Solution Properties of Polysaccharides - ACS Publications - American

14 Structural Characterization of Proteoglycan Subunit from Nasal Septum by Laser Light Scattering Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 31, 2018 | https://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch014

H. REIHANIAN, A. M . JAMIESON, and J. B L A C K W E L L Department of Macromolecular Science, Case Western Reserve University, Cleveland, OH 44106 L. H . T A N G and L . ROSENBERG Connective Tissue Research Laboratories, Montefiore Hospital, The Bronx, NY 10467

Proteoglycan subunit (PGS) is a class of macromolecular species which is found i n mammalian connective tissue. Chemi c a l l y , they consist of a linear protein backbone to which are grafted linear glycosaminoglycan side-chains. Physically, they are believed to exist in their native form as complexes linked to hyaluronic acid as shown i n F i g . 1. The complex or proteogly can aggregate (PGA) is stabilized by a highly insoluble protein called link protein. PGS is isolated in purified form by u l t r a centrifugation in CsCl density gradients f i r s t under associative conditions as aggregate, PGA, and subsequently as PGS under dis sociative conditions. Purified fractions are labelled A1-D1, A1D1-D1 etc. Recently, we have carried out a series of studies of the hydrodynamic properties of solutions of proteoglycan species iso lated from bovine nasal septum . We reported a limiting sedi mentation coefficient, S° = 23.4S, diffusion coefficient D°t = 3.32 x 10 cm /sec and i n t r i n s i c viscosity [η] = 861.4 ml/g, for an A1-D1-D1 proteoglycan subunit fraction (PGS). These results lead to the c o n c l u s i o n that the weight-average molecular weight M = 3.97 x 10 . Several interesting effects were observed in the light scat tering properties of PGS at f i n i t e concentration . In aqueous NaCl, the concentration-dependence of D is strongly negative. This result, coupled to reports of a large negative second osmo tic virial coefficient suggests a self-association mechanism for PGS which i s rather surprising in view of i t s high charge density. It was suggested that the locus for self-association may l i e at the hook region where polysaccharide chains are absent (Fig. 1). It was further r e p o r t e d that, unlike the intermolecular aggregation of PGS with HA in the absence of link protein, the self-association of PGS is intensified with increase of temp erature. In the absence of added salt, anomalous light scattering properties of PGS solutions were observed. The apparent D , calculated from the f i r s t moment of the photon correlation func1

2-4

-8

2

2,4

6

W

2-4

t

5,6

2-4

2-4

t

0097-615 6/81/0150-0201$05.00/ 0 © 1981 American Chemical Society Brant; Solution Properties of Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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t i o n s e x h i b i t e d a s t r o n g dependence on s c a t t e r i n g a n g l e . I t was s u g g e s t e d t h a t t h e s e p r o p e r t i e s c o u l d be t h e r e s u l t o f a t e n d e n c y f o r i n t e r m o l e c u l a r o r d e r i n g o f t h e expanded m a c r o i o n s u n d e r t h e c o n g e s t e d c o n d i t i o n s w h i c h o c c u r a t v e r y low i o n i c s t r e n g t h . F i n a l l y , i t was d e m o n s t r a t e d t h a t D of a d i l u t e s o l u t i o n of PGS a t p h y s i o l o g i c a l i o n i c s t r e n g t h e x h i b i t e d a s i g m o i d a l dec r e a s e upon t i t r a t i o n w i t h HA i n d i c a t i n g t h e f o r m a t i o n o f PGA. In the t r a n s i t i o n r e g i o n , h i g h l y non-exponential c o r r e l a t i o n funct i o n s w e r e o b s e r v e d b u t i t was n o t p o s s i b l e t o r e s o l v e t h e s e d a t a into i n d i v i d u a l components " . I n t h i s p a p e r , we p r e s e n t a d d i t i o n a l e v i d e n c e c o n c e r n i n g these e a r l i e r observations. F i r s t , we d e s c r i b e a l i g h t s c a t t e r i n g s t u d y o f t h e s o l u t i o n p r o p e r t i e s o f a PGS sample ( A l D l f r a c t i o n ) t r e a t e d w i t h c y a n o g e n b r o m i d e (CNBr) t o s e l e c t i v e l y , and i r r e v e r s i b l y , d e s t r o y t h e n a t i v e s t r u c t u r e o f t h e hook region. These show t h e a b s e n c e o f any s e l f - a s s o c i a t i o n b e h a v i o r . Then, a t o t a l i n t e n s i t y s t u d y o f t h e l i g h t s c a t t e r i n g p r o p e r t i e s o f PGS i n s a l t - f r e e s o l u t i o n s was c a r r i e d o u t . A comparison of these r e s u l t s w i t h the e a r l i e r d i f f u s i o n data under such c o n d i t i o n s supports the c o n c l u s i o n t h a t i n t e r m o l e c u l a r o r d e r i n g i s p r e s e n t u n d e r t h e s e c o n d i t i o n s . F i n a l l y , we p r e s e n t r e s u l t s o f a d y n a m i c l i g h t s c a t t e r i n g s t u d y o f t h e t i t r a t i o n o f PGS w i t h HA i n w h i c h an a t t e m p t t o s e p a r a t e PGA and r e s i d u a l PGS monomer was made. 2 - 4

