l u m n a n d allowed to d r a i n i n t o t h e resin b e d u n d e r g r a v i t y flow. E l u t i o n of c a r b o h y d r a t e w a s achieved using a g r a d i e n t p r o d u c e d by r u n n i n g IOO ml of 3.o M NaC1-3.o M HC! (I" I, v/'v) into a m i x i n g vessel fitted w i t h a m a g n e t i c stirrer a n d c o n t a i n i n g 4oo ml of o.I M N a C l - o . I M HC1 ( i ' I , v/v). T h e e l u a t e from t h e cohtmn was collected in 5-ml fractions a n d t h e c a r b o h y d r a t e c o n t e n t of t h e s e d e t e r m i n e d w i t h the p h e n o l - s u l p h u r i c acid reaction~L On a p r e p a r a t i v e scale, t h e polysaccharides (3-4 g) were c o n v e r t e d to the free acids as before, a n d t h e n a d d e d to an Ecteola-cellulose e o l u n m (6o cm × 3-5 cm). T h e c o l u m n w a s t h e n eluted in a stepwise m a n n e r using t h e a p p r o p r i a t e c o n c e n t r a t i o n s of t h e N a C 1 - H C I (i "i, v/v) m i x t u r e i n d i c a t e d b y t h e a n a l y t i c a l e x p e r i m e n t s . For t h e isolation of t h e polysaccharides from t xe eluates, fractions corresponding to h o m o g e n e o u s elution p e a k s were pooled a n d c o n c e n t r a t e d to 25o ml by r o t a r y e v a p o r a t i o n u n d e r reduced pressure at 37 °. T h e solutio/ls were t h e n dialysed e x h a u s tively against w a t e r for 24 h at 4 °. T h e dialysate was t h e n c o n c e n t r a t e d to IOO ml b y r o t a r y e v a p o r a t i o n , passed t h r o u g h a Dowex-5 o c o l u m n (H + form) ,tnd t h e acid e l u a t e a n d washings a d j u s t e d to p H 7.2 w i t h aqueous K O H before freeze-drying.
Biochim. Biophys. Acta, 69 (x963) 496--504
498
.~.. ~G. ~D0)~-IDL, IK.. s . D O D G S O N ,
R. G. PRICE
Ethanol fractionation For ethanol fractio~a~0~ ~ s t h e a m i n o p o i y s a c c h a r i d e s u l p h a t e s (x.5-2.o g in 3o m l of water) were c o ~ a a - ~ tto, ~Y~efree acid f o r m b y p a s s i n g t h r o u g h a D o w e x - 5 o c o l u m n (H + form, 5o-~,oo ~ % ~ _ Tlk~ e I ~ a t e a n d w a s h i n g s w e r e d i l u t e d to x2o m l b y t h e a d d i t i o n of wa~er 'be[ ,o~e a ~ ] ~ 6,_9, rni of glacial a c e t i c acid a n d 6_3 g of c a l c i u m acetate. Ethanol was a ~ tt~o flt~ m~_.~tttre in a d r o p w i s e m a n n e r x~'ith s t i r r i n g at r o o m t e m p e r a t u r e t o gi~-e ~ ~ 0 ~ a ~ r t of 37 % (v/v). F r e c i p i t a t i o n s t a r t i n g a t th~s e t h a n o l c o n c e n t r a t i o n ,~vas ~ ~ ~ e d i b v t h e f u r t h e r a d d i t i o n of e t h a n o l t o 4 ° °o ~v/v} a n d k e e p i n g t h e mixtaar, e ~ t 4 : g~¢ ~@ ~- A f t e r s e p a r a t i n g b y c e n t r i f u g i n g , t h e precipitate was washed by st~l~~ ~m 4~'% {v/v) e t h a n o l twice, w i t h a b s o l u t e ethar~ol a n d w i t h e t h e r u s i n g intea-me~mt~ ¢ ~ ~ , before d r y i n g in vactto o v e r CaCI z. T o t h e 4 ° % (v/v) ~ ~ e r m ~ t ~ n t w a s a d d e d e t h a n o l as before t o give a final c o n c e n t r a t i o n of 5 o % i(~'/*-))- _~tter ~ w i n g t h e m i x t u r e to s t a n d , p r e c i p i t a t e d m a t e r i a l w a s collected ~ ~vaz-~dl sm:cessively w i t h 5 ° o/ /o (v/v) a q u e o u s e t h a n o l (twice), a b s o l u t e ethatro] ~ d ~ ~ a ~ d r i e d as d e s c r i b e d a b o v e . T h e c a l c i u m salts off ~ gm~_'s~e ,hari'de s u l p h a t e s o b t a i n e d b y e t h a n o l fraction a t i o n w e r e c o n v e r t e d t,o ~ salts after passing through a Dowex-5o column (H + form) a n d t h e n ~ r ~ ~ f~ ltl~e p r e s e n c e of 80 % (v/v) e t h a n o l , c e n t r i f u g i n g a n d d r y i n g as a l r e a d y d l ~
Analytical met,hods T h e e s t e r s u l p l ~ t e ~on~e~tt$ ,0,if~ p r e p a r a t i o n s were d e t e r m i n e d g r a v i m e t n c a l l y m a f t e r h y d r o l y s i s in 3.5 N HCll a~t x ~ ? [or 2o h or s p e c t r o p h o t o m e t r i c a l l y m a f t e r sealedt u b e h y d r o l y s i s in x N I~IC~~ t ~x~;= ffvv $ h. U r o n i c acid was d e t e r m i n e d b y t h e c a r b a zole m e t h o d z° a n d ~ ~ ~c~0~g to LEVY AXD MCALLA.X~a a f t e r hx-drolvs~s in 3-5 N HCI at i o o ° ~ _~o ~. ~filt~0ge~ e s t i m a t i o n s w e r e m a d e with t h e m i c r o - K ] e I d a h l a p p a r a t u s ~" a x ~ ~ , ~ m ~ ~ t h e flame p h o t o m e t e r . A n a l y s i s figures are c o r r e c t e d for loss in w e i g h t ~ d~_a]~ t h e p o l y s a c c h a r i d e s at 80 ~ in vacuo o v e r P~,O~.
Infrared spectroscopy S p e c t r a w e r e meamar~-~l ~ ~e Perkin-Elmer Infracord spectrophotometer. All c o m p o u n d s w e r e ~ ~ m ~ % in Nujol. [~:rL-~EN'~'AL
AND
RESULTS
The heterogeneity of cra~e a~d~,o,podysa¢charide sulphate preparations A n a l y t i c a l f a g ~ e s ff~r t h e ~ s s 2 m a x salts of t h e c r u d e a r n i n o p o t y s a c c h a r i d e s u l p h a t e p r e p a r a t i o n s ffaxma lMme z~Jh~rtk, dogfish, s k a t e a n d fin w h a l e are r e c o r d e d in T a b l e I. T h e a n a l y ~ ff~r ~ ,,ti ~-hese p r e p a r a t i o n s are close t o t h e o r e t i c a l for " c h o n d r o i t i n StdlY~e-t~]l ~"° p ~ l ~ ~ e x c e p t for t h e c h a r a c t e r i s t i c p r e s e n c e of " e x c e s s " s u l p h a t e ha ~ M l~Ea~es h a r k , s k a t e a n d d o g ~ . ~ . However, paper d ~ m m ~ ~ a a a l ~ s i s of t h e acid h y d r o l y : =:tes of e a c h of t h e c r u d e p r e p a r a t i o n s ( T a t d e ll~ ~ e d ~ t h a t e a c h m a t e r i a l w a s c o m p o s e d of m o r e t h a n o n e m o l e c u l a r species_ T h e ~ ~ r ~ t i o n of D-glucuronic acid a n d D-gahcte~sarrfine w a s in a c c o r d w~_'.h t h e ~ m e a c h p r e p a r a t i o n of p o l y s a c c h a r i d e m a t e r i a l similar to chondroh~a ~ . ~ ff~e d e m o n s t r a t i o n of D-galactose, D - g l u c o s a m i n e Bioghim, Biopkys. .4cta, 69 (x96.-3~, 4 _ ~ - - 5-.,~4
AMINOPOLYSACCHARIDE TABLE FIGL~RES
ANALYTICAL
FOR
CRUDE
Nitrogen (",,J
Origin
Blue shark Skate I ) o g fish I:in w h a l e
I
AMINOPOLYSACCHARIDE
Hexosamine (%)
2.2
22.6
z.5 ".3 -'-3
28.I 29.2 22.o
CONNTITUENTS
OF
SULPHATE
Uronic acid (?6)
PREPARATIONS
Ester sulphate (%)
Potassium
I6.7 I7.o I9.o t3.7
xz.8 9.7
36.5 37.6 3o.I 37.9
TABLE MONOSACCHARIDI':
499
SULPHATES
(%)
x4.o i 1.5
II
AMINOPOLYSACCHARIDE
SULPHATE
PREPARATIONS
Glucuron ic
Galaetos
Glucosa m ine
Galactose
Fucose
Skate
+ +
+ +
+ +
+ +
+
Dogfish
+ +
+ 4-
+ +
+ +
+
['nfractionated 0 _ 4 0 o~/o f r a c t i o n 4o-5o % fraction
+ + + + --
+ + + + --
+ + -+ +
+ + -+ +
+ -+
Fin w h a l e Unfractionated o-4o % fraction 4 0 - 5 ° °/o f r a c t i o n
+ + + + +
+ + + + +
+ ~ +
Origin
acid
amine
Blue shark
+ -+
Trace --
Trace
and a trace of m e t h y l p e n t o s e suggested that the crude cartilage extracts also contain a molecular species corresponding to keratosulphate. In addition, analytical scale fractionation of each of the polysaccharides on Ecteola-cellulose was marked by the elution of carbohydrate at widely differing molarities of the NaC1-HC1 ( I : I , v]v) mixture (see Fig. x). The heterogeneity was most pronounced in the case of the blue shark preparation, but progressively less in the series skate, dogfish and fin whale.
