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The conformation of hyaluronic acid and chondroitin sulfate C: The metachromatic reaction
42
BIOCHIMICA ET BIOPHYSICA ACTA BBA 4 2 5 2
T H E C O N F O R M A T I O N OF H Y A L U R O N I C
ACID AND CHONDROITIN
S U L F A T E C: T H E M E T A C H R O M A T I C R E A C T I O N M. D. S C H O E N B E R G
AND I~. D. M O O R E
The Institute of Pathology, Western Reserve University, Cleveland, Ohio (U.S.A.) (Received J u l y 8th, 19(~3)
SUMMARY
The absorption characteristics of complexes of hyaluronic acid and chondroitin sulfate C with toluidine blue were studied. From the physical requirements necessary to form the metachromatic complexes, suggestions have been made concerning the conformation of the two polysaccharides.
INTRODUCTION
In dilute aqueous solution the interaction of a variety of planar cationic dyes with anionic macromolecules having regular-spaced polar groups results in changes in the absorption spectrum of the dye. The change in the absorption spectrum, the metachromatic effect, consists of the formation of new absorption m a x i m a at wavelengths shorter than the absorption m a x i m u m of the dye in the non-complexed form l-S°. The spectral changes are a consequence of a relatively precise orientation of neighboring dye molecules and reflect the steric arrangement of the polar residues on the macromolecule that are available for interaction with the dye. In this respect a study of the metachromatic effect m a y provide a basis for the study of the conformation of polar macromolecules, particularly those where the polar groups are regularly spaced. This paper deals with the changes in the absorption characteristics of H A - T B complexes and CSC-TB complexes in dilute solution. The results are considered in relation to the kinds of interaction that occur between the dye and substrate and their possible significance in determining the conformation of HA and CSC. MATERIALS AND METHODS
Hyaluronic acid was prepared from fresh acetone-dried h u m a n umbilical cords 21. IOO g of finely divided cord were extracted with a 5 % solution of sodium xylene sulfonate for 24 h at 4 °. The preparation was centrifuged for 30 rain at 4000 rev./min at 4 ° and the sediment discarded. The supernatant was adjusted to p H 2.0 with HC1 and allowed to stand for 15 rain in the cold. The precipitate that was formed was removed b y centrifugation. The supernatant was slowly added with stirring to 3-4 vol. of cold acetone. A white fibrous precipitate essentially free of protein was formed containing HA and sulfate-containing mucopolysaccharide(s). In order to remove the sulfated A b b r e v i a t i o n s : H A , h y a l u r o n i c acid; CSC, c h o n d r o i t i n sulfate C; TB, toluidine blue.
Biochim. Biophyr. ,4cta, 83 (1964) 42 51
POLYSACCHARIDE-DYE
43
INTERACTIONS
polysaccharide(s) a 0.25 % solution was made in distilled water and the p H adjusted to 2.0 with HC1. Enough cetyltrimethylammonium chloride was added to make the solution IO % (v/v). The precipitate that was formed was removed by centrifugation and the supematant slowly added to 3.5 vol. of cold acetone with stirring. A white fibrous precipitate resulted which was dried with acetone and then in air. The sodium salt was formed by dissolving the product in 0.5 M sodium acetate and precipitating in cold isopropanol. This was repeated twice. The final preparation contained no detectable protein or nucleic acid. The preparation was homogeneous by Tiselius electrophoresis and in the ultracentrifuge. Chondroitin sulfate C was obtained from a commercial preparation from the cartilage of bovine nasal septa (Nutritional Biochemicals, Cleveland, Ohio). The original material contained some protein which was removed by treatment with sodium xylene sulfonate. The final preparation was made as the sodium salt and contained no detectable protein. Toluidine blue (National Aniline-Certified Stain NU-I2) was twice recrystalllzed from hot water and ethanol. The product was homogeneous by chromatographic analysis. Stock solutions of TB were made in distilled water. The concentration of dye was determined by micro-Kjeldahl analysis. Stock solutions of H A and CSC containing o.Ioo g per IOO ml were prepared in distilled water. At least 2 days were allowed for solution in the cold. The stock solutions were stable and did not change in the measured properties when stored at 4 ° for as long as 2 weeks. The solutions for spectrophotometric and viscosimetric measurements were made by dilution of the stock with the appropriate concentration of dye or solvent. Absorption spectra were measured with a thermostated Beckman DU spectrophotometer in I-cm quartz cells after equilibration for 20 min. During the course of a determination the solution in the sample compartment was maintained within ± I o of the desired temperature. TABLE MOLECULAR E X T I N C T I O N C O E F F I C I E N T S OF
I
HA-TB AND CSC-TB COMPLEXES Wavelength
Metackromatie peak
(x so-")
560 mg ( I sosbestic point)
6~o ml*)
(x to-')
( x ,o-')
TB in distilled water
lO.4
21.5
47.5
HA-TB complex
3o.5 (54° m/~)
21.5
lO.2
CSC-TB complex
28.0 (53° ml,)
21.5
9.5
Molar extinction coefficients of TB at the maxima were calculated by the procedures of RABINOWITCHAND EPSTEIN5 and HARDY AND YOUNGz2. The determinations by the two methods agreed closely ( i 3 %) (Table I). Viscosity measurements were made with Ostwald viscometers of standard design. The temperatures for the viscosity and spectrophotometric measurements were the same (-4- I°). Biochim. Biophys. Acta, 83 (1964) 42-51
44
M.
D.
5CHOENBERG,
R.
MOORE
D,
RESULTS
The interaction of T B with H A and CSC results in significant changes in the absorption spectrum of the dye in the c o m p l e x e d state. The spectral changes are sensitive to alterations in concentration, ionic strength, temperature, dielectric constant and pH. Fig. I illustrates the nature of the changes in the absorption spectrum for a 1.8 4" IO -5 M solution of TB with H A in distilled water at 18 ° over a COO concentration from 1.32. IO-6 to 5.5" IO -~ M with respect to glucuronic acid. A n e w absorption m a x i m u m is found as a result of the formation of a c o m p l e x between TB and HA. The magnitude of the peak is a function of the extent of interaction between H A and TB. The value of the extinction coefficient at the m a x i m u m is diminished (4.7" l°4 at 62o m F to 3.o5" IOa at 54 ° m ~ ) . There is an isosbestic point at 56o m ~ indicating an equilibrium between the m e t a c h r o m a t i c form and the dissociated or normal form of
0.8
07
0.6t
g
0.5[
'i
/
i J
0.4~0,4
/
~
~
~ ~
: , . y ...................::, ;, / ,'~,# ".\ 03
/
,'
/, ,'/
, ;.
g
\
~
,' /
/*
C
. . . . . . . . ...
,
." ,
\
'\ ,\
o
,4
!
0.2 ',~
\ -
\
.~.',
\\.
~, ~",: \ ',\: \
'\
""
~o
:<~:_~.."
0.3
.
\,,:
o. z// i ,'/ // OI
,
\
0.1
%
,520 54o
56o 58o
Wavelength
60o
6~
64o
C
500
66o 6co
;
X--
?4, 2 . 6 8 - I O - - 6 M
i
540
58o
wavelength
(m)u)
Fig. i. C h a n g e s in t h e a b s o r p t i o n s p e c t r u m of i .84. I o -5 M s o l u t i o n of T B w i t h H A in d i s t i l l e d w a t e r a t 18 ° o v e r a C O O - c o n c e n t r a t i o n f r o m 1.32. i o -e M t o 5.5" I ° - 5 M w i t h r e s p e c t t o g l u c u r o n i c acid. -. . . . . , TB; O--O, 1.32"Io-~M COO
i
COO~;
.......