t

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 31, 2018 | https://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch014

2

M e t h o d s and

4

Materials

Materials H i g h l y p u r i f i e d p r o t e o g l y c a n f r a c t i o n s A l - D l - D l and A l - D l i s o l a t e d f r o m b o v i n e n a s a l c a r t i l a g e was u s e d i n t h i s e x p e r i m e n t . The s a m p l e s had b e e n d e t e r m i n e d t o be f r e e o f c o n t a m i n a t i o n by h y a l u r o n i c a c i d o r l i n k p r o t e i n . D e t a i l s o f i s o l a t i o n and p u r i f i c a t i o n of these f r a c t i o n s are d e s c r i b e d i n the l i t e r a t u r e ' . H y a l u r o n i c a c i d s a m p l e f r o m R o o s t e r ' s Comb was g e n e r o u s l y s u p p l i e d by D r . Swann, H a r v a r d M e d i c a l S c h o o l . An A l - D l s u b u n i t f r a c t i o n w h i c h had b e e n t r e a t e d w i t h CNBr was s u p p l i e d by D r . E. N. J a y n e s . The CNBr c l e a v a g e i n t h e s e s a m p l e s had b e e n p e r f o r m e d a s d e s c r i b e d by E. G r o s s . I t i s anticipated that mild t r e a t m e n t o f PGS w i t h CNBr w i l l d e s t r o y p r i n c i p a l l y t h e g l o b u l a r hook r e g i o n l e a v i n g t h e r e m a i n d e r o f t h e c o r e e s s e n t i a l l y i n t a c t . S o l u t i o n s p r e p a r e d u s i n g t h e s e samples were a l l b u f f e r e d w i t h 0.01M 2 - ( N - m o r p h o l i n o ) e t h a n e s u l f o n i c a c i d (MES) t o pH 7.0. All s o l u t i o n s w e r e p r e p a r e d f o r l i g h t s c a t t e r i n g by M i l l i p o r e f i l t e r a t i o n and a d j u d g e d s u i t a b l e f o r l i g h t s c a t t e r i n g when t h e i n t e n s i t y o f l i g h t s c a t t e r i n g was c o n s t a n t t o w i t h i n 1%. 7

8

9

1 0

Quasielastic Laser Light The

Scattering

l i g h t s c a t t e r i n g s t u d i e s were c a r r i e d o u t u s i n g

two

Brant; Solution Properties of Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

op-

REIHANIAN

14.

ET AL.

Proteoglycan

Subunit

203

t i c a l m i x i n g s p e c t r o m e t e r s . The f i r s t has b e e n w e l l d e s c r i b e d elsewhere ' and was u s e d t o s t u d y t h e s p e c t r u m o f s c a t t e r e d l i g h t i n t h e a n g u l a r r a n g e 30° < θ < 75°. The s e c o n d s p e c t r o meter i s d e s i g n e d s p e c i f i c i a l l y f o r photon c o r r e l a t i o n a n a l y s i s and a g a i n h a s b e e n d e s c r i b e d e l s e w h e r e . A n a l y s i s o f t h e c o r r e l a t i o n f u n c t i o n s o b t a i n e d b y t h i s i n s t r u m e n t was p e r f o r m e d b y t h e method o f m o m e n t s u s i n g t h e w e i g h t i n g p r o c e d u r e o f Brown e t al. i n which a polynomial f u n c t i o n i s f i t t e d t othe points i n the p l o t o f l n c ( x ) a g a i n s t t i m e : 1 1

1 2

3

1 3


1

4

l n | c ( x ) | - Β = -Γτ + — ^ | ( Γ τ ) 2! Γ

2

(1)