Ethanol fractionation of aminopolysaccharide sulphate preparations from blue shark and fin whale Fractionation of the aminopolysaccharide sulphate mixture from the blue shark between the limits of ethanol concentration of o - 4 0 % (v/v) and 4 0 - 5 o % (v/v) yielded I. 15 g and o.144 g of polysaccharide material respectively. W h e n treated in an identical manner the fin whale preparation gave I.I g and 0.o8 g of polysaccharide fractions respectively. Analysis figures for these preparations are given in Table III. E x a m i n a t i o n of the monosaccharide constituents of the blue shark fractions revealed the separation of the original material into two polymer types. The fraction precipitating between o - 4 o % (v/v) ethanol was found to contain only D-glucuronic acid and D-galactosamine while the 4o-5o % fraction was characterised by its content of D-galactose and D-glucosamine with a trace of material corresponding in chromatographic mobility with L-fucose (see Table II). W h e n examined similarly the o - 4 o % Biochim.
Biophys.
A v t a , 69 ( I 9 6 3 ) 4 9 6 - - 5 0 4
500
A. G. L L O Y D ,
K. S. D O D G S O N ,
R. G. P R I C E
fraction from the fin w h a l e poly'saccharide m i x t u r e was found to contain o n l y i / glucuronic acid a n d n - g a i a c t o s a m i n e but the 4o-5 ° 7.o e t h a n o l fraction c o n t a i n e d l,-galactose, I)-glucosamine a n d L-fucose as well as trace a m o u n t s of D-glucuronic acid a n d D-galactosamine (Table l l).
,7[ 1.5
1 . 7 ~-
N
1-5 !
1.3 1.1
0.9 0.7 0.5
1Df
A
!i
.
2.0
/i I///t/1/i1 I::: /
0.3 100
200
300
400 / / i
~q _
... L
i
0.5
1.6 1.2 0.8 0.4
o.,
500
J
2.0
O.
t°
~'~.~
K3.7
B
1,1
oo
-12.0 1.6
200
P-:qnrtrqH,H H, ~ n 300
400
0.'7
t
"
O9 f
/
0-°-,7
0.4 L
2o0 30O E!utlon votume [ml]
'll 4OO I
i
500
C~
..
", 100
1.(5
~ ""
i 0.8
~°,
,~
200 Elution
5O0 12.0
~.."
0.3 0.I
/
/
D
0.8
10o
,
,
300 volume
,
400
°°
.
500
[ml]
F i g . x. A n a l v t i c a l s c a l e f r a c t i o n a t i o n o f c r u d e a m i n o p o l y s a c c h a r i d e s u l p h a t e p r e p a r a t i o n s o n E c t e o l a - c e l l u i o s e . R i g h t h a n d o r d i n a t e , e x t i n c t i o n s of f r a c t i o n s a t 485 m / , i n t h e p h e n o l - s u l p h u r i c a c i d r e a c t i o n . L e f t h a n d o r d i n a t e , m o l a r i t y of t h e N a C I - H C 1 ( I : i , v / v ) m i x t u r e . A, b l u e - s h a r k p r e p a r a t i o n ; B, s k a t e p r e p a r a t i o n : C, d o g f i s h p r e p a r a t i o n ; D, f i n - w h a l e p r e p a r a t i o n .
Infrared spectra of aminopolysaccharide s~dphate preparations T h e results of t h e i n f r a r e d spectroscopic e x a m i n a t i o n of t h e c r u d e a m i n o p o l v s a c c h a r i d e s u l p h a t e s f r o m blue shark, s k a t e a n d dogfish a n d of t h e c o r r e s p o n d i n g d e s u l p h a t e d p o l y m e r s in relation to t h a t of m a m m a l i a n c h o n d r o i t i n s u l p h a t e s A a n d C TABLE
III
A~'ALYTICAL rIGt:RES FOR 0--40°0 AND 4 0 - - 5 0 % ETHANOL FRACTIONS FRO.%! BLL'E SHARK AND FIN %VHALE Origin
Nitrogen F°;,)
Hexosarnine (",,/
Uro~li~acid (%)
Ester sulphate (% )
Blue shark o--4o ~/o 40-5 ° %
2.4 2-3
2,.x 27-5
32.z 7. i
*6.7 IS.8
I2.0 8. 4
Fin whale 0--40 % 4 0 - 5 ° °o
--.2 2.3
23.6 26.4
34-5 3,.2
I3. i I29
io.2 Io.9
l~iochim. Biophys..4cta,
Potassium (% )
60 ( I 0 6 3 ) 4 9 6 - 5 0 4
AMINOPOLYSACCHARIDE SULPHATES
501
have a l r e a d y been describedL The infrared s p e c t r u m of t h e crude aminopolysacclaaride sulphate from fin-whale cartilage (Fig. 2A) exhibits t h e c h a r a c t e r i s t i c a b s o r p t i o n at z24o cm -t and in the range 8oo-86o cm -1 a t t r i b u t a b l e to vibrations involving t h e S - O and C - O - S linkages respectively. However, it is a p p a r e n t t h a t this s p e c t r u m bears a Wave number" (lOOcrn -I) ~4 ~3 i
12
I
t
.13 ....
lo i
i
9
@
7
•
i
!
E
~}
9
10 " lt1'
12
I'3
1~; I~
W a v e l e n g t h (}J)
Fig. 2. I n f r a r e d s p e c t r a of p o l y s a c c h a r i d e s from fin w h a l e a n d b l u e s h ~ r k . . ~ , C r u d e f i n - w h a l e p o l y s a c c h a r i d e s ; B, c h e m i c a l l y d e s u l p h a t e d f i n - w h a l e p o l y s a c c h a r i d e ; C. f i n - w h a l e o--4o°~ e t h a n o l f r a c t i o n " I), f i n - w h a l e 4 0 - 5 0 % e t h a n o l f r a c t i o n ; 12_ b l u e - s h a r k o - 4 o ° o e t h a n o l f r a c t i o n " F, blueshark 4o-5o~ ethanol fraction.