640
i
ooo
(m/u)
Iqg. 2, C h a n g e s in t h e a b s o r p t i o n s p e c t r a as a f u n c t i o n of t e m p e r a t u r e • L 8 4 " I o - s M T B a n d 9.3"io
4M
CO0
ete,
, 6.6.Io6M
, 2.68"1o s M CO()
_
COO-;
; eoe, 5.49-io
.
...---,
36.5°; ....... '..
sMCO()
6.2°; , 55".
, 1.3z'lo
---,
SM
18°;
CO()
;
.
13iochim. lJiophys .-Ieta,
83 ( 1 9 6 4 ) 't2 51
POLYSACCHARIDE-DYE INTERACTIONS the dye. Long-wavelength
45
transitions were not found at any
HA to TB
r a t i o 2~.
T a b l e I I s h o w s t h e e x t e n t of i n t e r a c t i o n b e t w e e n H A a n d T B a s a f u n c t i o n of c o n c e n tration and temperature. CSC i n t e r a c t s w i t h T B i n a s i m i l a r m a n n e r . T h e m a j o r d i f f e r e n c e is i n t h e p o s i t i o n of t h e n e w m a x i m u m a t 530 m/~. A s w i t h t h e H A - T B s y s t e m t h e v a l u e of t h e e x t i n c t i o n c o e f f i c i e n t a t t h e m a x i m u m is d i m i n i s h e d (4.7" lO4 a t 620 m ~ t o 2 . 8 . lO 4 a t 530 m ~ ) . T h e i s o s b e s t i c p o i n t r e m a i n s a t 5 6 0 m / , . T h e s e r e s u l t s a r e q u a l i t a t i v e l y t h e s a m e a s t h o s e r e p o r t e d b y LEVlNE AND SCHUBERT6 f o r t h e i n t e r a c t i o n of o t h e r thiazine dyes with chondroitin sulfate. Long-wavelength transitions were not found. T A B L E II INTERACTION
Temp.: COOmoles[ l
CO0-[TB
1.22" lO-3 1.o7" lO -3 9.3 " lO-4 6.71" IO- t 5-37" lO-4 4.o3- lO -4 2.68. IO-4 1.32" lO-4 5.49" lO-5 2.68- lO -5 1.32. lO-5 6.6 • IO-6 2.68- IO-e 1.32. IO 5
66.4 58.3 5°.6 36-5 29.2 21.9 14.6 7.13 3.00 1.5 0.72 0.36 o.14 0.077
OF
1.84" lO-5
MOLES
6.2 ° moles T B bound
1.72. lO-5 1.75" lO -5 1.77" lO-5 1.77" lO-5 1.77. lO-5 1.82- lO-5 1.82" lO-5 1.72" lO-6 1.68" lO-5 . . 1.22" lO-5 7.76. IO-e . . __
OF
TB
WITH
z8 ° % TB bound
93.2 93.3 96.1 96.1 96.I 99.o 99.0 93.2 91.3 . ----
moles T B bound
1.56" lO-5 1.59" IO-s 1.61" lO-5 1.63' lO-5 1.66. lO-5 1.68" lO-5 1.7o. lO-5 1.64 • lO-5 1.68. lO-5 . 9.89" lO -6 5.76. IO-e . . 3.8 • IO-e
H A IN
DISTILLED
36.5 °
WATER
55 °
% TB bound
moles T B bound
% TB bound
moles T B bound
% TB bound
84. 7 86.4 87. 5 88.6 90. 3 91.3 92. 3 88.6 91. 3
1.28" IO-s 1.31" lO-5 1-42" 1°-5 1.46" lO-5 1.49. IO-5 1.53" IO-s 1.56" IO-s 1.52. lO-5 1.18" lO-5 8.84" IO-e 8.55" IO-e 6.66' lO-5 . --
69.5 71.2 77 .2 79.2 81.o 83.2 85.0 83.2 64.1 47.9 ---
6.06. lO-s 6.86. IO-e 8.71" I o-e 9.87" IO-e 1.o5" lO-5 1.15" lO-5 1.18" lO -5 1.18. lO-5 9.18" IO-e 5.16" lO-8 3.66" lO-5 1.76" IO-e 2.24" IO-s 1.6 • IO-5
32.9 37.3 47-3 53.7
--. --
--
57.3 62. 3 64.1 64.1 49.9 28.1 -----
F i g . 2 i l l u s t r a t e s t h e c h a n g e s i n t h e a b s o r p t i o n s p e c t r a a s a f u n c t i o n of t e m p e r ature. With increasing temperature
t h e r e is a g r a d u a l t r a n s i t i o n of t h e a b s o r p t i o n
s p e c t r u m t o t h a t of t h e d y e i n t h e n o n - c o m p l e x e d f o r m , i n d i c a t i n g a d i s r u p t i o n of t h e metachromatic
array
(see b e l o w ) a n d / o r a d i s s o c i a t i o n b e t w e e n t h e d y e a n d p o l y -
saccharide. The changes with the CSC-TB system are qualitatively the same with the e x c e p t i o n t h a t t h e C S C - T B s y s t e m is l e s s s e n s i t i v e t o c h a n g e s i n t e m p e r a t u r e . T h e t e m p e r a t u r e e f f e c t s a r e r e v e r s i b l e . T h e b i n d i n g d a t a a s a f u n c t i o n of t e m p e r a t u r e i n c l u d e d in T a b l e II for t h e H A - T B s y s t e m . T h e d e p e n d e n c e of t h e H A - T B
are
s y s t e m o n i o n i c s t r e n g t h is s h o w n i n F i g . 3 a s a
f u n c t i o n of N a + c o n c e n t r a t i o n s . T h e d e p e n d e n c e o n c o u n t e r - i o n c o n c e n t r a t i o n w a s shown to be reversible both by dilution and temperature reversibility, Table III s h o w s e x a m p l e s of t h e b i n d i n g of T B t o H A i n t h e m e t a c h r o m a t i c various temperatures and Na + concentrations.
complex form at
S e v e r a l p o i n t s s h o u l d b e n o t e d . I n d i s t i l l e d w a t e r a t c o n c e n t r a t i o n s of T B i n e x c e s s , t h e C O O - i n H A o r C O O - a n d - O S O 3- s i t e s i n CSC a r e S a t u r a t e d a t t h e l o w e r t e m p e r a t u r e s a n d t h e b o u n d T B is e n t i r e l y i n t h e m e t a c h r o m a t i c f o r m . W h e n t h e
Biochim. Biophys. Act(*, 83 (1964) 42-51
40
M.D. SCHOENBERG, R. I). MOORE
c o n c e n t r a t i o n of t h e p o l a r g r o u p s o n t h e p o l y s a c c h a r i d e s is in e x c e s s , t h e d y e is also b o u n d in t h e m e t a c h r o m a t i c form. It m u s t be i n f e r r e d t h a t t h e n e a r e s t n e i g h b o r b i n d i n g s i t e is p r e f e r r e d . R a n d o m i n t e r a c t i o n s a r e p r o b a b l y r a r e o v e r t h e c o n c e n t r a tion range considered.
0.5
/
/ 0.4
\
\ \
/
/.;.;.-
>' 0 . 3 u C 0 .0 L
y
",,
o
0.2 <
0.1
0 480
520 560 600 Wavelength (rnN)
640
Fig. 3- Dependence of the HA TB system on Na + concentration. - - , distilled water; . . . . 0.o025 M NaC1; . . . . . . , o.olo M NaC1; • - - , 0.030 M NaCl.
,
TABLE lII NaG1 C O N C E N T R A T I O N O N W S - H a B I N D I N G TB concentration, 1.83. lO ~ M; COO- concentration, 9.6" io 4 M.
