15 Rheological Studies on Proteoglycan Solution Properties of Polysaccharides Downloaded from pubs.acs.org by IMPERIAL COLLEGE LONDON on 11/27/18. For personal use only.
GO MATSUMURA School of Pharmaceutical Science, Showa University, Hatanodai 1-5-8, Shinagawa, Tokyo 142, Japan
The physical and mechanical properties of cartilage are certainly important for the physiological role of this tissue. Proteoglycan in connection with collagen and other constituents of this tissue may affect its physical properties. Cartilage proteoglycan is a complex species. To a protein core, many chondroitin sulfate and keratan sulfate chains are attached. The molecular weight of a proteoglycan monomer prepared from bovine nasal septa has been reported as two to three million daltons (1), for example. This monomer is bound to a hyaluronic acid backbone reversibly and noncovalently. The gigantic proteo glycan thus formed is stabilized by link proteins. In the present study, we attempted to elucidate the rheolog i c a l behavior of proteoglycan in viscous solution, with special regard to the differences between free monomer and aggregate. Materials Proteoglycan monomer. From whale nasal cartilage proteo glycan was extracted with 0.5M LaCl in the presence of protease inhibitors (38mM α-aminohexanoic acid, 5mM benzamidine HCl and 10mM EDTA-2Na). From bovine nasal cartilage proteoglycan was recovered in Fraction Ρ (Scheme 1) in monomeric form binding with link protein(s) (2). However, most proteoglycan in Fraction Ρ was characterized as aggregated in the present preparation by ultracentrifugation (Figure 1, top) and gel f i l t r a t i o n profiles. So the preparation was reprecipitated in the presence of 2M urea in addition to LaCl3. In this precipitate, proteoglycan was recovered mostly as monomer (Figure 1, middle). By polyacryl amide gel electrophoresis in the presence of sodium dodecylsulfate, the existence of link proteins was suggested. The presence of some contaminating proteins was also suggested by this electrophoresis and by the high protein content of the preparation. 3
An example o f t h e chemical and p h y s i c a l a n a l y s i s o f t h i s preparation follows: glucuronic acid 2 9 % , h e x o s e 7.1%,
0097-6156/81/0150-0213$06.25/0 © 1981 American Chemical Society
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Scheme 1 P r e p a r a t i o n o f P r o t e o g l y c a n Monomer f r o m Whale N a s a l C a r t i l a g e C a r t i l a g e as s m a l l c u b e s ( 5 0 g ) 0.5M L a C l ( 1 L ) O v e r n i g h t a t 4°C Extract D i l u t e w i t h w a t e r (9 v o l u m e s ) 72 h o u r s a t 4°C 3
I
Precipitate j EDTA s o l u t i o n (300ml) I Overnight Solution Sodium a c e t a t e (15g) S t i r f o r 6 hours E t h y l a l c o h o l (900ml) Precipitate W a t e r (200ml) D i a l y s i s a g a i n s t 0.01M EDTA and t h e n w a t e r Lyophylized F r a c t i o n _P
Supernatant ( F r a c t i o n S)
0.5M L a C l - 2 M u r e a (240mg i n 24ml) S t i r f o r 1 h o u r a t 4°C D i l u t e w i t h w a t e r (9 v o l u m e s ) E t h y l a l c o h o l and s o d i u m a c e t a t e Precipitate EDTA s o l u t i o n (20ml) D i a l y s i s against water Lyophylized P r o t e o g l y c a n Monomer p r e p a r a t i o n (125mg) 3