closer resemblance to t h a t given b y m a t e r i a l of bovine origin r a t h e r t h a n t h e corresponding m a t e r i a l from blue s h a r k in t h a t t h e 85o cm -l b a n d ( a x i ~ C - O - S ) is most p r o n o u n c e d while t h e 89o cm -x b a n d (equatorial C - O - S ) is o n l y of limited intensity. T h a t a b s o r p t i o n at 124o cm -x, 85o c m -1 a n d 82o c m -x is associated w i t h t h e ester ~ulphate group h a s been d e m o n s t r a t e d b y t h e d i s a p p e a r a n c e of these b a n d s from t h e infrared s p e c t r u m of a whale polysaccharide p r e p a r a t i o n d e s u l p h a t e d chemically b y t h e m e t h o d of KANTOI~ AND SCHUBERT~"a (Fig. 2B). T h e infrared s p e c t r u m of t h e o-4o % fraction from t h e whale polysaccharide (Fig. 2C) is m a r k e d b y intensification of t h e b a n d a t 85o cm - t a n d t h e absence of a b a n d a t 820 cm -~ while t h e s p e c t r u m of t h e 4o-5 ° % fraction h a s a n intensified b a n d at 820 c m -x an d only limited absorption a t 85o e r a - ' (Fig. zD). I n c o n t r a s t to the correspon "ding p r e p a r a t i o n s from fin whale polysaccharide, t h e s p e c t r a of t h e blue s h a r k o-4o % a n d 4o-5o % fractions b o t h exhibit a b s o r p t i o n a t t r i b u t a b l e to t h e C - O - S v i b r a t i o n only a t 82o c m - t (Fig. 2E a n d 2F). However, t h e a n a l y t i c a l scale f r a c t i o n a t i o n of b o t h of these materiai.n on Ecteola-cellulose s h o w e d Biochim. Biophys. Acta. 69 (x963) 496---504
502
A. G. L L O Y D ,
K. ~. D O D G S O N ,
R. G. P R I C E
t h a t each p r e p a r a t i o n was still a p p a r e n t l y composed of p o l y m e r s of differing s u l p h a t e c o n t e n t (Fig. 3). I t has been d e m o n s t r a t e d previously t h a t the degree to which polys~ccharide sulphates are r e t a i n e d on Ecteola-cellulose is r e l a t e d directly to the ester s u l p h a t e c o n t e n t of the p r e p a r a t i o n 16. To establish w h e t h e r this was a c t u a l l y so in the present instance, 4 g of a 0-40 % (v/v) ethanol fr~ction f r o m the blue s h a r k was f u r t h e r f r a c t i o n a t e d on Ecteola-cellulose on a p r e p a r a t i v e scale. The elution p a t t e r n 1.3 1.1 "2.O 0.9
/
-1.6
0.7 A4B5
.~
0.5
C | - [M]
1.2
,/
0.8 0.3
0.4
0.1 100
200
Elution
volume
1.3[
300
4OO
I
5OO
[ml]
B
1.1
.j1 .s'*" i
O'g f
%85 0 . 7
c,-[M] o 1~;~ ~ " • -d~
[
i
i
100
"
--I
I
2CX~
h
i
I
1
400
300
I
500
Elutlon volume [ml] F i g . 3- A n a l y t i c a l s c a l e f r a c t i o n a t i o n o f b l u e - s h a r k o - 4 o % f r a c t i o n ( h i s t o g r a m A) a n d 40--5 ° % f r a c t i o n ( h i s t o g r a m B) o n E c t e o l a - c e l l u l o s e . O r d i n a t e s a s in F i g . z.
0.3M
,m
O.TM
1.OM m
T
1.6i
2.0M -T-
I t i i
Relotive A485
t I I l I I
1.~
1.0
| I I
0.8 0.6 0.4
[ I 4 I
0.2
| i I
I
0.2
o.a
--=~
0.6
0.8
-
[
1.0
t2 Elutlon
.a
1.6
1.8
2.0
2.2
24
9.6
2.8 3.0
3.2 3.4
3.6
3.8
v o l u m e [litre&]
F i g . 4- P r e p a r a t i v e s c a l e f r a c t i o n a t i o n o f b l u e - s h a r k 0 - 4 ° % e t h a n o l f r a c t i o n o n E c t e o l a - c e l l u l o s e .
Bioghim. Biophys. Acta, 69 (x963) 4 9 6 - - 5 0 4
A a M I N O P O L Y S A C C H A R I D E SULPHATE.'2,
503
•ob'ta~ned ,0m t t ~ . , ~ t~e column in a stepwise m a n n e r with the N a C I - H C I (I : I, v/v) mix-~re :art ,~0~m~mtt~kms of o.3 M, 0. 7 M, I.O M and 2.o M is shown in Fig. 4. The anat.~,-~icaU t f ~ m ~ $0tr tfl~e potassium salts of the polysaccharides isolated from eluates corres.po~diim~ t~0,n l ~ e molarities (Table IV) reflect the increasing sulphate contents of .~¢_hea~dh,tmlharr .,s~eeiJes in the preparation. TABLE ~iLxYut~Is2=[L
l~,IXg,+~'lt~S
FOR
POL%'SACCHARIDE
~CII~IIIL~X"XON.ATION
Eluaut
OF
THE
SULPHATES
BLUE-SHARK
ISOLATED o/ /o
0--40
ETHANOL
AFTER
THE
FRACTION
Nitrogen
Hexosamine
Uronic acid
Ester sulphate
Potassium
~'g,)
C:~
("'o)
(%)
(%)
(%)
(%)
o.3 0.77
ot.T'-'
2.6 2.2 5
20.4 2 3.o
34.5 39.5
*4- t 17.4 x 8. 3
,, .6 i 4.0
--
22.6
--
;o 2:0
~'~
IV
"-,~'4+
o~.P,oS~
2, 5
--
o~og2E
--
--
DISCUSSION
]~t is ,aa~mttm~ t~0tm tlke present work t h a t the aminopolysaccharide sulphate esters cons~ ttttm, m n ~ i ~ of cartilage from blue shark, dogfish a n d skate differ subs t a n t i a ~ ttmrm ¢ . r r e s p o n d i n g polymers present in the cartilage of b o t h terrestrial a n d a q u a ~ c ~ m r r ~ l k . lP~o~'mer species similar to m a m m a l i a n chondroitin sulphate and kera,~~t~e Ika~e, been d e m o n s t r a t e d b u t these are eharacterised b y their widely diff~ ~ ~ .tI ester sulphate. Although the existence in elasmobranch cartilage of ~ :~3alliptk~e@potysaccharides has formed the subject of earlier reports =-s, little is t~¢.am w ~ ~ the effect of ester sulphate content on the properties of these m a t emi,a~. ~ mmtlk~d:4 presently described permit the isolation of polysaccharide species ,~f ~¢medl s ~ p h a t e c o n t e n t suitable for further structural and enzymological ~tud,v..