EFFECT
OF TEMPERATURE
AND
N a C l concn. (M)
0 0.0025 O.OLO 0.030 o.o8o
Moles T B bound]l ( x l o ~) 6o
25 °
37 °
58
1.75 1.27 I. I8 0.96 0.83
1.38 0.96 0.89 0.58 0.43
1.03 o.71 0.52 0.46 0.44
0.60 0.40 0.42 0.42 0.39
p H dependence T h e d e p e n d e n c e on p H was c o n s i d e r e d for b o t h t h e H A - T B a n d C S C - T B s y s t e m s . A s a f u n c t i o n of p H a t c o n s t a n t t e m p e r a t u r e a n d c o n s t a n t c o u n t e r - i o n c o n c e n t r a t i o n i t w a s f o u n d t h a t t h e i n t e r a c t i o n of T B w i t h H A t o f o r m m e t a c h r o m a t i c a b s o r b i n g c o m p l e x e s d e c r e a s e d p r e c i p i t o u s l y f r o m p H 4 . o t o p H 3.0. A t t h e l o w e r p H
Biochint. Biophys..q cta, 83 (~964) 42 51
47
POLYSACCHARIDE--DYE INTERACTIONS
a metachromatic complex could not be detected under the experimental conditions. These results are in keeping with the values reported for the pKa of glucuronic acid (pK = 3.33) 24 in the HA moiety. In contrast to this, the change in the metachromatic properties of the CSC-TB system were different. At constant concentration of TB and CSC, temperature and counter-ion concentration there was no alteration of the absorption spectra of CSC-TB solutions from pH 4.0 to p H 7.0. Between p H 4.0 and p H 3.0 there was a precipitous change in the absorption characteristics of these solutions. The intensity of the short-wavelength maximum decreased to approx, one-half its original value. However, a distinct metachromatic effect persisted to p H 2.0. Measurements below this pH were not made. These results are again consistent with the pKa for the dissociation of the C O 0 - groups on the glucuronic acid residues and with the dissociation of sulfate half-ester groups that are readily ionizable at pH 2.0 (see ref. 24). Of importance in analyzing the pI-[ dependence is that above pH 4.0 CSC-TB complexes show an absorption maximum at 530 m/z while below p H 3.0 the absorption maximum has shifted to 520 m/z.
Viscosity measurements In order to determine whether there were any macroscopic changes in the structure of these polysaccharides their viscous behavior was measured with and without TB as a function of ionic strength, concentration and temperature. The viscosity of HA in distilled water at several temperatures with and without TB is shown in Table IV. The addition of TB did not change the viscosity of HA at the concentrations used, although the binding of TB to HA was extensive. There was no dependence of the viscosity of HA or HA-TB solutions on temperature. In contrast to the constant viscosity with temperature andTB concentration the absorption spectrum was temperature dependent. TABLE
IV
EFFECT OF TEMPI~RATURE AND DYE ON RELATIVE VISCOSITY OF HYALURONATE IN DISTILLED WATER T B c o n c e n t r a t i o n 2. 5. lO -5 M. HA conch. (g/~oo ml)
28.5 ° No dye
37.5 ° Plus dye
No dye
58.8 ° Plus dye
No dye
Plus dye
0.00208
I.O9
I.O 9
I. I2
I. 13
I. I I
I. IO
o.oo625
1.31
1.34
1.36
1.36
1.35
1.34
O.OLO4
1.5I
1.52
1.59
1.57
1.57
1.54
0.0208
2.08
2.o8
2.24
2.36
2.2 t
2.1o
2.63
2.57
2.72
2.61
o.o412
3.38
3.27
3.4 °
3.29
3.37
3.32
o.o516
3.56
3.62
o.o31o
o.o625
4.o4
3.92
3.83
4.39
4.19
4.57
3.69 4.28
The change in viscosity as a function of Na + concentration is shown in Fig. 4The shape of the curves for the effect of Na+ on metachromasy and viscosity is similar. There was no effect of temperature on the viscous properties in the presence of Na +. Biochim.
B i o p h y s . A c l a , 83 (1964) 4 2 - 5 1
4~
M.D.
SCHOENBEIRG, R. I). MOORE
In addition to these experiments in dilute solution observations were made on tissue sections rich in these polysaccharides as a function of pH and ionic strength. The observations on the tissue sections were qualitatively the same as those found in solution. 5.0
4.(
"" 3.0
oo
o~ "~, o_> 2.0
"~-~-~m~R~__
I.C
°o
i
i
o.ol
o:o3
i
o.bs'
olo7
tonic strength
Fig. 4. C h a n g e in v i s c o s i t y a n d m e t a c h r o m a s y as a f u n c t i o n of N a + c o n c e n t r a t i o n , o = q, × = R, w h e r e R is t h e r a t i o of t h e a b s o r p t i v i t y a t 54 ° m f f t o t h a t a t 62o m / , .
DISCUSSION
Spectrophotometric measurements show extensive interaction between TB and both HA and CSC. A model of a co-parallel complex between polyions with specific geometric conformation and planar dyes can be postulated to account for the short-wavelength transitions in the absorption spectra (Fig. 5). Theoretical and experimental consideraLONG RANGE FORCES
H
H
H S
I
~
C
I
C
N+
N +
¢t
I
C
coo
o
I
C
N+ j
'
~
N.-p POLAR
BOND~
c..osS,
coo
0 NHA¢
NHAc
Fig. 5. S c h e m a t i c i l l u s t r a t i o n of a m e t a c h r o m a t i c c o m p l e x .
lYiochim. Biophys.
/Iota, 83 (z904) 4 ~ 51
POLYSACCHARIDE--DYE
INTERACTIONS
49
tions indicate that the changes in the absorption spectra are a result of the formation of a complex having a linear arrangement of regularly spaced, extensively conjugated dye molecules, so aligned, that their planes are parallel and approximately normal, or inclined toward the long axis of the chainS, 7-11,13,14. Steric considerations limit the kinds of molecules that can form this array to planar or very nearly planar molecules or to those which can assume a planar configuration. The array is most likely stabilized by Van der Waals-type interactions between neighboring dye molecules. The attractive forces between the dye molecules are long range and additive z~, 36. In view of the experimental data using aqueous solutions, stabilization by hydrogen bonding through water molecules included between the dye planes should be considered. In order to arrive at the spectralchanges the interplanar distance between adjacent dye molecules must be sufficiently close so that electronic interaction (~ electron) between neighbors is permissible. Interdye distances between 3.5 and 7.0/k are possible for TB when Van der Waals interactions are considered or if hydrogen bonding through a water molecule occurs. A polyion having a functional-group identity within these limits, or which can asslmae a configuration so that the distance between the functional groups falls within these limits would be a reasonable template for the formation of a metachromatic complex between the dye and the polyion. Two mutually dependent interactions are necessary in the formation of a metachromatic complex of the kind described. (I) Polar interaction between the dye and consecutive groups on the polyanion (2) interaction of the Van der Waals type between adjacent dye molecules. The extent to which the metachromatic complex is formed and remains stable is governed by the degree to which these interactions can mutually exert themselves and maintain the consecutive order of the dye molecules. In dilute aqueous solution HA and CSC are highly charged linear molecules. The dye is exposed to long segments of the polysaccharides that have a number of regularly or nearly regularly spaced sites for polar interaction. Chains of optically coupled dye molecules can be and are formed using these polysaccharides as a template. The results show that the formation of a metachromatic complex between HA and TB and CSC and TB is not the result of a random positioning of the dye along the polysaccharide chain. The evidence is in favor of preferential binding to the site next to an already polar-bound neighbor. It is apparent from the temperature dependence that the stabilizing interactions extend over a number of bound dye molecules. A combination of chemical and enzymic analyses have established the basic structures of HA and CSC. HA is a fl-I,4-1inked unbranched polymer of the disaccharide fl-I,3-glucopyranosyl-2-deoxy-2-N-acetylglucosamine, and CSC is a similarly unbranched polymer of fl-I,3-glucopyranosyl-2-deoxy-2-N-acetylgalactosamine-6-sulfate2~-3~. The structures of HA and CSC are almost the same with the exception of the sulfate half-ester group on Ca in the galactosamine residue and the position of the hydroxyl group on C4. The similarities permit comparisons between the two. On the basis of the structure of the disaccharide of HA as its repeat unit then in a linear-extended polymer with maximal repulsion between COO- units the COO-C 0 0 - distance would be in the order of IO •. With TB, whose maximum dimension is 12.5 A, a COO--COO- separation of IO A between dye planes would be too large for sufficient inter-dye interaction and z electron overlap to establish the co-parallel arrangement to give the metachromatic effect. Since the results show that HA forms a Biochim. Biophys. Acta, 83 (1964) 42-51