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protein 18.6%, d i s a c c h a r i d e c o n s t i t u e n t o f c h o n d r o i t i n s u l f a t e 6 - s u l f a t e / 4 - s u l f a t e = 1/3, i n t r i n s i c v i s c o s i t y 2 . 9 d l / g , sedimentation constant 14.7s (main) a n d I l l s ( m i n o r ) . Physical measurements were c a r r i e d o u t w i t h s o l u t i o n s i n 0.1M T r i s - H C l b u f f e r , pH 7, c o n t a i n i n g 0.05M N a C l . By t h e a d d i t i o n o f h y a l u r o n i c a c i d , t h e v i s c o s i t y o f t h e p r o t e o g l y c a n s o l u t i o n was i n c r e a s e d . H y a l u r o n i c a c i d i n an amount e q u a l t o one t w e n t i e t h o f t h e w e i g h t o f p r o t e o g l y c a n seemed t o be enough t o r e a c h t h e maximum v a l u e ( F i g u r e 2 ) . By u l t r a c e n t r i f u g a t i o n ( F i g u r e 1, b o t t o m ) and g e l f i l t r a t i o n most p r o t e o g l y c a n appeared t o e x i s t as a g g r e g a t e u n d e r t h e s e c o n d i tions . Hyaluronic acid. From a w a t e r e x t r a c t o f human u m b i l i c a l c o r d s h y a l u r o n i c a c i d was p u r i f i e d b y t h e metnod o r i g i n a l l y r e p o r t e d f o r b o v i n e s y n o v i a l f l u i d ( 3 ) . The p r e p a r a t i o n was p r a c t i c a l l y f r e e o f p r o t e i n and s u l f a t e d g l y c o s a m i n o g l y c a n s . The m o l e c u l a r w e i g h t was e s t i m a t e d a s a b o u t one and a h a l f m i l l i o n by e q u i l i b r i u m c e n t r i f u g a t i o n . Thus t h e a g g r e g a t e u s e d i n t h e p r e s e n t s t u d y was n o t t h e n a t i v e a g g r e g a t e o f t h e c a r t i l a g e t i s s u e , b u t a n a r t i f i c i a l one. Methods V i s c o s i t y of d i l u t e solutions. A c a p i l l a r y viscometer (Cannon-Manning s e m i m i c r o , No. 100) was used f o r d e t e r m i n a t i o n o f t h e i n t r i n s i c v i s c o s i t y and f o r s t u d y o f e n z y m a t i c d e g r a d a t i o n . Measurements were c a r r i e d a t 37°C. F o r t h e l a t t e r s t u d y s p e c i f i c f l u i d i t i e s , t h e r e c i p r o c a l o f s p e c i f i c v i s c o s i t y , were p l o t t e d a g a i n s t r e a c t i o n t i m e . W i t h random d e g r a d a t i o n o f a c h a i n p o l y m e r a s t r a i g h t l i n e i s o b t a i n e d b y t h i s p l o t t i n g , and t h e slope o f the l i n e i s p r o p o r t i o n a l t o the r e a c t i o n r a t e constant (4). V i s c o s i t y w i t h s t a t i o n a l flow. A cone-plate type r o t a t i n g r h e o m e t e r ( S h i m a d z u , RM-1, e q u i p p e d w i t h a r e d u c t i o n g e a r , RDG-1) was employed. The r a t e o f s h e a r a v a i l a b l e r a n g e d f r o m 7 . 4 8 x 1 0 " t o 7 4 . 8 / s e c . The a p p a r e n t v i s c o s i t y a t a g i v e n r a t e o f s h e a r was c a l c u l a t e d f r o m t h e r a t e o f s h e a r and t h e o b s e r v e d s h e a r s t r e s s . Samples were d i s s o l v e d i n t h e b u f f e r s o l u t i o n m e n t i o n e d b e f o r e at 2 o r 4% c o n c e n t r a t i o n and measured a t room t e m p e r a t u r e (22+l°C). 3
Dynamic v i s c o e l a s t i c i t y . From t h e o s c i l l a t o r y r o t a t i o n o f t h e p l a t e dynamic v i s c o e l a s t i c i t y was e s t i m a t e d w i t h t h e same e q u i p m e n t . The maximum s h e a r was 0.250, a n d t h e a n g u l a r f r e q u e n c i e s employed r a n g e d f r o m 5 . 8 2 x 1 0 " t o 5 . 8 2 x 1 0 " r a d / s e c . The r o t a t i o n a n g l e o f t h e p l a t e and t h e t w i s t o f t h e cone were r e c o r d e d w i t h a n X-Y p l o t t e r . From t h e h y s t e r e s i s l o o p o b t a i n e d t h e s t o r a g e modulus o r dynamic e l a s t i c i t y ( G ) a n d t h e l o s s 3
1
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Figure 1. Sedimentation profile of pro teoglycan: (top) Fraction Ρ (see Scheme 1), 0.2%, 51,200 rpm, 6 min; (middle) Proteoglycan monomer, 0.3%, 60,000 rpm, 6 min; (bottom; aggregate, 0.3% proteoglycan monomer in the presence of 0.015%) hyaluronic acid, 51,200 rpm, 6 min.
