Aoba Et Al [1987] Selective Adsorption of Porcine-Amelogenins Onto Hydroxyapatite and Their...

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9 1987 Springer-VerlagNew York Inc.

S e le c ti ve A d s o r p t i o n o f P o r c in e A m e l o g e n i n s o n t o H y d r o x y a p a t i te a n d t h e i r

I n h i b i t o r y A c t i v i t y o n H y d r o x y a p a t i t e G r o w t h i n S u p e r s a t u r a t e d S o l u t i o n s

T . A o b a , 1 M . F u k a e , 2 T . T a n a b e , 2 M . S h i m i z u , 2 a n d E . C . M o r e n o 1

1 F o r s y t h D e n t a l C e n t e r , B o s t o n , M a s s a c h u s e t t s ; a n d 2 Tr ur um i U n i v e r s i t y , S c h o o l o f D e n t a l M e d i c i n e , D e p a r t m e n t o f B i o c h e m i s t r y ,

T s u r u m i , Y o k o h a m a , J a p a n

S u m m a r y . T h e s e l e c t iv e a d s o r p t i o n o f a m e l o g e n i n s

o n t o s y n t h e t i c h y d r o x y a p a t i t e ( H A ) a n d t h e i r i n hi b -

i t o r y a c t i v i t y o n t h e s e e d e d H A c r y s t a l g r o w t h

w e r e i n v e s t i g a t e d u s i n g e n a m e l p r o t e i n s o b t a i n e d

f r o m t h e o u t e r l a y e r o f i m m a t u r e p o r c i n e - e n a m e l

( s o f t, c h e e s e l i k e i n c o n s i s t e n c y ) o f d e v e l o p i n g p e r -

m a n e n t i n c i s o r s . S p e c i a l i n t e r e s t s w e r e p a i d t o t h e

e f f e c t o f a p o s t s e c r e t o r y d e g r a d a t i o n o f t h e o r ig i na l

a me l o g e n i n ( s ) o n t h e i r a d s o r p t i o n a n d i n h i b i t o r y a c -

t i v i t y . I n t h e a d s o r p t i o n s t u d i e s , i t w a s a p p a r e n t

t h a t t h e o r i g i n a l l y s e c r e t e d a me l o g e n i n ( 2 5 K ) , p r o -

l i n e , a n d h i s t i d i n e - r i c h p r o t e i n ( 2 a ) , a s w e l l a s t h e

h i g h e r m o l e c u l a r w e i g h t c o m p o n e n t s ( 6 0 - 9 0 K ) ,

s h o w e d a s t r o n g a d s o r p t i o n a f f i n i t y o n t o t h e H A .

T h i s a d s o r p t i o n o f p r o t e i n 2 a w a s r e l a t e d t o i t s i n hi -

b i t io n o f t h e c r y s t a l g r o w t h o f s e e d e d H A i n a d i lu t e

s u p e r s a t u r a t e d s o l u ti o n . O n t h e o t h e r h a n d , t h e

p a r t ia l l y d e g r a d e d p r o d u c t ( 2 0 K ) o f a m e l o g e n i n s ,

p r o t e i n 2 b , l o s t t h e h i g h a d s o r p t i o n a f f i n i t y o n t o t h e

H A , a n d c o n s e q u e n t l y s h o w e d n o s i g n if ic a n t in h ib i -

t o r y a c t i v i t y . T h e o b s e r v e d s e l e c t i v e a d s o r p t i o n o f

p r o t e in 2 a o n t o H A w a s a p p a r e n t a t p H 6 .0 a nd p H

7 . 4 e v e n i n t h e p r e s e n c e o f d is s o c i a t iv e s o l v e n t s ,

s u c h a s 3 M u r e a o r 2 a n d 4 M g u a n i d i n e - H C l ; h o w -

e v e r , t h i s s e l e c t i v e b e h a v i o r w a s s e n s i t i v e t o

c h a n g e s i n p H , a n d w a s n o t d i s p l a y e d a t p H v a l u e s

o f 7 .8 o r 1 0 .8 . T h e r e s u l t s s u g g e s t t h a t t h e o r i g i n a l ly

s e c r e t e d a m e l o g e n i n 2 a m a y p l a y a n a c t i v e r o l e i n

a m e l o g e n e s i s , a n d t h a t e n a m e l m i n e r a l i za t i on c o u l d

b e r e g u l a t e d b y t h e s e c r e t i o n o f a m e l o g e n i n s a n d

t h e i r i n a c t i v a t i o n t h r o u g h p a r t i a l e n z y mi c d e g r a d a -

t io n , p r i o r t o t h e i r c o m p l e t e r e m o v a l .