5O
M, D. S C H O E N B E R G , R. t). M O O R E
m e t a c h r o m a t i c complex w i t h TB it m u s t lye a s s u m e d t h a t t h e COO - -COO- distance between successive C O O - groups is smaller than I o A. The relative s t a b i l i t y of the complex as a fimction of increasing t e m p e r a t u r e also suggests t h a t the d y e molecules m u s t be closely disposed to one another. If the molecule was e x t e n d e d in solution with the io-z~ spacing between COO - groups as a n t i c i p a t e d from its disaccharide structure, the f o r m a t i o n of a m e t a c h r o m a t i c complex would necessitate size a n d shape changes of the p o l y s a c c h a r i d e molecule r e p r e s e n t e d as a contraction of t h e molecule. These alterations should be reflected in the viscosity of HA--TB complexes. No differences were found. T h e independence of the viscosity with extensive H A - T B interaction indicates at least no gross change in the configuration of the polysaccharide as a result of the f o r m a t i o n of the m e t a c h r o m a t i c complex. On the basis of these observations the conformation of H A in aqueous solution m u s t fulfill the following r e q u i r e m e n t s : (x) the s e p a r a t i o n between the C O O - groups on neighboring disaccharide residues m u s t be in the order of 7 k or less a n d have a configuration so t h a t t h e planes of b o u n d dye molecules can overlap (z) the molecules m u s t be sufficiently rigid so t h a t its viscous b e h a v i o r is n o t altered as a function of i n t e r a c t i o n with the dye or as a flmction of t e m p e r a t u r e . E x a m i n a t i o n of molecular models shows t h a t there is sufficient r o t a t i o n a b o u t the glycosidic bonds to reduce the charge s e p a r a t i o n between C O O - groups to t h e order of 7 A. Distances m u c h closer t h a n this a p p e a r to result in b o n d distortions. The m o s t likely possibility for the conformation of H A is t h a t of a fairly" rigid, linear chain, where neighboring C O O groups are close together. This model is consistent w i t h the results of viscosity, lights c a t t e r i n g a n d flow-birefringence m e a s u r e m e n t s sv .m. The changes in the l n e t a c h r o m a t i c properties as a result of the a d d i t i o n of N a + are m o s t likely the result of the successful c o m p e t i t i o n between the m e t a l ion a n d the dye. The fall in viscosity on the a d d i t i o n of electrolyte is c o m p a r a b l e to the b e h a v i o r of DNA. In the case of CSC similar p o s t u l a t e s concerning its configuration t a i l be made,, p a r t i c u l a r l y because of its b e h a v i o r with TB between p H 2.o a n d 4.o. The m e t a c h r o m a t i c effect o b s e r v e d above p H 4.0 is the result of the i n t e r a c t i o n of the dye w i t h b o t h the C O 0 - a n d OSO 3- groups. The persistence of the m e t a c h r o m a t i c effect below p H 3.o, shows t h a t at the higher H + c o n c e n t r a t i o n the m e t a c h r o m a t i c effect is the result of t h e interaction with. the sulfate half-ester groups. I t has a l r e a d y been p o i n t e d out t h a t the C O 0 - groups are n o t dissociated at the lower p H ' s . The change in the m a g n i t u d e a n d location of the m e t a c h r o m a t i c p e a k below p H 3.o shows t h a t there are two stacks of dye molecules. One s t a c k corresponds to the C O O - a n d one s t a c k corresponds to the OSO3-. In view of the m e t a c h r o m a t i c effect below p H 3.o it m u s t be a s s u m e d t h a t the COO - a n d OSO a - groups are not disposed on the same side of the p o l y m e r chain. I t has been shown bv MATHEWS AN1) DORFMAN'~4 t h a t the presence of t h e sulfate half-ester groups does n o t m o d i f y the dissociation c o n s t a n t of t h e CO0-groups. I t m u s t be inferred t h a t t h e y are well s e p a r a t e d . Other evidence has shown t h a t the molecule retains its s y m m e t r y over the same range of p H . It is a t t r a c t i v e to conclude t h a t CSC has a conformation similar to t h a t p r o p o s e d for HA. In t e r m s of biological significance it can be shown t h a t identical m e t a c h r o m a t i c changes occur in tissues. Tissues t h a t are rich in H A i.e. W h a r t o n ' s jelly show an identical d e p e n d e n c e of m e t a c h r o m a s y on p H a n d ionic strength. Similarly, tissues t h a t are rich in CSC also show the p H a n d ionic s t r e n g t h dependence found i~f vitro Hiochbn. Hiophy,~..Icla, 83 (1904) 42 5~
POLYSACCHARIDE--DYE INTERACTIONS
51
(i.e. cartilage). In these tissues the metachromatic effect persists below pH 3.o with a similar diminution in the intensity of the reaction. It is therefore likely that the configuration of these polysaccharides in vivo is the same as those suggested in dilute solution. ACKNOWLEDGEMENTS
This work was supported in part by grant AI9o 9 and R G 720o from the U.S. Public
Health Service. M.D.S. is a Research Career Development Awardee, U.S. Public Health Service. REFERENCES i B. SYLV]~N, Quart. J. Microscop. Sci., 95 (1954) 327. 2 M. SCHUBERT AND D. HAMERMAN, J. Histochem. Cytochem., 4 (1956) 158. s j . A. BERGERON AND M. SINGER, J. Biophys. Biochem. Cytol., 4 (1958) 433. 4 B. SYLV/~NAND H. MALMGREN, Lab. Invest., I (1952) 413 . 6 E. RAmNOWlTCH AND L. F. EPSTEIN, J. Am. Chem. Soc., 63 (1941) 69. s A. LEVlNE AND M. SCHUBERT, J. Am. Chem. Soc., 74 (1952) 91. G. SCHEIBE, Kolloid-Z., 82 (1938) I. 8 F. VICKERSTAFF AND D. R. LEMIN, Nature, 157 (1946) 373. G. SCHEME A~D V. ZAB.KER, Z. Physik, 123 (1952) 244. 10 G. SCHEIBE, Angew. Chem., 5 ° (1937) 212. 11 G. SCHEIBE, Angew. Chem., 52 (1939) 631. 1~ M. SCHUBERT AND A. LEVlNE, J. Am. Chem. Soc., 77 (1955) 4197 • 13 S. E. SHEPPARD AND A. L. GEDDES, J. Am. Chem. Soc., 66 (1944) 1995. 14 S. E. SHEPPARD AND A. L. GEDDES, J. Am. Chem. Soc., 66 (1944) 2oo3. 16 H. E. SPEAS, Phys. Rev., 31 (1928) 569. 16 O. BANK AND H. G. BUNGEB.BERG DE JOB'G, Protoplasma, 32 (1939) 489 • IT E. E. JELLEY, Nature, 138 (1936) lOO9. is E. E. JELLEY, Nature, 139 (1937) 631. IS L. LlSON, Arch. Biol. Liege, 46 (1935) 599s0 S. E. SHEPPARD, Rev. Mod. Phys., 14 (1942) 303 . 21 N. S. SIMMONS, personal communication. 22 A. C. HARDY AND F. M. YOUNG, J. Opt. Soc. Am., 38 (1948) 854. W. HOPPE, Kolloid-Z., lO9 (1944) 21. 2, M. D. MATHEWS AND A. DORFMAN, Arch. Biochem. Biophys., 42 (1954) 41. 2, H. KUHN, J. Chem. Phys., 17 (1949) 1198. zs C. A. COULSOI~AND P. L. DAVIES, Trans. Faraday Soc., 48 (1952) 777. 27 M. M. RAPPORT, K. MEYER AND A. LINKER, J. Am. Chem. Soc., 73 (1951) 2416. 22 B. WEISSMAN, K. MEYER, P. SAMPSON AND A. LINKER, J. Biol. Chem., 208 (1954) 417 • 29 B. WEISSMAN AND K. MEYER, J. Am. Chem. Soc., 76 (1954) 1753. so A. LINKER, K. MEYER A~rD B. WEISSMAN, J. Biol. Chem., 312 (1955) 237. 21 K. MEYER, A. LIZCKER AND M. M. RAPPORT, J. Biol. Chem., 192 (1951) 275. 22 E. A. DAVIDSOB.AND K. MEYER, J. Biol. Chem., 21I (1954) 605. s3 M. M. RAPPORT, n . WEISSMAB., A. LINKER AIqD K. MEYER, Nature, 168 (1951) 996. 24 E. A. DAVIDSON"AB.D K. MEYER, J. Am. Chem. Soc., 76 (1954) 5686. 3, B. WEISSMAB., M. M. RAPPORT, A. LINKER AND K. MEYER, J. Biol. Chem., 205 (1953) 205s* B. WEISSMAN" AND K. MEYER, J. Am. Chem. Soc., 74 (1952) 4729 • s~ B. S. BLUMBXRG AN'D G. OSTER, Science, 12o (1954) 432. 38 F. C. LAURENT AND J. GERGELY, J. Biol. Chem., 212 (1955) 325. z9 A. G. OGSTON AND J. E. STANIER, Biochem. J., 49 (1951) 585. 40 B. JAcoBsoN AND F. C. LAURENT, f . Colloid. Sci., 9 (1954) 36.