o.i
0
0.001 0.002 Concentration of Hyaluronic Acid (%)
0.003
Figure 2. Effect of hyaluronic acid on the viscosity of proteoglycan monomer. Concentration of monomer: (upper curve) 0.1 %; (lower curve,) 0.05%o.
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modulus (G") w e r e c a l c u l a t e d . The l o s s modulus can b e c o n v e r t e d t o the dynamic v i s c o s i t y by d i v i d i n g by the a n g u l a r f r e q u e n c y . Results V i s c o s i t y w i t h s t a t i o n a l f l o w . As i s u s u a l f o r s o l u t i o n s o f h i g h p o l y m e r i c compounds, t h e a p p a r e n t v i s c o s i t y o f p r o t e o g l y c a n s o l u t i o n s depended s t r o n g l y o n t h e r a t e o f s h e a r ; t h e h i g h e r v i s c o s i t y was o b s e r v e d a t t h e l o w e r r a t e o f s h e a r ( F i g u r e 3 ) . The c o n c e n t r a t i o n o f p r o t e o g l y c a n was 2% i n e a c h s o l u t i o n . To p r e p a r e t h e a g g r e g a t e s o l u t i o n h y a l u r o n i c a c i d was added t o t h e f i n a l c o n c e n t r a t i o n o f 0.1%. The a g g r e g a t e showed h i g h e r v i s c o s i t y t h a n monomer a t any r a t e o f s h e a r . B u t t h e d i f f e r e n c e was more r e m a r k a b l e a t h i g h s h e a r r a t e s . For comparison the r e s u l t w i t h h y a l u r o n i c a c i d o f t h e same c o n c e n t r a t i o n (2%) i s a l s o g i v e n in this figure with solid triangles. T h i s s o l u t i o n behaved l i k e a N e w t o n i a n l i q u i d a t s h e a r r a t e s l e s s t h a n 10"" / s e c . On t h e o t h e r h a n d , p r o t e o g l y c a n monomer seemed t o r e m a i n i n t h e power l a w r e g i o n t h r o u g h o u t t h e r a n g e examined ( 5 ) . 1
Dynamic v i s c o e l a s t i c i t y . W i t h same s o l u t i o n s u s e d f o r t h e measurements o f F i g u r e 3 d y n a m i c v i s c o e l a s t i c i t y was e x a m i n e d . I t i s c l e a r that both moduli of proteoglycan increased s i g n i f i c a n t l y upon f o r m a t i o n o f a g g r e g a t e w i t h h y a l u r o n i c a c i d ( F i g u r e 4). T h i s e f f e c t was more p r o n o u n c e d a t h i g h e r f r e q u e n c i e s a n d f o r t h e s t o r a g e m o d u l u s . Thus t h e l o s s t a n g e n t , w h i c h i s t h e r a t i o o f t h e l o s s modulus t o t h e s t o r a g e m o d u l u s , was d e c r e a s e d by t h e p r e s e n c e o f h y a l u r o n i c a c i d ( F i g u r e 5 ) . Though t h e l o s s t a n g e n t o f p r o t e o g l y c a n monomer was d e c r e a s e d b y i n c r e a s i n g t h e a n g u l a r f r e q u e n c y , t h a t o f a g g r e g a t e r e m a i n e d c o n s t a n t . As i s w e l l known, h y a l u r o n i c a c i d c a n n o t b e r e p l a c e d b y any o t h e r l i n e a r a c i d p o l y s a c c h a r i d e f o r aggregate formation. I n the r h e o l o g i c a l e x a m i n a t i o n s m e n t i o n e d above o n l y h y a l u r o n i c a c i d c o u l d a f f e c t t h e b e h a v i o r o f p r o t e o g l y c a n monomer s o l u t i o n . The v i s c o e l a s t i c i t y o f h y a l u r o n i c a c i d s o l u t i o n was much more i n f l u e n c e d t h a n p r o t e o g l y c a n , e i t h e r monomer o r a g g r e g a t e , by t h e a n g u l a r f r e q u e n c y o f t h e p l a t e . Thus t h e o r d i n a