2

T h i s g e n e r a t e s t h e f i r s t moment T, C(T)

from which the z-averaged

c i e n t Dt,z

o f the c o r r e l a t i o n

translational diffusion

function coeffi

computed Γ = D

where Κ t h e s c a t t e r i n g

t ) Z

K2

(2)

vector Κ = 47r/Xsin0/2

(3)

w h e r e λ i s t h e w a v e l e n g t h o f l i g h t i n t h e medium and θ i s t h e s c a t t e r i n g a n g l e . The s e c o n d moment i s a l s o computed w h i c h i s a measure o f d e v i a t i o n s from s i n g l e e x p o n e n t i a l b e h a v i o r . To i n v e s t i g a t e t h e p o s s i b i l i t y o f i n t e r m o l e c u l a r o r d e r i n g a t l o w i o n i c s t r e n g t h s o l u t i o n , we d e t e r m i n e d t h e a p p a r e n t s t r u c t u r e f a c t o r from the r e l a t i o n 1 3

2

= NM BP(K)S(K)

(4)

w h e r e P ( K ) i s t h e p a r t i c l e s c a t t e r i n g f u n c t i o n , Ν i s t h e number o f p a r t i c l e s p e r u n i t v o l u m e , M i s t h e p a r t i c l e w e i g h t and Β i s o p t i c a l c o n s t a n t . The a p p a r e n t s t r u c t u r e f a c t o r S ( K ) , c h a r a c t e r i z e s the angular v a r i a t i o n of the i n t e n s i t y of s c a t t e r e d l i g h t as a r e s u l t of intermolecular interference. For macroion s o l u t i o n s w i t h l o n g - r a n g e i n t e r m o l e c u l a r o r d e r i n g , S(K) i s r e l a t e d t o t h e r e l a x a t i o n t i m e s TfT- f o r c o n c e n t r a t i o n f l u c t u a tions b y ' 1

1 5

1 6

r

w h e r e D'(K)

D

= D'OO^CK)-

1

(5)

i s the " s e l f p a r t i c l e " t r a n s l a t i o n a l d i f f u s i o n coef

ficient. S c a t t e r e d i n t e n s i t i e s w e r e d e t e r m i n e d f r o m mean s c a t t e r i n g c o u n t r a t e s u s i n g a n O r t e c #9315 P h o t o n C o u n t e r . Measurements o f t h e p o l a r i z e d and d e p o l a r i z e d s c a t t e r i n g components o f p u r e benzene were used t o c a l i b r a t e t h e photometer.


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Results a)

S e l f - A s s o c i a t i o n o f PGS

I n F i g . 2, we d i s p l a y r e s u l t s o f a p h o t o n c o r r e l a t i o n s t u d y o f s o l u t i o n s o f A l - D l PGS i n 0.15M N a C l a t pH 7.4 a t t e m p e r a t u r e s Τ = 25°C and 37°C. A t t h e s e c o n c e n t r a t i o n s , t h e c o r r e l a t i o n f u n c t i o n s a r e c l o s e t o s i n g l e e x p o n e n t i a l d e c a y s {^l^ ^ 0.01) and t h e d a t a a r e w e l l c h a r a c t e r i z e d b y a s i n g l e p a r a m e t e r , t h e mean t i m e c o n s t a n t T. A s d e s c r i b e d i n e a r l i e r w o r k " " , D decreases as c o n c e n t r a t i o n i n c r e a s e s , r e f l e c t i n g an i n c r e a s e i n p a r t i c l e s i z e because o f s e l f - a s s o c i a t i o n . For comparison, i n F i g s . 3 and 4, we show t h e c o n c e n t r a t i o n - d e p e n d e n c e o f D ( = Γ/Κ ) and t h e r e d u c e d s c a t t e r i n g i n t e n s i t y o f t h e CNBrtreated subunit. From t h e l a t t e r we compute ^ = 3.0 + 0.3 χ 1 0 g m o l e " w h i c h compares f a v o r a b l y w i t h ^ = 3.2 ± 0.3 χ 1 0 g/mole f o r n a t i v e PGS A l - D l deduced f r o m t h e Zimm p l o t shown b e l o w ( F i g . 5) and = 3.97 ± 0.2 χ 1 0 g/mole f o r a n A l - D l - D l f r a c t i o n reported e a r l i e r ' . We t h e r e f o r e deduce t h a t t h e CNBrt r e a t m e n t h a s d e g r a d e d t h e s u b u n i t p r i m a r i l y i n t h e 'hook r e g i o n a t t h e c h a i n end r a t h e r t h a n i n t h e c o r e r e g i o n . I t i s n o t e d f r o m F i g s . 3 and 4 t h a t t h e c o n c e n t r a t i o n - d e p e n d e n c e o f D and t h e s e c o n d v i r i a l c o e f f i c i e n t a r e e a c h p o s i t i v e , d i s p l a y i n g o n l y a modest e x c l u d e d v o l u m e e f f e c t . This r e s u l t contrasts w i t h t h e l a r g e n e g a t i v e c o n c e n t r a t i o n dependence o b s e r v e d f o r the i n t a c t subunit. F i n a l l y , i t i s p e r t i n e n t to note t h a t , as shown i n F i g . 2, t h e c o n c e n t r a t i o n - d e p e n d e n c e o f D f o r PGSA1D1D1 i n 0.15M N a C l i s more n e g a t i v e a t Τ = 37°C t h a n i t i s a t 25°C. 2