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D r . T . A o b a , F o r s y t h D e n t a l C e n t e r ,

1 40 F e n w a y , B o s t o n , M a s s a c h u s e t t s 0 2 11 5 .

K e y w o r d s A m e l o g e n e s is - - A m e l o g e n i ns - - H y -

d r o x y a p a t it e - - A d s o r p t i o n - - C r y s t a l g r o w th .

I n t h e p r o c e s s o f b i o lo g i c a l m i n e r a l i z a t i o n , i t i s i n -

v a r i a b l y o b s e r v e d t h a t t h e p r e s e n c e o f a n o r g a n ic

m a t r i x i s a s s o c i a t e d w i t h m i n e r a l d e p o s i t i o n . S u c h

a c o n s i s t e n t o b s e r v a t i o n h a s l e d t o t h e a s s u m p t i o n

[ 1 ] t h a t t h e ma t r i x a c t s a s a s e e d i n g ma t e r i a l t h a t

d e s t r o y s t h e s t a t e o f s t a b le s u p e r s a t u r a t i o n ( w it h

r e s p e c t t o t h e p r e c i p it a t in g s o li d p h a s e ) p r e s u m a b l y

e x i s t i n g i n t h e f l u i d f r o m w h i c h t h e mi n e r a l p r e c i p i -

t a t e s . A d i s t i n c t p o s s i b i l i t y , h o w e v e r , i s t h a t t h e o r -

g a n i c m a t r i x ( o r s o m e o f it s c o n s ti t u e n t s ) m a y a c t

a s i n h i b i to r s o f c r y s t a l g r o w t h , t h u s r e g u l a t i n g t h e

r a t e o f m i n e r al a c c r e t i o n . S u c h a m o d u l a t i o n o f t h e

m i n e r a l i z a ti o n p r o c e s s c o u l d b e r e l a t e d , f o r e x-

a m p l e , t o t h e p r e s e n c e ( a n d re m o v a l ) o f n o n c o l-

l a g e n o u s p r o t e i n s i n th e c a s e o f b o n e f o r m a t i o n

[2, 3].

I n a m e l o g e n e s i s , t w o k i n d o f p r o t e i n s h a v e b e e n

c h a r a c t e r i z e d [ 4 - 8 ] i n t h e e a r l y s t a g e s o f t h e p r o -

c e s s a n d d e s i g n a t e d a s a m e l o g e n i n s a n d e n a m e l i n s .

P u b l i s h e d r e p o r t s s u g g e s t t h a t t h e e n a m e l i n s a r e

s t r o n g ly a d s o r b e d o n t o t h e e n a m e l c r y s t a l s a n d th a t

t h e y p l a y a m a j o r r o l e i n t h e r e g u l a t i o n o f t h e

g r o w t h o f t h e e n a m e l a p a t i t e c r y s t a l s [ 7 , 9 , 1 0 ] a n d

i n t h e f o r m a t i o n o f t h e u l tr a s t r u c t u ra l f e a t u r e s s u c h

a s t h e r o d a n d i n t e r r o d e n a me l [ 4 , 1 1 ] . Re c e n t l y ,

J o d a i k i n e t a l. [ 1 2] s h o w e d t h a t o n l y e n a m e l i n

a d o p t a [ 3 - sh e e t c o n f o r m a t i o n , s u g g e s t i n g a p r e -

f e r r e d a l i g n m e n t o n t h e c r y s t a l s u r f a c e o f e n a m e l

a p a t i t e . H a y a s h i e t a l . [ 1 3 ] a l s o d e mo n s t r a t e d i n a n

i m m u n o h i s t o c h e m i c a l s t u d y t h a t t h e e n a m e l i n s c a n

b e c o n s t i t u e n t s o f th e c r y s t a l g h o s t s . Su r p r i s i n g l y ,

n o c l e a r f u n c t i o n h a s b e e n a d v a n c e d f o r t h e a m e l o -


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2 8 2 T . A o b a e t a l. : A d s o r p t i o n o f P o r c i n e - A m e l o g e n i n s o n t o H y d r o x y a p a t i t e

g e n i n s i n s p i t e o f t h e f a c t t h a t t h e y a r e t h e d o m i n a n t

p r o t e i n s i n t h e s e c r e t o r y s t a g e o f e n a m e l f o r m a t i o n .