t e o f F i g u r e 6, w h i c h r e p r e s e n t s b o t h m o d u l i , i s e x p r e s s e d l o g a r i t h mically. I n c o n t r a s t t o p r o t e o g l y c a n a g g r e g a t e t h e l o s s modulus was b i g g e r t h a n t h e s t o r a g e modulus e s p e c i a l l y a t l o w e r f r e q u e n cies . E f f e c t o f Streptomyces h y a l u r o n i d a s e . The e f f e c t s o f some d e p o l y m e r i z i n g enzymes on t h e v i s c o e l a s t i c p r o p e r t i e s o f p r o t e o g l y c a n were e x a m i n e d . The f i r s t example was t h a t o f S t r e p t o m y c e s h y a l u r o n i d a s e ( h y a l u r o n a t e l y a s e , EC 4 . 2 . 2 . 1 . ) , w h i c h i s shown as s t r i c t l y s p e c i f i c t o h y a l u r o n i c a c i d ( 6 ) . Thus t h i s enzyme can d e g r a d e o n l y t h e h y a l u r o n i c a c i d b a c k b o n e o f a g g r e g a t e a n d s h o u l d n o t a t t a c k t h e p r o t e o g l y c a n monomer. To 0.1% s o l u t i o n o f a g g r e g a t e 0.05TRU o f S t r e p t o m y c e s h y a l u r o n i d a s e was added. A s
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Figure 3. Rheogram of proteoglycan and hyaluronic acid: (Φ) monomer, 2%; (O) aggregate (2% monomer in the presence of 0.1% hyaluronic acid); (A) hya luronic acid, 2 %.
H -2 Log(Angu1ar
1 -1 Frequency)
1
ο
Figure 4. Dynamic viscoelasticity of proteoglycan: (Ο,Φ) monomer; (A, A)' aggregate (see Figure 3 for concentration); (Φ, A) storage modulus; (O, A) loss modulus.
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+2
Log
Figure 5.
(Angular Frequncy)
Loss tangent of proteoglycan: (%) monomer; (O) aggregate (see Figure 3 for concentration).
Log(Angular Frequency)
Figure 6.
Dynamic viscoelasticity of hyaluronic acid: (%) storage modulus; (O) loss modulus (see Figure 3 for concentration).
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shown i n F i g u r e 7, t h e s p e c i f i c f l u i d i t y o f t h e a g g r e g a t e i n c r e a s e d g r a d u a l l y by t h i s e n z y m a t i c t r e a t m e n t . This increase i n s p e c i f i c f l u i d i t y seemed t o l e v e l o f f a f t e r some i n c u b a t i o n p e r i o d , s u g g e s t i n g t h a t m a c r o m o l e c u l a r p r o t e o g l y c a n monomer r e m a i n e d even a f t e r p r o l o n g e d d i g e s t i o n . W i t h o u t t h e p r e s e n c e of h y a l u r o n i c a c i d , however, the s p e c i f i c f l u i d i t y of p r o t e o g l y c a n monomer s o l u t i o n a l s o i n c r e a s e d a f t e r t r e a t m e n t w i t h t h i s enzyme. The p l a t e a u v a l u e was s i m i l a r t o t h a t o b t a i n e d i n t h e presence of h y a l u r o n i c a c i d . As shown i n F i g u r e 1 ( b o t t o m ) , t h i s p r e p a r a t i o n c o n t a i n e d some p r o t e o g l y c a n i n t h e a g g r e g a t e d f o r m , b u t t h e amount o f t h e a g g r e g a t e was t o o s m a l l t o e x p l a i n t h e d e c r e a s e i n v i s c o s i t y p r o d u c e d by t h i s e n z y m a t i c d i g e s t i o n . To 4% s o l u t i o n of p r o t e o g l y c a n , e i t h e r i n t h e p r e s e n c e o f o r i n t h e a b s e n c e o f h y a l u r o n i c a c i d , 1TRU o f t h e enzyme was added. The v i s c o e l a s t i c i t y o f t h e s e r e a c t i o n m i x t u r e s were measured w i t h a p p r o p r e a t e i