2

4


t

2

t

6

1

6

6

3

4

1

t

t

b)

PGS i n t h e A b s e n c e o f Added S a l t

F i g u r e 5 i s a Zimm p l o t o f t h e t o t a l i n t e n s i t y o f l i g h t s c a t t e r e d b y n a t i v e PGS A l - D l i n w a t e r i n t h e a b s e n c e o f added salt. E x t r a p o l a t i o n o f t h e s e d a t a i n t j i e l i m i t c -> 0, θ -> 0 l e a d s t o 1 ^ = 3.2 χ 1 0 g/mole and ^ = 1450A. The p r i m e m o t i v a t i o n f o r t h i s s t u d y was t o a t t e m p t t o o b s e r v e i n t e r m o l e c u l a r i n t e r f e r e n c e e f f e c t s a s embodied i n t h e p a r a m e t e r S ( K ) i n e q . ( 4 ) . From F i g . 5, t h e Zimm p l o t o s t e n s i b l y a p p e a r s q u i t e n o r m a l , p e r m i t t i n g i n t e r p o l a t i o n o f ^ and R | z « We draw a t t e n t i o n , however, t o t h e c u r v a t u r e e v i d e n t a t l o w c o n c e n t r a t i o n s . We h a v e e s t i m a t e d t h e s t a t i c s t r u c t u r e f a c t o r S ( K ) , u s i n g eq. ( 4 ) , based on t h e assumption t h a t t h e e x t r a p o l a t e d v a l ues a t c = 0 o f t h e Zimm p l o t a r e a v a l i d r e p r e s e n t a t i o n f o r P ( K ) , i . e . ( c / R ) c - K ) = l / k M P ( K ) . The r e s u l t s a r e shown i n F i g . 8. A l s o i n c l u d e d a r e a s e t o f S ( K ) v a l u e s computed f r o m o u r e a r l i e r s m a l l - a n g l e h e t e r o d y n e s t u d y o f D t ( c ) f o r PGS i n s a l t free water " . These S(K) v a l u e s were c a l c u l a t e d u s i n g e q . ( 5 ) . I t i s p o s s i b l e t h a t e r r o r s may e x i s t i n t h e e x t r a p o l a t i o n t o c = 0 i n t h i s s t r o n g l y - i n t e r a c t i n g s y s t e m . However, we n o t e o

6

e

2

4


REIHANIAN E T A L .

Proteoglycan

205

Subunit

Subunits - Core Protein


Chondroitin Sulfate Attachment Region

Keratan Sulfate Attachment Region Hyaluronic Acid

-Chondroitin Sulfate Chains '~Γ-

Τ

- Keratan Sulfate Chains ^A=^

i - v HA-binding inding r ~ \ Region

7

. Link b

Link a * * 50-80 Disaccharides

Figure 1.

Currently accepted model for connective tissue proteoglycan aggregate (D

1.0

2.0

3.0

Concentration

4.0

5.0

(mg/ml)

Figure 2. Concentration dependence of the translational diffusion coefficient of PGS A1-D1-D1 in 0.15M NaCl/0.01M MES, pH 7.0 at 25°C (Φ) and at 37°C (A)



206

SOLUTION

PROPERTIES

O F POLYSACCHARIDES

rO

•9 CO

I

1

1

1

1

0

.5

I

1.5

2

Concentration (mg/ml) Figure 3. PGS Al-Dl

Concentration dependence of translational diffusion coefficient D of fraction treated with CNBr in 0.15M NaCl, pH 7.0 at a 40° scattering angle. t z

3r

Solution Properties of Polysaccharides - ACS Publications - American

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