I n f a c t , i n a r e c e n t p u b l i c a t i o n [ 1 4 ] i t w a s p r o p o s e d

t h a t t h e a m e l o g e n i n s m i g h t n o t b e d i re c t l y i n v o l v e d

i n e n a m e l m i n e r a li z a t io n b e c a u s e o n l y e n a m e l in l i k e

p r o t e i n s a r e s e c r e t e d b y r a b b i t s a m e l o b l a s t s . T h i s

l a t t e r v i e w c o n t r a s t s s h a r p l y w i t h t h e h y p o t h e s i s

[ 15 ] t h a t t h e e v o l u t i o n o f e n a m e l o i d t o e n a m e l m a y

h a v e r e q u i r e d a n e w s t r u c t u r a l p r o t e i n , t h e a m e l o -

g e n i n , t o f o r m l o n g e r c r y s t a l l i t e s i n e n a me l , s i n c e

o n e p r i n c ip a l d i s t in c t i o n b e t w e e n e n a m e l o i d a n d

e n a m e l i s th e s i z e o f t h e a p a t i t i c c r y s t a l l i t e s ; t h e y

a r e s ma l l e r a n d l e s s u n i f o r m i n s i z e i n e n a me l o i d .

B e f o r e a d v a n c i n g p o s s i b l e r o l e s f o r t h e a m e l o -

g e n i n s , i t s e e m e d d e s i r a b l e t o a s s e s s t h e i n t e r a c -

t io n s o f t h e s e p r o t e i n s w i t h m i n e r al s u r f a c e s o f

c r y s t a l s , s u c h a s h y d r o x y a p a t i t e ( H A ) , c o n s i d e r e d

a s p r o t o t y p e s o f t h e e n a m e l m i n e r a l. I n t hi s c o n -

n e c t i o n , S n e a d e t a l . [ 1 6 ] r e p o r t e d t h e c D N A s e -

q u e n c e o f m u r i n e a m e l o g e n i n , i n d i c a ti n g t h e p r e s -

e n c e o f a G l u an d A s p - r i c h s e g m e n t . T h e p u b l i s h e d

a m i n o a c i d s e q u e n c e s o f a m e l o g e n i n s f r o m p o r c i n e

a n d b o v i n e [ 1 7 , 1 8 ] s h o w e d c o n s i d e r a b l e h o m o l o -

g i e s i n t h e N - t e r m i n a l s e g m e n t o f t h e c D N A s e -

q u e n c e o f m u r i n e a m e l o g e n i n , b u t a l a c k o f t h e G l u

a n d A s p - r i c h C - t e r mi n a l s e g m e n t . A n i n t ri g u in g h y -

p o t h e s i s d e r i v e d f r o m t h i s s e q u e n c e i s t h a t a n y

f u n c t i o n o f a m e l o g e n i n s c o u l d b e m o d u l a t e d b y th e

p o s t s e c r e t o r y d e g r a d a t i o n , i . e ., c l e a v a g e o f t h e c-

t e r m i n a l s e g m e n t a n d f u r t h e r d e g r a d a t i o n i n t o

s m a l l e r m o i e t i e s .

T o a s c e r t a i n t h e a b o v e h y p o t h e s i s , t h e p r e s e n t

s t u d y w a s d e s i g n e d w i t h a d o u b l e p u r p o s e : ( 1 ) t o

i n v e s t ig a t e t h e a d s o r p t i o n b e h a v i o r o f p o r c i n e a m e -

l o g e n i n s o n t o h y d r o x y a p a t i t e , a n d ( 2 ) t o d e t e r m i n e

t h e ir e f f e c t o n t h e s e e d e d c r y s t a l g r o w t h o f h y -

d r o x y a p a t i t e i n a w e l l - d e f i n e d s o l u t i o n s u p e r s a t u -

r a t e d w i t h r e s p e c t t o h y d r o x y a p a t i t e . A s m e n t i o n e d

a b o v e , s i n c e th e o r ig i na l ly s e c r e t e d a m e l o g e n i n a r e

s u b j e c t e d t o i n c r e a s in g d e g r a d a t i o n i n s i tu [ 1 9 - 2 5 ] ,

a n a t t e m p t w a s m a d e t o a s c e r t a i n t h e e f f e c t o f s u c h

a d e g r a d a t i o n o n a d s o r p t i o n a f f i n i t i e s o n t o H A a n d

o n t h e i r i n h i b i t o r y a c t i v i t y o f c r y s t a l g r o w t h .