Biochim. Biophys. Acta, 83 (1964) 42-51
BIOCHIMICA ET BIOPHYSICA ACTA BBA 4 2 5 2
T H E C O N F O R M A T I O N OF H Y A L U R O N I C
ACID AND CHONDROITIN
S U L F A T E C: T H E M E T A C H R O M A T I C R E A C T I O N M. D. S C H O E N B E R G
AND I~. D. M O O R E
The Institute of Pathology, Western Reserve University, Cleveland, Ohio (U.S.A.) (Received J u l y 8th, 19(~3)
SUMMARY
The absorption characteristics of complexes of hyaluronic acid and chondroitin sulfate C with toluidine blue were studied. From the physical requirements necessary to form the metachromatic complexes, suggestions have been made concerning the conformation of the two polysaccharides.
INTRODUCTION
In dilute aqueous solution the interaction of a variety of planar cationic dyes with anionic macromolecules having regular-spaced polar groups results in changes in the absorption spectrum of the dye. The change in the absorption spectrum, the metachromatic effect, consists of the formation of new absorption m a x i m a at wavelengths shorter than the absorption m a x i m u m of the dye in the non-complexed form l-S°. The spectral changes are a consequence of a relatively precise orientation of neighboring dye molecules and reflect the steric arrangement of the polar residues on the macromolecule that are available for interaction with the dye. In this respect a study of the metachromatic effect m a y provide a basis for the study of the conformation of polar macromolecules, particularly those where the polar groups are regularly spaced. This paper deals with the changes in the absorption characteristics of H A - T B complexes and CSC-TB complexes in dilute solution. The results are considered in relation to the kinds of interaction that occur between the dye and substrate and their possible significance in determining the conformation of HA and CSC. MATERIALS AND METHODS
Hyaluronic acid was prepared from fresh acetone-dried h u m a n umbilical cords 21. IOO g of finely divided cord were extracted with a 5 % solution of sodium xylene sulfonate for 24 h at 4 °. The preparation was centrifuged for 30 rain at 4000 rev./min at 4 ° and the sediment discarded. The supernatant was adjusted to p H 2.0 with HC1 and allowed to stand for 15 rain in the cold. The precipitate that was formed was removed b y centrifugation. The supernatant was slowly added with stirring to 3-4 vol. of cold acetone. A white fibrous precipitate essentially free of protein was formed containing HA and sulfate-containing mucopolysaccharide(s). In order to remove the sulfated A b b r e v i a t i o n s : H A , h y a l u r o n i c acid; CSC, c h o n d r o i t i n sulfate C; TB, toluidine blue.
Biochim. Biophyr. ,4cta, 83 (1964) 42 51
POLYSACCHARIDE-DYE
43
INTERACTIONS
polysaccharide(s) a 0.25 % solution was made in distilled water and the p H adjusted to 2.0 with HC1. Enough cetyltrimethylammonium chloride was added to make the solution IO % (v/v). The precipitate that was formed was removed by centrifugation and the supematant slowly added to 3.5 vol. of cold acetone with stirring. A white fibrous precipitate resulted which was dried with acetone and then in air. The sodium salt was formed by dissolving the product in 0.5 M sodium acetate and precipitating in cold isopropanol. This was repeated twice. The final preparation contained no detectable protein or nucleic acid. The preparation was homogeneous by Tiselius electrophoresis and in the ultracentrifuge. Chondroitin sulfate C was obtained from a commercial preparation from the cartilage of bovine nasal septa (Nutritional Biochemicals, Cleveland, Ohio). The original material contained some protein which was removed by treatment with sodium xylene sulfonate. The final preparation was made as the sodium salt and contained no detectable protein. Toluidine blue (National Aniline-Certified Stain NU-I2) was twice recrystalllzed from hot water and ethanol. The product was homogeneous by chromatographic analysis. Stock solutions of TB were made in distilled water. The concentration of dye was determined by micro-Kjeldahl analysis. Stock solutions of H A and CSC containing o.Ioo g per IOO ml were prepared in distilled water. At least 2 days were allowed for solution in the cold. The stock solutions were stable and did not change in the measured properties when stored at 4 ° for as long as 2 weeks. The solutions for spectrophotometric and viscosimetric measurements were made by dilution of the stock with the appropriate concentration of dye or solvent. Absorption spectra were measured with a thermostated Beckman DU spectrophotometer in I-cm quartz cells after equilibration for 20 min. During the course of a determination the solution in the sample compartment was maintained within ± I o of the desired temperature. TABLE MOLECULAR E X T I N C T I O N C O E F F I C I E N T S OF
I
HA-TB AND CSC-TB COMPLEXES Wavelength
Metackromatie peak
(x so-")
560 mg ( I sosbestic point)
6~o ml*)
(x to-')
( x ,o-')
TB in distilled water
lO.4
21.5
47.5
HA-TB complex
3o.5 (54° m/~)
21.5
lO.2
CSC-TB complex
28.0 (53° ml,)
21.5
9.5
Molar extinction coefficients of TB at the maxima were calculated by the procedures of RABINOWITCHAND EPSTEIN5 and HARDY AND YOUNGz2. The determinations by the two methods agreed closely ( i 3 %) (Table I). Viscosity measurements were made with Ostwald viscometers of standard design. The temperatures for the viscosity and spectrophotometric measurements were the same (-4- I°). Biochim. Biophys. Acta, 83 (1964) 42-51
44
M.
D.
5CHOENBERG,
R.