IPl~e ._~nl~,t~ectt a~i~opolysaccharides from fin-whale cartilage closely resemble prepa~.nnii0ms ~ . r ~ terrestrial m a m m a l s in t h a t although containing chondroitin s u l p h ~ e :+~_~$I k ~ a m G ~ h a t e , fractionation of the p r e p a r a t i o n on Ecteola-cellulose reveaAs ,~,m. ~ ib o n l y limited heterogeneity as regards ester sulphate contents of t h e ,oo~ii~i,m~mtiT~( ~ : . . ~ G E R T Z A N D REICRARD!6). In particular, the presence iv the inkawedi ~ o~ strong absorption at 85o cm -~ b o t h for crude preparations and t h e ,.o-#o'D, t ~ r ~ t i ~ suggests t h a t material identical in structure to chondroitin .% i~ tt~. ~ ":m~iple polysaccharide constituent of the original matrix, condr~ ~U#m~, ~ ~ k e r a t o s ~ p h a t e (both of which give rise to absorption at 82o ,<~m-~))~ I t m ~ n t in q u a n t i t a t i v e l y lesser amounts. ~e ~ 0 ~ d~monstration of a m e t h y l p e n t o s e similar to L-fucose as a mono.~aceh~e ~~,'~ in all of the crude preparations, and its specific association ~ / a ~ g a I l m ~ 0 ~ am~ D-glucosamine in the 40-50 % fraction from blue shark, deserves ~-pecfi~ ~ 0 m . ~ i a n keratosulphate preparations, having n-galactose and I~-gl~~ ~ ipri~iple monosaccharide constituents, have been reported conM s t ~ ~ ¢ ~ ~ traces (1-2 %) of a methylpentose component =~. In contrast. v~te~raI1 ,¢mltrb]t~ imm[uced to form in tissue cartilage and not containing keratoB i o c h i m . rCiophys. A c t a , 69 {x963) 4 9 6 - 5 0 4
50 4
G, LLOYD, K. S. DODGSON, R. G. PRICE
s u l p h a t e (as s h o w n b y t h e a b s e n c e o f D - g a l a c t o s e a n d o - g l u c o s a m i n e ) w a s a l s o ~vee f r o m m e t h y l p e n t o s e 25. A l t h o u g h these observations present evidence for the a s s o c i a t i o n of m e t h y l p e n t o s e components with keratosulphate f r a c t i o n s , i t is ~a~t known how these moieties are involved structurally in the polymer molecule ~. ACKNOWLEDGEMENTS T h i s w o r k h a s b e e n s u p p o r t e d b y a g r a n t (A 1 9 8 2 ) f r o m t h e A r t h r i t i s a n d M e t a b o l i c D i s e a s e s D i v i s i o n of t h e U.G. P~,blic H e a l t h S e r v i c e . W e w o u l d l i k e t o e x p r e s s o t t r t h a n k s t o D r . K . E N G E o f t h e U n i v e r s i t y of O s l o f o r s u p p l i e s of f i n - w h a l e c a r t i l a g e a n d t o t h e X V e l l c o m e T r u s t f o r t h e g i f t of a n i n f r a r e d s p e c t r o p h o t o m e t e r . O n e off n s ( R . G . P . ) is g r a t e f u l t o t h e D e p a r t m e n t of S c i e n t i f i c a n d I n d u s t r i a l R e s e a r c h f¢~r a studentship. REFERENCES t K. MEYER, ~llolecular Biology, Academic Press, London, [cj6o, p. 59. 2 K. NAGANIS~X, N. TAKAHASnI AND F. EGAMX, Bull. Ch~m. Soc. J a p a n , ,_t) (t95b) 434a j . ~v. LASH AND M. XV. XVHITEHOUSE, .4rch. Biochem. Biophys., 9o (I96o) I59. 4 M. B. MATHEWS AND M. INOUYE, Biochim. Biophys. Acta, 53 (I96I) 500. 5 M. ]3. M,~THEWS, Proceedings Vth Int. Congr. Biochemisto, , AIoscow, 1961, A b s t r a c t b.2~. a M. B. MATHE~'S, Nature, 193 (x962) 378. 7 $. SUZUKr, J . Biol. Chem., 235 (t96o) 358o. 0 T. FURUHASHI, J. Biochem, (Tokyo), 50 (I96I) 546. 9 A. G. LLOYD, K. S. DODGSON, R. G. PRICE AND F. A. ROSE, Biochim. Biophys..4cta, 46 (I96~i xo8. t0 I. SMITH, Chromatographic Techniqtws, W i l l i a m H e i n e m a n n Medical Books L t d . , London, p. I67. i1 S, M. PARTRIDGE, Nature, I64 (t949) 479. lS L . H O U G H , J . K . N . J O N E S AND ~V. H . *~VADMAN, f . Chem. Sot., ( I 9 5 0 ) 1r702. l* S, M . P A R T R I D G E , Biochem. J . , 42 ( I 9 4 8 ) 2 3 8 . 14 S. G A R D E L L , F . HI~IJKI~NSKJOLD AND A. R O C H - N O R L U N D , A c t a Chem. Stand., 4 (I95o) 9 7 o. t~ p . j. S T O F F Y N AND R . ~W. JEANLOZ, Arch. Biochem. Biophys., 52 (z954) 373te ~'NI. RINGERTZ AND P. REICHARD, Mcta Chem. Scand., t4 (t96o) 303. 17 R. MONTGOMERY, Biochim. Biophys. ,4cta, 48 (~901) 59I. i s j . W . H. LuG6, Biochem. J . , 32 (I938) 2114. a~ K. S. DODGSON Al~O R. G. PRICE, Biochem. J., 84 ([962) Io6. 20 Z. Dxscttv-, J . Biol, Chem., 167 (x947) I89. • l G. A. L~-vvY AND A. MCALLA~', Biochem. J.. 73 (x959) I27. z~ R. MARKHAM, Biochem. J . , 36 (t94 z) 79o. 2s T. G. KANTOR AND ~I. SCHUBERT, J. A m . Chem. So¢., 79 (I956) x52. t 4 0 . ROSEN, P. HOFFMAN AND K. MEYER, Federation Proc., 19 (I96O) 147. '~ j. w . LASH AND M. ~r. ~VHITEHOUSE, Arch. Biochem. Biophys., 90 (I96o) 159. t, S° HIRANO, P. HOFF.~tAI"~AND K. MEYER, f . OYg. Chem., 26 (I96I) 5064.
Biochim. Biophys. Acta, 69 (I963) 49tr--3-o4
BIOCHIMICA ET BIOPHYSICA ACTA
COMPARATIVE CARTILAGE I. P O L Y S A C C H A R I D E S
STUDIES
AMINOPOLYSACCHARIDE
ON SL'~L.~,~,~~
F R O M S H A R K , S K A T E , D O G F I S H A_.%-~-D~
~VftE~.EF.:
A. (5. LI.OYI), K. S. D()DGS()S AND R. G. PRICE
Department of Biochemistr.v, University of IVales. Cardiff (Great Britain) (Received June I8th, I 9 6 " ) SU~IMARY S t u d i e s on t h e p o l y s a c c h a r i d e c o n s t i t u e n t s of c a r t i l a g e f r o m a d u l t s p e c i ~ ~0ff5Ik~e s h a r k , s k a t e a n d dogfish r e v e a l t h e p r e s e n c e in e a c h of t h e s e prepar~.~i~m~,dfrm~e.fiat c o r r e s p o n d i n g t o c h o n d r o i t i n s u l p h a t e a n d k e r a t o s u l p h a t e . Howex, er. ,~m6h ,~t a m i n o p o l y s a c c h a r i d e s is c h a r a c t e r i s e d b y t h e p r e s e n c e of f r a c t i o n s ~¢ff~i~h~_" & ~ 5 ~ n g s u l p h a t e c o n t e n t . I n c o n t r a s t to t h e m a t e r i a l f r o m c a r t i l a g i n o u s ~ tt~e. ~.~tys a c c h a r i d e m i x t u r e from_ fin-whale c a r t i l a g e h a s c h o n d r o i t i n s u ~ ~ ~ ~ rn~jor constituent, both chondroitin sulphate C and keratosulphate being~r~ im.'~s t a n t i a l l y lower a m o u n t s . INTRODUCTION I t h a s n o w b e e n e s t a b l i s h e d t h a t m a m m a l i a n c a r t i l a g e s are chaxaa~emk~t 15~ r_he p r e s e n c e of t h r e e t y p e s of a r n i n o p o l y s a c c h a r i d e s u l p h a t e x. I n gemerat!, , d h 0 m ~ : ~ s u l p h a t e A ( r e p e a t i n g unit" 3-O-fl-D-glucuronido-N-acetyl-D-galactosamaineq4~d~tk~e) a n d c h o n d r o i t i n s u l p h a t e C ( r e p e a t i n g unit" 3 - O - f l - D - g l u c u r o n i d o - N - a c ~ ~ . ~ . ~ s a m i n e 6 - s u l p h a t e ) occur as m a i n c o n s t i t u e n t s , t o g e t h e r w i t h varbfiaag~rr~." , ~ 2 ~ r : ~ r n V ~ of k e r a t o s u l p h a t e ( r e p e a t i n g unit" 4-O-[3-D-galactosido-N-acety]-~nt~o~nrni_ne 6-sulphate). R e c e n t r e p o r t s 2-s reflect a s u b s t a n t i a l increase in i n t e r e s t i n ~Jae ~rrr/m~p)~Vs a c c h a r i d e c o n s t i t u e n t s of t h e c o n n e c t i v e tissues of c a r t i l a g i n o u s :fmh. ~nn r~ii~,~rrtText LLOYD et al. 9 r e p o r t e d t h e isolation a n d i n f r a r e d s p e c t r o s c o p i c exa,,m~rlii0m ,~fl poEy-s a c c h a r i d e s u l p h a t e s f r o m t h e c a r t i l a g e of t h e blue s h a r k (Carchart~,~s ~g~,aT~9),. t~e d o g f i s h (Scyllium canicula) a n d t h e s k a t e (Raja batis). T h e s e obserxr~tS~m~ lta~e, m~w b e e n e x t e n d e d t o i n c l u d e s t u d i e s on t h e c o m p o s i t i o n of t h e s e ma-~emia]~ :mx~i off ~ e s u l p h a t e d a m i n o p o l y s a c c h a r i d e s f r o m t h e c a r t i l a g e of t h e fin ~ 1 , ~ !g~h~n/hno~
physalus). MATERIAL~ AND .~IETHODS
A rninopolysacchari& sulphate preparat-ons P r e p a r a t i o n s of a m i n o p o l y s a c c h a r i d e ~ulphate m i x t u r e s were , 0 t ~ m ~ L p o t a s s i u m salts, f r o m a d u l t c a r t i l a g e s a m p l e s as d e s c r i b e d earlier.
a,s-v_tle
Biochim. Biophys. :4a;,. ,69 ((~sjt-5~4~-5o4!