n t e r v a l s ( F i g u r e 8 ) . W i t h t h e a g g r e g a t e s o l u t i o n t h e d e c r e a s e o f b o t h m o d u l i was o b s e r v e d . The d e c r e a s e o f s t o r a g e modulus seemed t o s t o p a f t e r some r e a c t i o n p e r i o d , b u t t h e d e c r e a s e o f l o s s modulus c o n t i n u e d f u r t h e r . A g a i n t h i s enzyme a f f e c t e d t h e v i s c o e l a s t i c p r o p e r t i e s o f t h e monomer s o l u t i o n . The e f f e c t on t h e l o s s modulus was l i t t l e , i f any. However, t h e s t o r a g e modulus i n c r e a s e d s i g n i f i c a n t l y . S i n c e t h e s e d i m e n t a t i o n c o n s t a n t o f t h e monomer p r e p a r a t i o n was n o t a f f e c t e d by t h i s e n z y m a t i c t r e a t m e n t , t h e d e p o l y m e r i z a t i o n o f p r o t e o g l y c a n was n o t l i k e l y . The i n c r e a s e o f s t o r a g e modulus i s d i f f i s u l t t o e x p l a i n i n terms o f d e g r a d a t i o n . One m i g h t s p e c u l a t e t h a t some p r o t e o g l y c a n monomer e x i s t e d as a complex w i t h s m a l l h y a l u r o n i c a c i d o l i g o m e r . The r e m o v a l o f t h i s o l i g o m e r m i g h t c a u s e some c o n f o r m a t i o n a l change i n t h e p r o t e o g l y c a n molecule t o i n c r e a s e the s p e c i f i c f l u i d i t y of d i l u t e s o l u t i o n and t h e s t o r a g e modulus o f c o n c e n t r a t e d s o l u t i o n . The p o s s i b i l i t y t h a t t h i s enzyme p r e p a r a t i o n was c o n t a m i n a t e d w i t h p r o t e o l y t i c enzymes was n o t e l i m i n a t e d . As w i l l be s t a t e d l a t e r , b o t h m o d u l i o f t h e p r o t e o g l y c a n monomer i n c r e a s e d w i t h p r o t e o l y t i c digestion. E f f e c t o f c h o n d r o i t i n a s e ABC. T h i s enzyme ( c h o n d r o i t i n ABC l y a s e , EC 4.2.2.4.) a l s o d e p o l y m e r i z e s t h e h y a l u r o n i c a c i d b a c k bone o f a g g r e g a t e . I n a d d i t i o n , i t can degrade c h o n d r o i t i n s u l f a t e c h a i n s o f monomer, b u t n o t i t s k e r a t a n s u l f a t e c h a i n s . To 0.1% s o l u t i o n o f p r o t e o g l y c a n Q.005U o f t h e enzyme was added. As shown i n F i g u r e 9, t h e s p e c i f i c f l u i d i t y o f t h e s o l u t i o n , e i t h e r i n the presence of or i n the absence of h y a l u r o n i c a c i d , i n c r e a s e d i n two s t a g e s . The f i r s t l i n e a r i n c r e a s e may i n d i c a t e t h e b r e a k down o f h y a l u r o n i c a c i d b a c k b o n e . Then upward i n c r e a s e w h i c h f o l l o w s may be due t o t h e s h o r t e n i n g o r r e m o v a l o f c h o n d r o i t i n s u l f a t e c h a i n s o f monomeric p r o t e o g l y c a n . The e f f e c t on v i s c o e a l s t i c p r o p e r t i e s o f c o n c e n t r a t e d s o l u t i o n ( 4 % p r o t e o g l y c a n and 1U c h o n d r o i t i n a s e ABC) was more o r l e s s s i m i l a r to that w i t h Streptomyces h y a l u r o n i d a s e treatment ( F i g u r e
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180
Reaction Time (min)
Figure 7. Effect of Streptomyces hyaluronidase on the specific fluidity of dilute solution of proteoglycan. Concentrations: proteoglycan, 0.1%; enzyme, 0.05 TRU; (Φ) monomer; (O) aggregate.