MOORE
D,
RESULTS
The interaction of T B with H A and CSC results in significant changes in the absorption spectrum of the dye in the c o m p l e x e d state. The spectral changes are sensitive to alterations in concentration, ionic strength, temperature, dielectric constant and pH. Fig. I illustrates the nature of the changes in the absorption spectrum for a 1.8 4" IO -5 M solution of TB with H A in distilled water at 18 ° over a COO concentration from 1.32. IO-6 to 5.5" IO -~ M with respect to glucuronic acid. A n e w absorption m a x i m u m is found as a result of the formation of a c o m p l e x between TB and HA. The magnitude of the peak is a function of the extent of interaction between H A and TB. The value of the extinction coefficient at the m a x i m u m is diminished (4.7" l°4 at 62o m F to 3.o5" IOa at 54 ° m ~ ) . There is an isosbestic point at 56o m ~ indicating an equilibrium between the m e t a c h r o m a t i c form and the dissociated or normal form of
0.8
07
0.6t
g
0.5[
'i
/
i J
0.4~0,4
/
~
~
~ ~
: , . y ...................::, ;, / ,'~,# ".\ 03
/
,'
/, ,'/
, ;.
g
\
~
,' /
/*
C
. . . . . . . . ...
,
." ,
\
'\ ,\
o
,4
!
0.2 ',~
\ -
\
.~.',
\\.
~, ~",: \ ',\: \
'\
""
~o
:<~:_~.."
0.3
.
\,,:
o. z// i ,'/ // OI
,
\
0.1
%
,520 54o
56o 58o
Wavelength
60o
6~
64o
C
500
66o 6co
;
X--
?4, 2 . 6 8 - I O - - 6 M
i
540
58o
wavelength
(m)u)
Fig. i. C h a n g e s in t h e a b s o r p t i o n s p e c t r u m of i .84. I o -5 M s o l u t i o n of T B w i t h H A in d i s t i l l e d w a t e r a t 18 ° o v e r a C O O - c o n c e n t r a t i o n f r o m 1.32. i o -e M t o 5.5" I ° - 5 M w i t h r e s p e c t t o g l u c u r o n i c acid. -. . . . . , TB; O--O, 1.32"Io-~M COO
i
COO~;
.......
640
i
ooo
(m/u)
Iqg. 2, C h a n g e s in t h e a b s o r p t i o n s p e c t r a as a f u n c t i o n of t e m p e r a t u r e • L 8 4 " I o - s M T B a n d 9.3"io
4M
CO0
ete,
, 6.6.Io6M
, 2.68"1o s M CO()
_
COO-;
; eoe, 5.49-io
.
...---,
36.5°; ....... '..
sMCO()
6.2°; , 55".
, 1.3z'lo
---,
SM
18°;
CO()
;
.
13iochim. lJiophys .-Ieta,
83 ( 1 9 6 4 ) 't2 51
POLYSACCHARIDE-DYE INTERACTIONS the dye. Long-wavelength
45
transitions were not found at any
HA to TB
r a t i o 2~.
T a b l e I I s h o w s t h e e x t e n t of i n t e r a c t i o n b e t w e e n H A a n d T B a s a f u n c t i o n of c o n c e n tration and temperature. CSC i n t e r a c t s w i t h T B i n a s i m i l a r m a n n e r . T h e m a j o r d i f f e r e n c e is i n t h e p o s i t i o n of t h e n e w m a x i m u m a t 530 m/~. A s w i t h t h e H A - T B s y s t e m t h e v a l u e of t h e e x t i n c t i o n c o e f f i c i e n t a t t h e m a x i m u m is d i m i n i s h e d (4.7" lO4 a t 620 m ~ t o 2 . 8 . lO 4 a t 530 m ~ ) . T h e i s o s b e s t i c p o i n t r e m a i n s a t 5 6 0 m / , . T h e s e r e s u l t s a r e q u a l i t a t i v e l y t h e s a m e a s t h o s e r e p o r t e d b y LEVlNE AND SCHUBERT6 f o r t h e i n t e r a c t i o n of o t h e r thiazine dyes with chondroitin sulfate. Long-wavelength transitions were not found. T A B L E II INTERACTION
Temp.: COOmoles[ l
CO0-[TB
1.22" lO-3 1.o7" lO -3 9.3 " lO-4 6.71" IO- t 5-37" lO-4 4.o3- lO -4 2.68. IO-4 1.32" lO-4 5.49" lO-5 2.68- lO -5 1.32. lO-5 6.6 • IO-6 2.68- IO-e 1.32. IO 5
66.4 58.3 5°.6 36-5 29.2 21.9 14.6 7.13 3.00 1.5 0.72 0.36 o.14 0.077
OF
1.84" lO-5
MOLES
6.2 ° moles T B bound
1.72. lO-5 1.75" lO -5 1.77" lO-5 1.77" lO-5 1.77. lO-5 1.82- lO-5 1.82" lO-5 1.72" lO-6 1.68" lO-5 . . 1.22" lO-5 7.76. IO-e . . __
OF
TB
WITH
z8 ° % TB bound
93.2 93.3 96.1 96.1 96.I 99.o 99.0 93.2 91.3 . ----
moles T B bound
1.56" lO-5 1.59" IO-s 1.61" lO-5 1.63' lO-5 1.66. lO-5 1.68" lO-5 1.7o. lO-5 1.64 • lO-5 1.68. lO-5 . 9.89" lO -6 5.76. IO-e . . 3.8 • IO-e
H A IN
DISTILLED
36.5 °
WATER
55 °
% TB bound
moles T B bound
% TB bound
moles T B bound
% TB bound
84. 7 86.4 87. 5 88.6 90. 3 91.3 92. 3 88.6 91. 3
1.28" IO-s 1.31" lO-5 1-42" 1°-5 1.46" lO-5 1.49. IO-5 1.53" IO-s 1.56" IO-s 1.52. lO-5 1.18" lO-5 8.84" IO-e 8.55" IO-e 6.66' lO-5 . --
69.5 71.2 77 .2 79.2 81.o 83.2 85.0 83.2 64.1 47.9 ---
6.06. lO-s 6.86. IO-e 8.71" I o-e 9.87" IO-e 1.o5" lO-5 1.15" lO-5 1.18" lO -5 1.18. lO-5 9.18" IO-e 5.16" lO-8 3.66" lO-5 1.76" IO-e 2.24" IO-s 1.6 • IO-5
32.9 37.3 47-3 53.7
--. --
--
57.3 62. 3 64.1 64.1 49.9 28.1 -----
F i g . 2 i l l u s t r a t e s t h e c h a n g e s i n t h e a b s o r p t i o n s p e c t r a a s a f u n c t i o n of t e m p e r ature. With increasing temperature
t h e r e is a g r a d u a l t r a n s i t i o n of t h e a b s o r p t i o n
s p e c t r u m t o t h a t of t h e d y e i n t h e n o n - c o m p l e x e d f o r m , i n d i c a t i n g a d i s r u p t i o n of t h e metachromatic
array
(see b e l o w ) a n d / o r a d i s s o c i a t i o n b e t w e e n t h e d y e a n d p o l y -
saccharide. The changes with the CSC-TB system are qualitatively the same with the e x c e p t i o n t h a t t h e C S C - T B s y s t e m is l e s s s e n s i t i v e t o c h a n g e s i n t e m p e r a t u r e . T h e t e m p e r a t u r e e f f e c t s a r e r e v e r s i b l e . T h e b i n d i n g d a t a a s a f u n c t i o n of t e m p e r a t u r e i n c l u d e d in T a b l e II for t h e H A - T B s y s t e m . T h e d e p e n d e n c e of t h e H A - T B
are
s y s t e m o n i o n i c s t r e n g t h is s h o w n i n F i g . 3 a s a
f u n c t i o n of N a + c o n c e n t r a t i o n s . T h e d e p e n d e n c e o n c o u n t e r - i o n c o n c e n t r a t i o n w a s shown to be reversible both by dilution and temperature reversibility, Table III s h o w s e x a m p l e s of t h e b i n d i n g of T B t o H A i n t h e m e t a c h r o m a t i c various temperatures and Na + concentrations.