AMINOPOLYSACCHARIDE
SULPHATES
497
31onosaccharide constituettts of polysaccharide preparations Portions (Ioo-2oo mg) of t h e polysaccharides dissolved in IO ml of I N H~SO4 were h e a t e d in sealed t u b e s for 4-5 h at IiO °. After cooling, t h e h y d r o l y s a t e s were n d-n.:l tre,,L,u with Ba(OH)o to p H 8.0 a n d t h e n c e n t r i f u g e d to remove BaSO4. Excess B a ~+ in t h e s u p e r n a t a n t s was t h e n precipiti~ted b y gassing with CO2 a n d t h e BaCO3 precipitates r e m o v e d b y centr!fuging. The p r o d u c t s were t h e n c o n c e n t r a t e d to d r y n e s s b y r o t a r y e v a p o r a t i o n u n d e r reduced pressure a t 35 ° and the residues redissolved in o.5-ml portions of water. T h e c o n c e n t r a t e d h y d r o l y s a t e s were c h r o m a t o g r a p h e d on W h a t m a n No. I or 3MM p a p e r s using d o w n w a r d irrigation w i t h p h e n o l - a m m o n i a - w a t e r t° or e t h y l a c e t a t e - p y r i d i n e - w a t e r (I2O'5O:4o, v/v) as solvents. The results of u n i - d i m e n s i o n a l s e p a r a t i o n s were checked b y t w o d i m e n s i o n a l c h r o m a t o g r a p h 3, using c o m b i n a t i o n s of p h e n o l - w a t e r - a m m o n i a 1°, e t h y l a c e t a t e - p y r i d i n e - w a t e r or b u t a n . . I - o l - a c e t i c a c i d - w a t e r (5o" I 2 : 2 5 , v/v) as developing solvents. S e p a r a t e d c o m p o n e n t s were d e t e c t e d w i t h aniline h y d r o g e n p h t h a l a t e n or p - a n i s i d i n O 2 sprays for r e d u c i n g s u b s t a n c e s a n d w i t h E l s o n - M o r g a n r e a g e n t s Is for hexosamines. T h e n a t u r e of t h e h e x o s a m i n e c o m p o n e n t s was also a s c e r t a i n e d by conversion to t h e c o r r e s p o n d i n g p e n t o s e d e r i v a t i v e s ~4, ~5 Chromatography on Ecleola-cdlttlose Commercial g r a d e Ecteola-ceilulose (Brown Co., Berlin, N.Y.) was s u s p e n d e d in o.5 M NaCI and, after p a c k i n g in a glass column, t r e a t e d successively w i t h o.5 M N a O H , 3.o M NaC1 a n d o.I M N a C l - o . I M HCI ( I ' I , v/v), according to RlXGEI~TZ AND
RE1CHARD
16.
For a n a l y t i c a l s e p a r a t i o n s t h e modified cellulose was p a c k e d u n d e r g r a v i t y flow in a glass t u b e (15 cm i cm) fitted at t h e lower end w i t h a f r i t t e d glass disc. T h e column was t h e n w a s h e d w i t h 5 h o l d - u p v o l u m e s of t h e o.I M N a C I - o . I M HC1 (i :I, vlv ) m i x t u r e . F o r t h e s e p a r a t i o n t h e p o l y s a c c h a r i d e ( I 5 - 2 o rag), dissolved in 2-4 ml of w a t e r , was c o n v e r t e d t.'~ t h e free acid b y passing t h r o u g h a Dowex-5o c o l u m n (H + 2orm, 5o-xoo mesh) a n d t h e acid e l u a t e a n d w a s h i n g s c o n c e n t r a t e d to I ml by freeze-drying. T h e sample was t h e n a d d e d to t h e c o l u m n a n d allowed to d r a i n i n t o t h e resin b e d u n d e r g r a v i t y flow. E l u t i o n of c a r b o h y d r a t e w a s achieved using a g r a d i e n t p r o d u c e d by r u n n i n g IOO ml of 3.o M NaC1-3.o M HC! (I" I, v/'v) into a m i x i n g vessel fitted w i t h a m a g n e t i c stirrer a n d c o n t a i n i n g 4oo ml of o.I M N a C l - o . I M HC1 ( i ' I , v/v). T h e e l u a t e from t h e cohtmn was collected in 5-ml fractions a n d t h e c a r b o h y d r a t e c o n t e n t of t h e s e d e t e r m i n e d w i t h the p h e n o l - s u l p h u r i c acid reaction~L On a p r e p a r a t i v e scale, t h e polysaccharides (3-4 g) were c o n v e r t e d to the free acids as before, a n d t h e n a d d e d to an Ecteola-cellulose e o l u n m (6o cm × 3-5 cm). T h e c o l u m n w a s t h e n eluted in a stepwise m a n n e r using t h e a p p r o p r i a t e c o n c e n t r a t i o n s of t h e N a C 1 - H C I (i "i, v/v) m i x t u r e i n d i c a t e d b y t h e a n a l y t i c a l e x p e r i m e n t s . For t h e isolation of t h e polysaccharides from t xe eluates, fractions corresponding to h o m o g e n e o u s elution p e a k s were pooled a n d c o n c e n t r a t e d to 25o ml by r o t a r y e v a p o r a t i o n u n d e r reduced pressure at 37 °. T h e solutio/ls were t h e n dialysed e x h a u s tively against w a t e r for 24 h at 4 °. T h e dialysate was t h e n c o n c e n t r a t e d to IOO ml b y r o t a r y e v a p o r a t i o n , passed t h r o u g h a Dowex-5 o c o l u m n (H + form) ,tnd t h e acid e l u a t e a n d washings a d j u s t e d to p H 7.2 w i t h aqueous K O H before freeze-drying.
Biochim. Biophys. Acta, 69 (x963) 496--504
498
.~.. ~G. ~D0)~-IDL, IK.. s . D O D G S O N ,
R. G. PRICE
Ethanol fractionation For ethanol fractio~a~0~ ~ s t h e a m i n o p o i y s a c c h a r i d e s u l p h a t e s (x.5-2.o g in 3o m l of water) were c o ~ a a - ~ tto, ~Y~efree acid f o r m b y p a s s i n g t h r o u g h a D o w e x - 5 o c o l u m n (H + form, 5o-~,oo ~ % ~ _ Tlk~ e I ~ a t e a n d w a s h i n g s w e r e d i l u t e d to x2o m l b y t h e a d d i t i o n of wa~er 'be[ ,o~e a ~ ] ~ 6,_9, rni of glacial a c e t i c acid a n d 6_3 g of c a l c i u m acetate. Ethanol was a ~ tt~o flt~ m~_.~tttre in a d r o p w i s e m a n n e r x~'ith s t i r r i n g at r o o m t e m p e r a t u r e t o gi~-e ~ ~ 0 ~ a ~ r t of 37 % (v/v). F r e c i p i t a t i o n s t a r t i n g a t th~s e t h a n o l c o n c e n t r a t i o n ,~vas ~ ~ ~ e d i b v t h e f u r t h e r a d d i t i o n of e t h a n o l t o 4 ° °o ~v/v} a n d k e e p i n g t h e mixtaar, e ~ t 4 : g~¢ ~@ ~- A f t e r s e p a r a t i n g b y c e n t r i f u g i n g , t h e precipitate was washed by st~l~~ ~m 4~'% {v/v) e t h a n o l twice, w i t h a b s o l u t e ethar~ol a n d w i t h e t h e r u s i n g intea-me~mt~ ¢ ~ ~ , before d r y i n g in vactto o v e r CaCI z. T o t h e 4 ° % (v/v) ~ ~ e r m ~ t ~ n t w a s a d d e d e t h a n o l as before t o give a final c o n c e n t r a t i o n of 5 o % i(~'/*-))- _~tter ~ w i n g t h e m i x t u r e to s t a n d , p r e c i p i t a t e d m a t e r i a l w a s collected ~ ~vaz-~dl sm:cessively w i t h 5 ° o/ /o (v/v) a q u e o u s e t h a n o l (twice), a b s o l u t e ethatro] ~ d ~ ~ a ~ d r i e d as d e s c r i b e d a b o v e . T h e c a l c i u m salts off ~ gm~_'s~e ,hari'de s u l p h a t e s o b t a i n e d b y e t h a n o l fraction a t i o n w e r e c o n v e r t e d t,o ~ salts after passing through a Dowex-5o column (H + form) a n d t h e n ~ r ~ ~ f~ ltl~e p r e s e n c e of 80 % (v/v) e t h a n o l , c e n t r i f u g i n g a n d d r y i n g as a l r e a d y d l ~
Analytical met,hods T h e e s t e r s u l p l ~ t e ~on~e~tt$ ,0,if~ p r e p a r a t i o n s were d e t e r m i n e d g r a v i m e t n c a l l y m a f t e r h y d r o l y s i s in 3.5 N HCll a~t x ~ ? [or 2o h or s p e c t r o p h o t o m e t r i c a l l y m a f t e r sealedt u b e h y d r o l y s i s in x N I~IC~~ t ~x~;= ffvv $ h. U r o n i c acid was d e t e r m i n e d b y t h e c a r b a zole m e t h o d z° a n d ~ ~ ~c~0~g to LEVY AXD MCALLA.X~a a f t e r hx-drolvs~s in 3-5 N HCI at i o o ° ~ _~o ~. ~filt~0ge~ e s t i m a t i o n s w e r e m a d e with t h e m i c r o - K ] e I d a h l a p p a r a t u s ~" a x ~ ~ , ~ m ~ ~ t h e flame p h o t o m e t e r . A n a l y s i s figures are c o r r e c t e d for loss in w e i g h t ~ d~_a]~ t h e p o l y s a c c h a r i d e s at 80 ~ in vacuo o v e r P~,O~.