0
30
60 90 Reaction Time (min)
120
Figure 8. Effect of Streptomyces hyaluronidase on the dynamic viscoelasticity of proteoglycan. Concentrations: proteoglycan, 4%?; enzyme, 1 TRU. Angular fre quency: 0.349 rad/s; (0,%) monomer; (A, A) aggregate; (Φ, A) storage modu lus; (O, A) loss modulus.
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Figure 9. Effect of chondroitinase ABC on the specific fluidity of dilute solution of proteoglycan. Concentrations: proteoglycan, 0.1%; enzyme, 0.005 U; (Φ) mono mer; (O) aggregate.
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100 R e a c t i o n T i m e (min)
Figure 10. Effect of chondroitinase ABC on the dynamic viscoelasticity of proteo glycan. Concentrations: proteoglycan, 4%; enzyme, 1 U. Angular frequency: 0.349 rad/s; (Ο, Φ) monomer; (A, A) aggregate; (Φ, storage modulus; (O, A) loss modulus.
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£20
0
100
Reaction T i m e (min)
200
Figure 11. Effect of trypsin on the dynamic viscoelasticity of proteoglycan. Con centrations: proteoglycan, 4%; enzyme, 1 U. Angular frequency: 0.349 rad/s; (Φ, Φ) monomer; (A, A) aggregate; (Φ, A) storage modulus; (O, A) loss modu lus.
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15
__| Log
J
I 2
-
1
0
(Angular Frequncy)
Figure 12. Dynamic viscoelasticity of proteoglycan monomer treated with trypsin for 2 h: (Φ) storage modulus; (O) loss modulus (see Figure 11 for concentrations).
1
-0.2
•
-0.1
•
0
I
0.1
I—
0.2
Shear
Figure 13. Hysteresis loop of proteoglycan monomer: ( ) after 5 min; ( after 1 h (see Figure 11 for concentrations and angular frequency).
)
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OF
POLYSACCHARIDES
10). A f t e r e x h a u s t i v e d i g e s t i o n , h o w e v e r , v i s c o e l a s t i c i t y was almost completely a b o l i s h e d . Effect of trypsin. T h i s enzyme (EC 3.4.21.4) c a n d e g r a d e the p r o t e i n core o f p r o t e o g l y c a n . But i n the aggregated form the h y a l u r o n i c a c i d l i n k a g e r e g i o n o f core p r o t e i n and l i n k p r o t e i n s a r e s a i d n o t t o be a t t a c k e d b y t h i s p r o t e o l y t i c enzyme ( 7 ) . W i t h a r e a c t i o n m i x t u r e o f 4% p r o t e o g l y c a n a n d 1U o f t r y p s i n the e f f e c t o f t h i s enzymatic treatment on v i s c o e l a s t i c p r o p e r t i e s was e x a m i n e d ( F i g u r e 1 1 ) . W i t h a g g r e g a t e a r a p i d d e c r e a s e o f b o t h m o d u l i was o b s e r v e d a t e a r l y s t a g e s o f t h i s t r e a t m e n t ; t h e n s m a l l i n c r e a s e s f o l l o w e d . W i t h monomer s t e e p i n c r e a s e s o f b o t h m o d u l i were o b s e r v e d ; t h e n g r a d u a l d e c r e a s e s f o l l o w e d . The h i g h e s t v a l u e s were e v e n h i g h e r t h a n t h o s e o f u n t r e a t e d a g g r e g a t e . A two h o u r d i g e s t o f monomer was s u b j e c t e d t o measurement of the v i s c o e l a s t i c i t y a t v a r i o u s a n g u l a r f r e q u e n c i e s ( F i g u r e 12). I n c o n t r a s t t o u n t r e a t e d a g g