a t e r ia l s a n d e t h o d s

n a m e l P r o t e in s a n d H y d r o x y a p a t i t e

E n a m e l p r o t e i n s u s e d a s a d s o r b a t e s w e r e o b t a i n e d f r o m t h e

o u t e r l a y e r ( a b o u t 1 00 ~ m i n d e p t h f r o m t h e s u r fa c e ) o f i m m a -

t u r e e n a m e l ( s o f t , c h e e s e l i k e i n c o n s i s t e n c y ) t i s s u e s i n t h e m a -

t r i x - f o r m a t i o n s t a g e o f d e v e l o p i n g p e r m a n e n t i n c i s o r s i n 6 -

m o n t h - o l d p i g l e t s . P r e v i o u s s t u d i e s [ 2 2 ] d e m o n s t r a t e d t h a t t h i s

l a y e r c o n t a i n s m u c h o f t h e o r i g i n al l y s e cr e t e d a m e l o g e n i n p r o -

t e i n ( s ) , ( h e r e t o f o r e c a l l e d p r o t e i n 2 a ) , a s c o m p a r e d t o t h e u n d e r -

l y i n g i n n e r e n a m e l t i s su e s . A m i n o a c i d c o m p o s i t i o n a n d p a r t i a l

s e q u e n c e o f t h i s p r o t e i n h a v e b e e n r e p o r t e d [ 22 , 26 ]. B r i e f ly ,

p r o t e i n 2 a w a s c h a r a c t e r i z e d b y i t s h ig h c o n c e n t r a t i o n s o f p r o -

l i n e , g l u t a m i c a c i d , l e u c i n e , a n d h i s t i d i n e : t h e N - t e r m i n a l s e -

q u e n c e o f t h is p r o t e i n e n d e d i n M e t - P r o - L e u - P r o - P ro - H i s - a n d

t h e C - t e r m i n a l s e q u e n c e e n d e d i n - V a t - A s p . T h e C - t e r m i n a l s e -

q u e n c e w a s t h e s a m e a s t h a t o f m u r i n e am e l o g e n i n , w h i c h w a s

d e t e rm i n e d u s i n g c o m p l e m e n t a ry D N A t o t h e m e s s e n g e r R N A s

[ 16 ]. W e a l s o i n t e n d e d t o c o m p a r e t h e n a t u r e o f p r o t e i n 2 a a n d

t h e p a r t i a l d e g r a d e d p r o d u c t ( h e r e t o f o r e c a l l e d p r o t e i n 2 b ) .

T h e m o l e c u l a r w e i g h t s o f p r o t e i n s 2 a a n d 2 b w e r e 2 5 K a n d 2 0 K

d a l t o n , r e s p e c t i v e l y , a s o b t a i n e d b y t h e s o d i u m d o d e c y l s u l -

f a t e / p o l y a c r y l a m i d e - g e l e l e c t r o p h o r e s i s ( S D S - P A G E ) a c -

c o r d i n g t o t h e p r o c e d u r e o f L a e m m l i [ 27 ]. I t h a s b e e n r e p o r t e d

[2 6] t h a t t h e N - t e r m i n a l s e q u e n c e o f b o t h p r o t e i n s 2 a a n d 2 b w a s

t h e s a m e u p t o t h e 5 4 t h r e s i d u e , w h e r e a s t h e C - t e r m i n a l s e -

q u e n c e o f p r o t e i n 2 b w a s - P h y - S e r , r a t h e r t h a n - V a l - A s p o f p r o -

t e i n 2 a , s u g g e s t i n g t h a t 2 b o r i g i n a t e s f r o m 2 a b y c l e a v a g e o f a

m o l e c u l a r s e g m e n t .