Infrared spectroscopy S p e c t r a w e r e meamar~-~l ~ ~e Perkin-Elmer Infracord spectrophotometer. All c o m p o u n d s w e r e ~ ~ m ~ % in Nujol. [~:rL-~EN'~'AL
AND
RESULTS
The heterogeneity of cra~e a~d~,o,podysa¢charide sulphate preparations A n a l y t i c a l f a g ~ e s ff~r t h e ~ s s 2 m a x salts of t h e c r u d e a r n i n o p o t y s a c c h a r i d e s u l p h a t e p r e p a r a t i o n s ffaxma lMme z~Jh~rtk, dogfish, s k a t e a n d fin w h a l e are r e c o r d e d in T a b l e I. T h e a n a l y ~ ff~r ~ ,,ti ~-hese p r e p a r a t i o n s are close t o t h e o r e t i c a l for " c h o n d r o i t i n StdlY~e-t~]l ~"° p ~ l ~ ~ e x c e p t for t h e c h a r a c t e r i s t i c p r e s e n c e of " e x c e s s " s u l p h a t e ha ~ M l~Ea~es h a r k , s k a t e a n d d o g ~ . ~ . However, paper d ~ m m ~ ~ a a a l ~ s i s of t h e acid h y d r o l y : =:tes of e a c h of t h e c r u d e p r e p a r a t i o n s ( T a t d e ll~ ~ e d ~ t h a t e a c h m a t e r i a l w a s c o m p o s e d of m o r e t h a n o n e m o l e c u l a r species_ T h e ~ ~ r ~ t i o n of D-glucuronic acid a n d D-gahcte~sarrfine w a s in a c c o r d w~_'.h t h e ~ m e a c h p r e p a r a t i o n of p o l y s a c c h a r i d e m a t e r i a l similar to chondroh~a ~ . ~ ff~e d e m o n s t r a t i o n of D-galactose, D - g l u c o s a m i n e Bioghim, Biopkys. .4cta, 69 (x96.-3~, 4 _ ~ - - 5-.,~4
AMINOPOLYSACCHARIDE TABLE FIGL~RES
ANALYTICAL
FOR
CRUDE
Nitrogen (",,J
Origin
Blue shark Skate I ) o g fish I:in w h a l e
I
AMINOPOLYSACCHARIDE
Hexosamine (%)
2.2
22.6
z.5 ".3 -'-3
28.I 29.2 22.o
CONNTITUENTS
OF
SULPHATE
Uronic acid (?6)
PREPARATIONS
Ester sulphate (%)
Potassium
I6.7 I7.o I9.o t3.7
xz.8 9.7
36.5 37.6 3o.I 37.9
TABLE MONOSACCHARIDI':
499
SULPHATES
(%)
x4.o i 1.5
II
AMINOPOLYSACCHARIDE
SULPHATE
PREPARATIONS
Glucuron ic
Galaetos
Glucosa m ine
Galactose
Fucose
Skate
+ +
+ +
+ +
+ +
+
Dogfish
+ +
+ 4-
+ +
+ +
+
['nfractionated 0 _ 4 0 o~/o f r a c t i o n 4o-5o % fraction
+ + + + --
+ + + + --
+ + -+ +
+ + -+ +
+ -+
Fin w h a l e Unfractionated o-4o % fraction 4 0 - 5 ° °/o f r a c t i o n
+ + + + +
+ + + + +
+ ~ +
Origin
acid
amine
Blue shark
+ -+
Trace --
Trace
and a trace of m e t h y l p e n t o s e suggested that the crude cartilage extracts also contain a molecular species corresponding to keratosulphate. In addition, analytical scale fractionation of each of the polysaccharides on Ecteola-cellulose was marked by the elution of carbohydrate at widely differing molarities of the NaC1-HC1 ( I : I , v]v) mixture (see Fig. x). The heterogeneity was most pronounced in the case of the blue shark preparation, but progressively less in the series skate, dogfish and fin whale.
Ethanol fractionation of aminopolysaccharide sulphate preparations from blue shark and fin whale Fractionation of the aminopolysaccharide sulphate mixture from the blue shark between the limits of ethanol concentration of o - 4 0 % (v/v) and 4 0 - 5 o % (v/v) yielded I. 15 g and o.144 g of polysaccharide material respectively. W h e n treated in an identical manner the fin whale preparation gave I.I g and 0.o8 g of polysaccharide fractions respectively. Analysis figures for these preparations are given in Table III. E x a m i n a t i o n of the monosaccharide constituents of the blue shark fractions revealed the separation of the original material into two polymer types. The fraction precipitating between o - 4 o % (v/v) ethanol was found to contain only D-glucuronic acid and D-galactosamine while the 4o-5o % fraction was characterised by its content of D-galactose and D-glucosamine with a trace of material corresponding in chromatographic mobility with L-fucose (see Table II). W h e n examined similarly the o - 4 o % Biochim.
Biophys.
A v t a , 69 ( I 9 6 3 ) 4 9 6 - - 5 0 4
500
A. G. L L O Y D ,
K. S. D O D G S O N ,
R. G. P R I C E
fraction from the fin w h a l e poly'saccharide m i x t u r e was found to contain o n l y i / glucuronic acid a n d n - g a i a c t o s a m i n e but the 4o-5 ° 7.o e t h a n o l fraction c o n t a i n e d l,-galactose, I)-glucosamine a n d L-fucose as well as trace a m o u n t s of D-glucuronic acid a n d D-galactosamine (Table l l).
,7[ 1.5
1 . 7 ~-
N
1-5 !
1.3 1.1
0.9 0.7 0.5
1Df
A
!i
.
2.0
/i I///t/1/i1 I::: /
0.3 100
200
300
400 / / i
~q _
... L
i
0.5
1.6 1.2 0.8 0.4
o.,
500
J
2.0
O.
t°
~'~.~
K3.7
B
1,1
oo
-12.0 1.6
200
P-:qnrtrqH,H H, ~ n 300
400
0.'7
t
"
O9 f
/
0-°-,7
0.4 L
2o0 30O E!utlon votume [ml]
'll 4OO I
i
500
C~
..
", 100
1.(5
~ ""
i 0.8
~°,
,~
200 Elution
5O0 12.0
~.."
0.3 0.I
/
/
D
0.8
10o
,
,
300 volume
,
400
°°
.