complex form at
S e v e r a l p o i n t s s h o u l d b e n o t e d . I n d i s t i l l e d w a t e r a t c o n c e n t r a t i o n s of T B i n e x c e s s , t h e C O O - i n H A o r C O O - a n d - O S O 3- s i t e s i n CSC a r e S a t u r a t e d a t t h e l o w e r t e m p e r a t u r e s a n d t h e b o u n d T B is e n t i r e l y i n t h e m e t a c h r o m a t i c f o r m . W h e n t h e
Biochim. Biophys. Act(*, 83 (1964) 42-51
40
M.D. SCHOENBERG, R. I). MOORE
c o n c e n t r a t i o n of t h e p o l a r g r o u p s o n t h e p o l y s a c c h a r i d e s is in e x c e s s , t h e d y e is also b o u n d in t h e m e t a c h r o m a t i c form. It m u s t be i n f e r r e d t h a t t h e n e a r e s t n e i g h b o r b i n d i n g s i t e is p r e f e r r e d . R a n d o m i n t e r a c t i o n s a r e p r o b a b l y r a r e o v e r t h e c o n c e n t r a tion range considered.
0.5
/
/ 0.4
\
\ \
/
/.;.;.-
>' 0 . 3 u C 0 .0 L
y
",,
o
0.2 <
0.1
0 480
520 560 600 Wavelength (rnN)
640
Fig. 3- Dependence of the HA TB system on Na + concentration. - - , distilled water; . . . . 0.o025 M NaC1; . . . . . . , o.olo M NaC1; • - - , 0.030 M NaCl.
,
TABLE lII NaG1 C O N C E N T R A T I O N O N W S - H a B I N D I N G TB concentration, 1.83. lO ~ M; COO- concentration, 9.6" io 4 M.
EFFECT
OF TEMPERATURE
AND
N a C l concn. (M)
0 0.0025 O.OLO 0.030 o.o8o
Moles T B bound]l ( x l o ~) 6o
25 °
37 °
58
1.75 1.27 I. I8 0.96 0.83
1.38 0.96 0.89 0.58 0.43
1.03 o.71 0.52 0.46 0.44
0.60 0.40 0.42 0.42 0.39
p H dependence T h e d e p e n d e n c e on p H was c o n s i d e r e d for b o t h t h e H A - T B a n d C S C - T B s y s t e m s . A s a f u n c t i o n of p H a t c o n s t a n t t e m p e r a t u r e a n d c o n s t a n t c o u n t e r - i o n c o n c e n t r a t i o n i t w a s f o u n d t h a t t h e i n t e r a c t i o n of T B w i t h H A t o f o r m m e t a c h r o m a t i c a b s o r b i n g c o m p l e x e s d e c r e a s e d p r e c i p i t o u s l y f r o m p H 4 . o t o p H 3.0. A t t h e l o w e r p H
Biochint. Biophys..q cta, 83 (~964) 42 51
47
POLYSACCHARIDE--DYE INTERACTIONS
a metachromatic complex could not be detected under the experimental conditions. These results are in keeping with the values reported for the pKa of glucuronic acid (pK = 3.33) 24 in the HA moiety. In contrast to this, the change in the metachromatic properties of the CSC-TB system were different. At constant concentration of TB and CSC, temperature and counter-ion concentration there was no alteration of the absorption spectra of CSC-TB solutions from pH 4.0 to p H 7.0. Between p H 4.0 and p H 3.0 there was a precipitous change in the absorption characteristics of these solutions. The intensity of the short-wavelength maximum decreased to approx, one-half its original value. However, a distinct metachromatic effect persisted to p H 2.0. Measurements below this pH were not made. These results are again consistent with the pKa for the dissociation of the C O 0 - groups on the glucuronic acid residues and with the dissociation of sulfate half-ester groups that are readily ionizable at pH 2.0 (see ref. 24). Of importance in analyzing the pI-[ dependence is that above pH 4.0 CSC-TB complexes show an absorption maximum at 530 m/z while below p H 3.0 the absorption maximum has shifted to 520 m/z.
Viscosity measurements In order to determine whether there were any macroscopic changes in the structure of these polysaccharides their viscous behavior was measured with and without TB as a function of ionic strength, concentration and temperature. The viscosity of HA in distilled water at several temperatures with and without TB is shown in Table IV. The addition of TB did not change the viscosity of HA at the concentrations used, although the binding of TB to HA was extensive. There was no dependence of the viscosity of HA or HA-TB solutions on temperature. In contrast to the constant viscosity with temperature andTB concentration the absorption spectrum was temperature dependent. TABLE
IV
EFFECT OF TEMPI~RATURE AND DYE ON RELATIVE VISCOSITY OF HYALURONATE IN DISTILLED WATER T B c o n c e n t r a t i o n 2. 5. lO -5 M. HA conch. (g/~oo ml)
28.5 ° No dye
37.5 ° Plus dye
No dye
58.8 ° Plus dye
No dye
Plus dye
0.00208
I.O9
I.O 9
I. I2
I. 13
I. I I
I. IO
o.oo625
1.31
1.34
1.36
1.36
1.35
1.34
O.OLO4
1.5I
1.52
1.59
1.57
1.57
1.54
0.0208
2.08
2.o8
2.24
2.36
2.2 t
2.1o
2.63
2.57
2.72
2.61
o.o412
3.38
3.27
3.4 °
3.29
3.37
3.32
o.o516
3.56
3.62
o.o31o
o.o625
4.o4
3.92
3.83
4.39
4.19
4.57
3.69 4.28
The change in viscosity as a function of Na + concentration is shown in Fig. 4The shape of the curves for the effect of Na+ on metachromasy and viscosity is similar. There was no effect of temperature on the viscous properties in the presence of Na +. Biochim.
B i o p h y s . A c l a , 83 (1964) 4 2 - 5 1
4~
M.D.
SCHOENBEIRG, R. I). MOORE
In addition to these experiments in dilute solution observations were made on tissue sections rich in these polysaccharides as a function of pH and ionic strength. The observations on the tissue sections were qualitatively the same as those found in solution. 5.0
4.(
"" 3.0
oo
o~ "~, o_> 2.0
"~-~-~m~R~__
I.C
°o
i
i
o.ol
o:o3
i
o.bs'
olo7
tonic strength
Fig. 4. C h a n g e in v i s c o s i t y a n d m e t a c h r o m a s y as a f u n c t i o n of N a + c o n c e n t r a t i o n , o = q, × = R, w h e r e R is t h e r a t i o of t h e a b s o r p t i v i t y a t 54 ° m f f t o t h a t a t 62o m / , .
DISCUSSION
Spectrophotometric measurements show extensive interaction between TB and both HA and CSC. A model of a co-parallel complex between polyions with specific geometric conformation and planar dyes can be postulated to account for the short-wavelength transitions in the absorption spectra (Fig. 5). Theoretical and experimental consideraLONG RANGE FORCES
H
H
H S
I
~
C
I
C
N+
N +
¢t
I
C
coo
o
I
C
N+ j
'
~
N.-p POLAR
BOND~
c..osS,
coo
0 NHA¢
NHAc
Fig. 5. S c h e m a t i c i l l u s t r a t i o n of a m e t a c h r o m a t i c c o m p l e x .
lYiochim. Biophys.