r e g a t e ( F i g u r e 4 ) , t h e l o s s modulus was b i g g e r t h a n t h e s t o r a g e m o d u l u s . The l o s s m o d u l u s was l e s s ^ a f f e c t e d by the angular frequency. The h y s t e r e s i s l o o p o f p r o t e o g l y c a n , e i t h e r a s monomer o r as a g g r e g a t e , was a t y p i c a l e l l i p s e s u g g e s t i n g n o r m a l v i s c o e l a s t i c behavior. As g i v e n i n F i g u r e 13 w i t h a b r o k e n l i n e , t h e r e a c t i o n m i x t u r e o f p r o t e o g l y c a n monomer a n d t r y p s i n showed a n e l l i p t i c l o o p a t f i v e m i n u t e s a f t e r a d d i t i o n o f t h e enzyme. A f t e r one h o u r i n c u b a t i o n , t h e h y s t e r e s i s l o o p was d i s t o r t e d t o a somewhat t e t r a g o n a l s h a p e . The d i r e c t i o n o f t h e l o o p i s c l o c k w i s e . Some p l a s t i c i t y m i g h t b e i n d u c e d i n p r o t e o g l y c a n monomer b y t h i s tryptic digestion. These anomalous r h e o l o g i c a l p r o p e r t i e s were f o u n d w i t h monomer b u t n o t w i t h a g g r e g a t e . Since the h y a l u r o n i c a c i d l i n k a g e r e g i o n o f p r o t e o g l y c a n was p r o t e c t e d f r o m t r y p t i c d i g e a s t i o n b y t h e f o r m a t i o n o f a g g r e g a t e , t h i s anomaly may b e t h e r e s u l t o f a c o n f o r m a t i o n a l change c a u s e d by p r o c e s s i n g o f c o r e p r o t e i n i n this region. Conclusion The a p p a r e n t v i s c o s i t y w i t h s t a t i o n a l f l o w a n d d y n a m i c v i s c o e l a s t i c i t y o f p r o t e o g l y c a n w e r e i n c r e a s e d by f o r m a t i o n o f a g g r e gate. E n z y m a t i c t r e a t m e n t w i t h h y a l u r o n i d a s e o r t r y p s i n on p r o t e o g l y c a n monomer c o u l d i n c r e a s e t h e v i s c o e l a s t i c i t y . The f o r m e r enzyme i n c r e a s e d t h e s t o r a g e m o d u l s more r e m a r k a b l y , w h e r e a s t h e l a t t e r a f f e c t e d t h e l o s s modulus more s i g n i f i c a n t l y . These e f f e c t s m i g h t b e u n d e r s t o o d a s c o n f o r m a t i o n a l changes i n t h e monomer m o l e c u l e . F u r t h e r s t u d i e s are under the p r o g r e s s .
Litereture cited 1. Rosenbergs L.; Choi, H . ; Pal, S.; Tang, L . "Carbohydrateprotein interaction (ACS Symposium Series 88)"; American Chemical Society: Washington, D . C . , 1979; p. 186.
15.
MATSUMURA
Rheological
Studies on
Proteoglycan
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2. Mason, R. W.; Roughley, P. J . Biochem. Soc. Trans., 1974, 2, 894. 3. Matsumura, G . ; De Salegui, M . ; Herp, Α . ; Pigman, W. Biochim. Biophys. Acta, 1963, 69, 574. 4. Matsumura, G . ; Pigman, W. Arch. Biochem. Biophys., 1965, 110, 526. 5. Lenk, R. S. "Plastic rheology"; Wiley Interscience: New York, 1968; p. 12. 6. Ohya, T . ; Kaneko, Y. Biochim. Biophys. Acta, 1970, 198, 607. 7. Heinegard, D.; Hascall, V. C. J. B i o l . Chem., 1974, 249, 4250. RECEIVED October 15,
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