500
[ml]
F i g . x. A n a l v t i c a l s c a l e f r a c t i o n a t i o n o f c r u d e a m i n o p o l y s a c c h a r i d e s u l p h a t e p r e p a r a t i o n s o n E c t e o l a - c e l l u i o s e . R i g h t h a n d o r d i n a t e , e x t i n c t i o n s of f r a c t i o n s a t 485 m / , i n t h e p h e n o l - s u l p h u r i c a c i d r e a c t i o n . L e f t h a n d o r d i n a t e , m o l a r i t y of t h e N a C I - H C 1 ( I : i , v / v ) m i x t u r e . A, b l u e - s h a r k p r e p a r a t i o n ; B, s k a t e p r e p a r a t i o n : C, d o g f i s h p r e p a r a t i o n ; D, f i n - w h a l e p r e p a r a t i o n .
Infrared spectra of aminopolysaccharide s~dphate preparations T h e results of t h e i n f r a r e d spectroscopic e x a m i n a t i o n of t h e c r u d e a m i n o p o l v s a c c h a r i d e s u l p h a t e s f r o m blue shark, s k a t e a n d dogfish a n d of t h e c o r r e s p o n d i n g d e s u l p h a t e d p o l y m e r s in relation to t h a t of m a m m a l i a n c h o n d r o i t i n s u l p h a t e s A a n d C TABLE
III
A~'ALYTICAL rIGt:RES FOR 0--40°0 AND 4 0 - - 5 0 % ETHANOL FRACTIONS FRO.%! BLL'E SHARK AND FIN %VHALE Origin
Nitrogen F°;,)
Hexosarnine (",,/
Uro~li~acid (%)
Ester sulphate (% )
Blue shark o--4o ~/o 40-5 ° %
2.4 2-3
2,.x 27-5
32.z 7. i
*6.7 IS.8
I2.0 8. 4
Fin whale 0--40 % 4 0 - 5 ° °o
--.2 2.3
23.6 26.4
34-5 3,.2
I3. i I29
io.2 Io.9
l~iochim. Biophys..4cta,
Potassium (% )
60 ( I 0 6 3 ) 4 9 6 - 5 0 4
AMINOPOLYSACCHARIDE SULPHATES
501
have a l r e a d y been describedL The infrared s p e c t r u m of t h e crude aminopolysacclaaride sulphate from fin-whale cartilage (Fig. 2A) exhibits t h e c h a r a c t e r i s t i c a b s o r p t i o n at z24o cm -t and in the range 8oo-86o cm -1 a t t r i b u t a b l e to vibrations involving t h e S - O and C - O - S linkages respectively. However, it is a p p a r e n t t h a t this s p e c t r u m bears a Wave number" (lOOcrn -I) ~4 ~3 i
12
I
t
.13 ....
lo i
i
9
@
7
•
i
!
E
~}
9
10 " lt1'
12
I'3
1~; I~
W a v e l e n g t h (}J)
Fig. 2. I n f r a r e d s p e c t r a of p o l y s a c c h a r i d e s from fin w h a l e a n d b l u e s h ~ r k . . ~ , C r u d e f i n - w h a l e p o l y s a c c h a r i d e s ; B, c h e m i c a l l y d e s u l p h a t e d f i n - w h a l e p o l y s a c c h a r i d e ; C. f i n - w h a l e o--4o°~ e t h a n o l f r a c t i o n " I), f i n - w h a l e 4 0 - 5 0 % e t h a n o l f r a c t i o n ; 12_ b l u e - s h a r k o - 4 o ° o e t h a n o l f r a c t i o n " F, blueshark 4o-5o~ ethanol fraction.
closer resemblance to t h a t given b y m a t e r i a l of bovine origin r a t h e r t h a n t h e corresponding m a t e r i a l from blue s h a r k in t h a t t h e 85o cm -l b a n d ( a x i ~ C - O - S ) is most p r o n o u n c e d while t h e 89o cm -x b a n d (equatorial C - O - S ) is o n l y of limited intensity. T h a t a b s o r p t i o n at 124o cm -x, 85o c m -1 a n d 82o c m -x is associated w i t h t h e ester ~ulphate group h a s been d e m o n s t r a t e d b y t h e d i s a p p e a r a n c e of these b a n d s from t h e infrared s p e c t r u m of a whale polysaccharide p r e p a r a t i o n d e s u l p h a t e d chemically b y t h e m e t h o d of KANTOI~ AND SCHUBERT~"a (Fig. 2B). T h e infrared s p e c t r u m of t h e o-4o % fraction from t h e whale polysaccharide (Fig. 2C) is m a r k e d b y intensification of t h e b a n d a t 85o cm - t a n d t h e absence of a b a n d a t 820 cm -~ while t h e s p e c t r u m of t h e 4o-5 ° % fraction h a s a n intensified b a n d at 820 c m -x an d only limited absorption a t 85o e r a - ' (Fig. zD). I n c o n t r a s t to the correspon "ding p r e p a r a t i o n s from fin whale polysaccharide, t h e s p e c t r a of t h e blue s h a r k o-4o % a n d 4o-5o % fractions b o t h exhibit a b s o r p t i o n a t t r i b u t a b l e to t h e C - O - S v i b r a t i o n only a t 82o c m - t (Fig. 2E a n d 2F). However, t h e a n a l y t i c a l scale f r a c t i o n a t i o n of b o t h of these materiai.n on Ecteola-cellulose s h o w e d Biochim. Biophys. Acta. 69 (x963) 496---504
502
A. G. L L O Y D ,
K. ~. D O D G S O N ,
R. G. P R I C E
t h a t each p r e p a r a t i o n was still a p p a r e n t l y composed of p o l y m e r s of differing s u l p h a t e c o n t e n t (Fig. 3). I t has been d e m o n s t r a t e d previously t h a t the degree to which polys~ccharide sulphates are r e t a i n e d on Ecteola-cellulose is r e l a t e d directly to the ester s u l p h a t e c o n t e n t of the p r e p a r a t i o n 16. To establish w h e t h e r this was a c t u a l l y so in the present instance, 4 g of a 0-40 % (v/v) ethanol fr~ction f r o m the blue s h a r k was f u r t h e r f r a c t i o n a t e d on Ecteola-cellulose on a p r e p a r a t i v e scale. The elution p a t t e r n 1.3 1.1 "2.O 0.9
/
-1.6
0.7 A4B5
.~
0.5
C | - [M]
1.2
,/
0.8 0.3
0.4
0.1 100
200
Elution
volume
1.3[
300
4OO
I
5OO
[ml]
B
1.1
.j1 .s'*" i
O'g f
%85 0 . 7
c,-[M] o 1~;~ ~ " • -d~
[
i
i
100
"
--I
I
2CX~
h
i
I
1
400
300
I
500
Elutlon volume [ml] F i g . 3- A n a l y t i c a l s c a l e f r a c t i o n a t i o n o f b l u e - s h a r k o - 4 o % f r a c t i o n ( h i s t o g r a m A) a n d 40--5 ° % f r a c t i o n ( h i s t o g r a m B) o n E c t e o l a - c e l l u l o s e . O r d i n a t e s a s in F i g . z.
0.3M
,m
O.TM
1.OM m
T
1.6i
2.0M -T-
I t i i
Relotive A485
t I I l I I
1.~
1.0
| I I
0.8 0.6 0.4
[ I 4 I
0.2
| i I
I
0.2
o.a
--=~
0.6
0.8
-
[
1.0
t2 Elutlon
.a
1.6
1.8
2.0
2.2
24
9.6
2.8 3.0
3.2 3.4
3.6
3.8
v o l u m e [litre&]
F i g . 4- P r e p a r a t i v e s c a l e f r a c t i o n a t i o n o f b l u e - s h a r k 0 - 4 ° % e t h a n o l f r a c t i o n o n E c t e o l a - c e l l u l o s e .