I n t h e p r e s e n t s t u d y , m a n d i b l e s o f f r e s h l y s l a u g h t e r e d p i g l e t s ,

5 - 6 m o n t h s o l d , w e r e c o l l e c te d a t a lo c a l sl a u g h t e r h o u s e , a n d

w e r e b r o u g h t t o t h e l a b o r a t o r y o n i c e . T h e i n c i s o r s , e m b e d d e d

i n th e m a n d i b l e s , w e r e d i s s e c t e d l e a v i n g t h e e n a m e l o r g a n

t i s s u e s a t t a c h e d , a n d p l a c e d i n a g l a s s b e a k e r w h i c h w a s c o o l e d

w i t h i c e - c o l d w a t e r . F o l l o w i n g t h e r e m o v a l o f c o v e r i n g s o f t

t i s s u e s , t h e s u r f a c e o f t h e c h e e s e l i k e e n a m e l w a s r i n s e d w i t h

c o l d s a l in e , w i p e d g e n t l y w i t h a p a p e r t i s su e , a n d t h e n t h e o u t e r

e n a m e l t i s s u e w a s s c r a p p e d c a r e f u l l y w i t h t h e a i d o f a r a z o r

b l a d e . T h e o b t a i n e d p i e c es o f c h e e s e l ik e e n a m e l w e r e s u s p e n d e d

i n 0 .5 M a c e t i c a c i d ( 1 0 m g s o l id / 1 5 m l a c id s o l u t i o n ) a n d h o m o g -

e n i z e d w i t h a P o l y t r o n h o m o g e n i z e r . T h e s u s p e n s i o n w a s c e n t r i -

f u g e d a t 1 0,0 0 0 r p m f o r 10 m i n a t 4 ~ t h e s u p e r n a t a n t w a s r e-

c o v e r e d , a n d t h e n a n e w a c i d s o l u t i o n w a s a d d e d t o t h e t u b e

c o n t a i n i n g th e r e s i d u e s . T h e s a m e p r o c e d u r e w a s r e p e a t e d ( a t

l e a s t 3 ti m e s ) u n t il n o C a w a s d e t e c t e d i n t h e s u p e r n a t a n t . A t t h e

e n d o f t h e d i s s o lu t i o n , n o r e s i d u e s w e r e a p p a r e n t ; n e v e r t h e l e s s ,

t h e t u b e w a s w a s h e d w i t h 1 S D S - T r i s / E D T A b u f f e r ( p H 8 .0 )

[2 7] a n d a n a l y z e d b y S D S - P A G E t o a s c e r t a i n t h a t n o s i g n i f ic a n t

a m o u n t s o f p r o t e i n s r e m a i n e d i n i n so l u b l e f o r m s i n t h e t u b e . T h e

a c i d s o l u t i o n c o n t a i n e d p r o t e i n a s e a n d p h o s p h a t a s e i n h i b i t o r s

( 5 0 m M e - a m i n o c a p r o i c a ci d , 2 . 5 m M b e n z a m i d i n e , 1 m M p h e n -

y l m e t h y l s u l p h o n y l fl u o r i d e, 0 . 5 m M N - e t h y l m a l e i m i d e , 1 0 m M

N a F ) . A l l s t e p s w e r e c a r r i e d o u t a t 4 ~ o r a t i c e - c o l d t e m p e r a -

t u r e . T h e r e s u l t i n g s a m p l e s w e r e c o n c e n t r a t e d a n d d e s a l t e d

t h r o u g h u l t r a f i lt r a t io n u s i n g a n Y M - 5 m e m b r a n e ( A m / c o n ) , a n d

t h e n f r e e z e - d r i e d . T h e s o l i d s a m p l e s o b t a i n e d w e r e s t o r e d a t

- 2 5 ~ u n t i l u s e d f o r f u r t h e r p u ri f i ca t i o n o r f o r t h e r e q u i re d e x -

p e r i m e n t s .