/Iota, 83 (z904) 4 ~ 51
POLYSACCHARIDE--DYE
INTERACTIONS
49
tions indicate that the changes in the absorption spectra are a result of the formation of a complex having a linear arrangement of regularly spaced, extensively conjugated dye molecules, so aligned, that their planes are parallel and approximately normal, or inclined toward the long axis of the chainS, 7-11,13,14. Steric considerations limit the kinds of molecules that can form this array to planar or very nearly planar molecules or to those which can assume a planar configuration. The array is most likely stabilized by Van der Waals-type interactions between neighboring dye molecules. The attractive forces between the dye molecules are long range and additive z~, 36. In view of the experimental data using aqueous solutions, stabilization by hydrogen bonding through water molecules included between the dye planes should be considered. In order to arrive at the spectralchanges the interplanar distance between adjacent dye molecules must be sufficiently close so that electronic interaction (~ electron) between neighbors is permissible. Interdye distances between 3.5 and 7.0/k are possible for TB when Van der Waals interactions are considered or if hydrogen bonding through a water molecule occurs. A polyion having a functional-group identity within these limits, or which can asslmae a configuration so that the distance between the functional groups falls within these limits would be a reasonable template for the formation of a metachromatic complex between the dye and the polyion. Two mutually dependent interactions are necessary in the formation of a metachromatic complex of the kind described. (I) Polar interaction between the dye and consecutive groups on the polyanion (2) interaction of the Van der Waals type between adjacent dye molecules. The extent to which the metachromatic complex is formed and remains stable is governed by the degree to which these interactions can mutually exert themselves and maintain the consecutive order of the dye molecules. In dilute aqueous solution HA and CSC are highly charged linear molecules. The dye is exposed to long segments of the polysaccharides that have a number of regularly or nearly regularly spaced sites for polar interaction. Chains of optically coupled dye molecules can be and are formed using these polysaccharides as a template. The results show that the formation of a metachromatic complex between HA and TB and CSC and TB is not the result of a random positioning of the dye along the polysaccharide chain. The evidence is in favor of preferential binding to the site next to an already polar-bound neighbor. It is apparent from the temperature dependence that the stabilizing interactions extend over a number of bound dye molecules. A combination of chemical and enzymic analyses have established the basic structures of HA and CSC. HA is a fl-I,4-1inked unbranched polymer of the disaccharide fl-I,3-glucopyranosyl-2-deoxy-2-N-acetylglucosamine, and CSC is a similarly unbranched polymer of fl-I,3-glucopyranosyl-2-deoxy-2-N-acetylgalactosamine-6-sulfate2~-3~. The structures of HA and CSC are almost the same with the exception of the sulfate half-ester group on Ca in the galactosamine residue and the position of the hydroxyl group on C4. The similarities permit comparisons between the two. On the basis of the structure of the disaccharide of HA as its repeat unit then in a linear-extended polymer with maximal repulsion between COO- units the COO-C 0 0 - distance would be in the order of IO •. With TB, whose maximum dimension is 12.5 A, a COO--COO- separation of IO A between dye planes would be too large for sufficient inter-dye interaction and z electron overlap to establish the co-parallel arrangement to give the metachromatic effect. Since the results show that HA forms a Biochim. Biophys. Acta, 83 (1964) 42-51
5O
M, D. S C H O E N B E R G , R. t). M O O R E
m e t a c h r o m a t i c complex w i t h TB it m u s t lye a s s u m e d t h a t t h e COO - -COO- distance between successive C O O - groups is smaller than I o A. The relative s t a b i l i t y of the complex as a fimction of increasing t e m p e r a t u r e also suggests t h a t the d y e molecules m u s t be closely disposed to one another. If the molecule was e x t e n d e d in solution with the io-z~ spacing between COO - groups as a n t i c i p a t e d from its disaccharide structure, the f o r m a t i o n of a m e t a c h r o m a t i c complex would necessitate size a n d shape changes of the p o l y s a c c h a r i d e molecule r e p r e s e n t e d as a contraction of t h e molecule. These alterations should be reflected in the viscosity of HA--TB complexes. No differences were found. T h e independence of the viscosity with extensive H A - T B interaction indicates at least no gross change in the configuration of the polysaccharide as a result of the f o r m a t i o n of the m e t a c h r o m a t i c complex. On the basis of these observations the conformation of H A in aqueous solution m u s t fulfill the following r e q u i r e m e n t s : (x) the s e p a r a t i o n between the C O O - groups on neighboring disaccharide residues m u s t be in the order of 7 k or less a n d have a configuration so t h a t t h e planes of b o u n d dye molecules can overlap (z) the molecules m u s t be sufficiently rigid so t h a t its viscous b e h a v i o r is n o t altered as a function of i n t e r a c t i o n with the dye or as a flmction of t e m p e r a t u r e . E x a m i n a t i o n of molecular models shows t h a t there is sufficient r o t a t i o n a b o u t the glycosidic bonds to reduce the charge s e p a r a t i o n between C O O - groups to t h e order of 7 A. Distances m u c h closer t h a n this a p p e a r to result in b o n d distortions. The m o s t likely possibility for the conformation of H A is t h a t of a fairly" rigid, linear chain, where neighboring C O O groups are close together. This model is consistent w i t h the results of viscosity, lights c a t t e r i n g a n d flow-birefringence m e a s u r e m e n t s sv .m. The changes in the l n e t a c h r o m a t i c properties as a result of the a d d i t i o n of N a + are m o s t likely the result of the successful c o m p e t i t i o n between the m e t a l ion a n d the dye. The fall in viscosity on the a d d i t i o n of electrolyte is c o m p a r a b l e to the b e h a v i o r of DNA. In the case of CSC similar p o s t u l a t e s concerning its configuration t a i l be made,, p a r t i c u l a r l y because of its b e h a v i o r with TB between p H 2.o a n d 4.o. The m e t a c h r o m a t i c effect o b s e r v e d above p H 4.0 is the result of the i n t e r a c t i o n of the dye w i t h b o t h the C O 0 - a n d OSO 3- groups. The persistence of the m e t a c h r o m a t i c effect below p H 3.o, shows t h a t at the higher H + c o n c e n t r a t i o n the m e t a c h r o m a t i c effect is the result of t h e interaction with. the sulfate half-ester groups. I t has a l r e a d y been p o i n t e d out t h a t the C O 0 - groups are n o t dissociated at the lower p H ' s . The change in the m a g n i t u d e a n d location of the m e t a c h r o m a t i c p e a k below p H 3.o shows t h a t there are two stacks of dye molecules. One s t a c k corresponds to the C O O - a n d one s t a c k corresponds to the OSO3-. In view of the m e t a c h r o m a t i c effect below p H 3.o it m u s t be a s s u m e d t h a t the COO - a n d OSO a - groups are not disposed on the same side of the p o l y m e r chain. I t has been shown bv MATHEWS AN1) DORFMAN'~4 t h a t the presence of t h e sulfate half-ester groups does n o t m o d i f y the dissociation c o n s t a n t of t h e CO0-groups. I t m u s t be inferred t h a t t h e y are well s e p a r a t e d . Other evidence has shown t h a t the molecule retains its s y m m e t r y over the same range of p H . It is a t t r a c t i v e to conclude t h a t CSC has a conformation similar to t h a t p r o p o s e d for HA. In t e r m s of biological significance it can be shown t h a t identical m e t a c h r o m a t i c changes occur in tissues. Tissues t h a t are rich in H A i.e. W h a r t o n ' s jelly show an identical d e p e n d e n c e of m e t a c h r o m a s y on p H a n d ionic strength. Similarly, tissues t h a t are rich in CSC also show the p H a n d ionic s t r e n g t h dependence found i~f vitro Hiochbn. Hiophy,~..Icla, 83 (1904) 42 5~
POLYSACCHARIDE--DYE INTERACTIONS
51
(i.e. cartilage). In these tissues the metachromatic effect persists below pH 3.o with a similar diminution in the intensity of the reaction. It is therefore likely that the configuration of these polysaccharides in vivo is the same as those suggested in dilute solution. ACKNOWLEDGEMENTS
This work was supported in part by grant AI9o 9 and R G 720o from the U.S. Public
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Biochim. Biophys. Acta, 83 (1964) 42-51