Bioghim. Biophys. Acta, 69 (x963) 4 9 6 - - 5 0 4
A a M I N O P O L Y S A C C H A R I D E SULPHATE.'2,
503
•ob'ta~ned ,0m t t ~ . , ~ t~e column in a stepwise m a n n e r with the N a C I - H C I (I : I, v/v) mix-~re :art ,~0~m~mtt~kms of o.3 M, 0. 7 M, I.O M and 2.o M is shown in Fig. 4. The anat.~,-~icaU t f ~ m ~ $0tr tfl~e potassium salts of the polysaccharides isolated from eluates corres.po~diim~ t~0,n l ~ e molarities (Table IV) reflect the increasing sulphate contents of .~¢_hea~dh,tmlharr .,s~eeiJes in the preparation. TABLE ~iLxYut~Is2=[L
l~,IXg,+~'lt~S
FOR
POL%'SACCHARIDE
~CII~IIIL~X"XON.ATION
Eluaut
OF
THE
SULPHATES
BLUE-SHARK
ISOLATED o/ /o
0--40
ETHANOL
AFTER
THE
FRACTION
Nitrogen
Hexosamine
Uronic acid
Ester sulphate
Potassium
~'g,)
C:~
("'o)
(%)
(%)
(%)
(%)
o.3 0.77
ot.T'-'
2.6 2.2 5
20.4 2 3.o
34.5 39.5
*4- t 17.4 x 8. 3
,, .6 i 4.0
--
22.6
--
;o 2:0
~'~
IV
"-,~'4+
o~.P,oS~
2, 5
--
o~og2E
--
--
DISCUSSION
]~t is ,aa~mttm~ t~0tm tlke present work t h a t the aminopolysaccharide sulphate esters cons~ ttttm, m n ~ i ~ of cartilage from blue shark, dogfish a n d skate differ subs t a n t i a ~ ttmrm ¢ . r r e s p o n d i n g polymers present in the cartilage of b o t h terrestrial a n d a q u a ~ c ~ m r r ~ l k . lP~o~'mer species similar to m a m m a l i a n chondroitin sulphate and kera,~~t~e Ika~e, been d e m o n s t r a t e d b u t these are eharacterised b y their widely diff~ ~ ~ .tI ester sulphate. Although the existence in elasmobranch cartilage of ~ :~3alliptk~e@potysaccharides has formed the subject of earlier reports =-s, little is t~¢.am w ~ ~ the effect of ester sulphate content on the properties of these m a t emi,a~. ~ mmtlk~d:4 presently described permit the isolation of polysaccharide species ,~f ~¢medl s ~ p h a t e c o n t e n t suitable for further structural and enzymological ~tud,v..
IPl~e ._~nl~,t~ectt a~i~opolysaccharides from fin-whale cartilage closely resemble prepa~.nnii0ms ~ . r ~ terrestrial m a m m a l s in t h a t although containing chondroitin s u l p h ~ e :+~_~$I k ~ a m G ~ h a t e , fractionation of the p r e p a r a t i o n on Ecteola-cellulose reveaAs ,~,m. ~ ib o n l y limited heterogeneity as regards ester sulphate contents of t h e ,oo~ii~i,m~mtiT~( ~ : . . ~ G E R T Z A N D REICRARD!6). In particular, the presence iv the inkawedi ~ o~ strong absorption at 85o cm -~ b o t h for crude preparations and t h e ,.o-#o'D, t ~ r ~ t i ~ suggests t h a t material identical in structure to chondroitin .% i~ tt~. ~ ":m~iple polysaccharide constituent of the original matrix, condr~ ~U#m~, ~ ~ k e r a t o s ~ p h a t e (both of which give rise to absorption at 82o ,<~m-~))~ I t m ~ n t in q u a n t i t a t i v e l y lesser amounts. ~e ~ 0 ~ d~monstration of a m e t h y l p e n t o s e similar to L-fucose as a mono.~aceh~e ~~,'~ in all of the crude preparations, and its specific association ~ / a ~ g a I l m ~ 0 ~ am~ D-glucosamine in the 40-50 % fraction from blue shark, deserves ~-pecfi~ ~ 0 m . ~ i a n keratosulphate preparations, having n-galactose and I~-gl~~ ~ ipri~iple monosaccharide constituents, have been reported conM s t ~ ~ ¢ ~ ~ traces (1-2 %) of a methylpentose component =~. In contrast. v~te~raI1 ,¢mltrb]t~ imm[uced to form in tissue cartilage and not containing keratoB i o c h i m . rCiophys. A c t a , 69 {x963) 4 9 6 - 5 0 4
50 4
G, LLOYD, K. S. DODGSON, R. G. PRICE
s u l p h a t e (as s h o w n b y t h e a b s e n c e o f D - g a l a c t o s e a n d o - g l u c o s a m i n e ) w a s a l s o ~vee f r o m m e t h y l p e n t o s e 25. A l t h o u g h these observations present evidence for the a s s o c i a t i o n of m e t h y l p e n t o s e components with keratosulphate f r a c t i o n s , i t is ~a~t known how these moieties are involved structurally in the polymer molecule ~. ACKNOWLEDGEMENTS T h i s w o r k h a s b e e n s u p p o r t e d b y a g r a n t (A 1 9 8 2 ) f r o m t h e A r t h r i t i s a n d M e t a b o l i c D i s e a s e s D i v i s i o n of t h e U.G. P~,blic H e a l t h S e r v i c e . W e w o u l d l i k e t o e x p r e s s o t t r t h a n k s t o D r . K . E N G E o f t h e U n i v e r s i t y of O s l o f o r s u p p l i e s of f i n - w h a l e c a r t i l a g e a n d t o t h e X V e l l c o m e T r u s t f o r t h e g i f t of a n i n f r a r e d s p e c t r o p h o t o m e t e r . O n e off n s ( R . G . P . ) is g r a t e f u l t o t h e D e p a r t m e n t of S c i e n t i f i c a n d I n d u s t r i a l R e s e a r c h f¢~r a studentship. REFERENCES t K. MEYER, ~llolecular Biology, Academic Press, London, [cj6o, p. 59. 2 K. NAGANIS~X, N. TAKAHASnI AND F. EGAMX, Bull. Ch~m. Soc. J a p a n , ,_t) (t95b) 434a j . ~v. LASH AND M. XV. XVHITEHOUSE, .4rch. Biochem. Biophys., 9o (I96o) I59. 4 M. B. MATHEWS AND M. INOUYE, Biochim. Biophys. Acta, 53 (I96I) 500. 5 M. ]3. M,~THEWS, Proceedings Vth Int. Congr. Biochemisto, , AIoscow, 1961, A b s t r a c t b.2~. a M. B. MATHE~'S, Nature, 193 (x962) 378. 7 $. SUZUKr, J . Biol. Chem., 235 (t96o) 358o. 0 T. FURUHASHI, J. Biochem, (Tokyo), 50 (I96I) 546. 9 A. G. LLOYD, K. S. DODGSON, R. G. PRICE AND F. A. ROSE, Biochim. Biophys..4cta, 46 (I96~i xo8. t0 I. SMITH, Chromatographic Techniqtws, W i l l i a m H e i n e m a n n Medical Books L t d . , London, p. I67. i1 S, M. PARTRIDGE, Nature, I64 (t949) 479. lS L . H O U G H , J . K . N . J O N E S AND ~V. H . *~VADMAN, f . Chem. Sot., ( I 9 5 0 ) 1r702. l* S, M . P A R T R I D G E , Biochem. J . , 42 ( I 9 4 8 ) 2 3 8 . 14 S. G A R D E L L , F . HI~IJKI~NSKJOLD AND A. R O C H - N O R L U N D , A c t a Chem. Stand., 4 (I95o) 9 7 o. t~ p . j. S T O F F Y N AND R . ~W. JEANLOZ, Arch. Biochem. Biophys., 52 (z954) 373te ~'NI. RINGERTZ AND P. REICHARD, Mcta Chem. Scand., t4 (t96o) 303. 17 R. MONTGOMERY, Biochim. Biophys. ,4cta, 48 (~901) 59I. i s j . W . H. LuG6, Biochem. J . , 32 (I938) 2114. a~ K. S. DODGSON Al~O R. G. PRICE, Biochem. J., 84 ([962) Io6. 20 Z. Dxscttv-, J . Biol, Chem., 167 (x947) I89. • l G. A. L~-vvY AND A. MCALLA~', Biochem. J.. 73 (x959) I27. z~ R. MARKHAM, Biochem. J . , 36 (t94 z) 79o. 2s T. G. KANTOR AND ~I. SCHUBERT, J. A m . Chem. So¢., 79 (I956) x52. t 4 0 . ROSEN, P. HOFFMAN AND K. MEYER, Federation Proc., 19 (I96O) 147. '~ j. w . LASH AND M. ~r. ~VHITEHOUSE, Arch. Biochem. Biophys., 90 (I96o) 159. t, S° HIRANO, P. HOFF.~tAI"~AND K. MEYER, f . OYg. Chem., 26 (I96I) 5064.
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