T h e a m e l o g e n i n s 2 a a n d 2 b w e r e p u r i f i e d u s i n g g e l f i l t r a t i o n

c h r o m a t o g r a p h y t h r o u g h a S e p h a d e x G - 1 0 0 c o l u m n ( 2 .6 9 0

c m ) e q u i l i b r a t e d w i t h 5 0 m M c a r b o n a t e b u f f e r ( p H 1 0 . 8 ) i n t h e

p r e s e n c e o f t h e p r o t e i n a s e a n d p h o s p h a t a s e i n h i b i to r s , a l t h o u g h

t h e c o n c e n t r a t i o n s o f t h e i n d i v i d u a l i n h i b i to r s w e r e r e d u c e d t o

o n e - t e n t h o f t h o s e l i s t e d a b o v e t o a v o i d i n t e r f e r e n c e w i t h t h e

m o n i t o r i n g a t 2 8 0 n m . E a c h p r o t e i n f r a ct i o n w a s e l u t e d w i t h t h e

s a m e a l k a l i n e b u f f e r , a n d c o n c e n t r a t e d u s i n g t h e Y M - 5 m e m -

b r a n e . F u r t h e r p u r i f ic a t i o n o f t h e a m e l o g e n i n s w a s c a r r ie d o u t

b y r e c y c l i n g t h e c h r o m a t o g r a p h y s e v e n t i m e s u n d e r t h e s a m e

c o n d i t i o n s . F o l l o w i n g t h e f i n a l c h r o m a t o g r a p h y , e l u e n t f r a c t i o n s

c o r r e s p o n d i n g t o t h e d e s i r e d p r o t e i n s w e r e p o o l e d , c o n c e n -

t r a t e d , a n d d e s a l t e d u s i n g u l t r a f i l t r a t i o n w i t h t h e Y M - 5 m e m -

b r a n e , a n d t h e n l y o p h i l i z e d f o r f u r t h e r a n a l y s i s . T h e p u r i t y o f


3/9

T . A o b a e t a l .: A d s o r p t i o n o f P o r c i n e - A m e l o g e n i n s o n t o H y d r o x y a p a t i t e 2 83

t h e s e p a r a t e d s a m p l e s w a s d e t e r m i n e d b y m e a n s o f S D S - P A G E

u s i n g 1 5 a c r y l a m i d e g el . T h e g el s w e r e r u n i n 0 . 0 5 M

T r i s /0 . 3 8 M g l y c i n e b u f f e r , p H 8 . 3 , c o n t a i n i n g 0 . 1 S D S , a n d

t h e n s t a i n e d w i t h C o o m a s s i e B r i l l i an t B lu e . M o l e c u l a r w e i g h t

s t a n d a r d s ( P h a r m a c i a ) w e r e p h o s p h o r y l a s e b ( 9 4 K ) , b o v i n e

s e r u m a l b u m i n ( 67 K ) , o v a l b u m i n ( 43 K ) , c a r b o n i c a n h y d r a s e ( 3 0

K ) , s o y b e a n t r y p s i n i n h i b i t o r ( 2 0. 1 K ) , a n d c ~ - l a c ta l b u m i n

(14 .4 K) .

T h e H A u s e d a s a n a b s o r b e n t w a s p r e p a r e d a n d c h a r a c t e r iz e d

i n o u r l a b o r a t o r y [ 2 8 ] . T h e s p e c i f i c s u r f a c e a r e a o f t h e c r y s t a l s

w a s 6 . 3 9 m 2 /g ( n i tr o g e n a b s o r p t i o n ; Q u a n t a c h r o m e ) . T h e C a / P

m o la r r a t io of the c rys ta l wa s 1 .67 +_ 0 .02 .

dsorp t ion Exper imen t s

S i n c e t h e e n a m e l p r o t e i n s a r e v e r y i n s o l u b le i n th e p r e s e n c e o f

h i g h e l e c t r o l y t e c o n c e n t r a t i o n s [ 2 9] , t h e i r s o l u t i o n s t o b e e q u i li -

b r a t e d w i t h H A w e r e c a r e f u l l y s e l e c t e d . T h e b a c k g r o u n d c o m -

p o s i t i o n o f t h e s o l u t i o n s u s e d w e r e ( 1 ) 5 0 m M a c e t a t e b u f f e r a t

p H 6 . 0 a t i c e - co l d t e m p e r a t u r e , ( 2) 5 0 m M T r i s b u f f e r i n 3 M u r e a

o r 2 a n d 4 M g u a n i d i n i u m c h l o r i d e a t p H 7 . 4 , ( 3 ) 5 0 m M a m -

m o n i u m b i c a r b o n a t e s o l u t i o n a t p H 7 . 8 - 8 . 0 , a n d ( 4 ) 5 0 m M c a r-

b o n a t e - b i c a r b o n a t e b u f f e r a t p H 1 0 .8 . I n a l l c a s e s , i t w a s p o s -

s i b l e t o p r e p a r e s o l u t i o n s o f c o n c e n t r a t i o n s u p t o 0 . 1 - 0 . 1 5

w t / v o f p r o t e i n w i t h o u t a n y c l o u d i n g . I n c o n t r a s t , i f t h e t e m p e r a -

t u r e o f t h e a c e t a t e b u f f e r i n c r e a s e d , t h e p r o t e i n s , w h e t h e r m i x -

t u r e s o r p u r i f i e d s a m p l e s , s t a r t e d a g g r e g a t i n g , r e s u l t in g i n

c l o u d i n g o f t h e s o l u t io n s .

F o r t h e a d s o r p t i o n e x p e r i m e n t s , a c c u r a t e l y w e i g h e d a m o u n t s

o f t h e a d s o r b e n t , 3 - 1 5 0 m g , w e r e a d d e d t o 2 m l o f e a c h o f th e

s o l u t i o n s c o n t a i n i n g 0 . 1 - 0 . 1 5 ( w t /v ) o f t h e a d s o r b a t e s . T h e

e q u i l i b r a t i o n w a s c o n d u c t e d ( r o t a t i n g e n d o v e r e n d ) , a t i c e - c o l d

t e m p e r a t u r e a n d p H 6 . 0 in t h e c a s e o f a c e t a t e b u f f er , a n d a t

r o o m t e m p e r a t u r e ( a b o u t 2 0 ~ i n t h e s o l u t i o n s ( 2 ) t h r o u g h (4 ).

A f t e r e q u i l i b r a ti o n f o r 1 h o u r , t h e s u s p e n s i o n w a s s e p a r a t e d b y

c e n t r i f u g a t i o n a t 1 0 ,0 0 0 g f o r 1 5 m i n , a t t h e e q u i l i b r a t i o n t e m p e r -

a t u r e s ( 0~ o r 2 0 ~ T h e s u p e r n a t a n t w a s r e m o v e d c a r e f u l l y

w i t h t h e a i d o f a P a s t e u r p i p e t t e . T h e s e p a r a t e d c r y s t a l s w e r e

w a s h e d w i t h 1 m l o f a so l u t i o n h a v i n g t h e s a m e b a c k g r o u n d

c o m p o s i t i o n a n d t e m p e r a t u r e a s t h e e q u i l i b r a t i n g s o l u t i o n , r e -

c e n t r i f u g e d , a n d t h e n t h e s u p e r n a t a n t w a s r e m o v e d . T h i s

w a s h i n g p r o c e d u r e w a s r e p e a t e d t h r e e t i m e s . F i n a ll y , t h e r e c o v -

e r e d c r y s t a l s w e r e d i s s o l v e d w i t h m i n i m u m v o l u m e s o f 0 . 5 M

a c e t ic a c i d . P r o t e i n c o n c e n t r a t i o n s o f t h e s u p e r n a t a n t a n d t h e

a c i d s o l u t i o n w e r e a n a l y z e d a c c o r d i n g t o L o w r y ' s [ 30 ] a n d

B r a d f o r d ' s [ 3 1 ] m e t h o d s . B o v i n e s e r u m a l b u m i n w a s u s e d a s

s t a n d a r d f o r t h e p r o t e i n a s s a y . I t w a s a l s o c o n f i r m e d t h a t t h e

a m o u n t s o f p r o t e i n s s o l u b i l i ze d b y t h e w a s h i n g s o l u t io n s w e r e

l e ss t h a n 1 0 o f t h e e q u i li b r iu m c o n c e n t r a t i o n s in th e s u p e r n a -

t a n t s . A f t e r d e t e r m i n i n g t h e t o t a l c o n c e n t r a t i o n s o f t h e a d s o r -

b a t e s i n b o t h t h e s u p e r n a t a n t a n d t h e a c i d s o l u ti o n , b o t h f l u id s

w e r e d e s a l t e d b y p a s s i n g t h r o u g h a P D - 1 0 c o l u m n ( P h a r m a c i a )

u s i n g 0 . 2 5 M a c e t i c a c i d a s a n e l u e n t , a n d t h e n f r e e z e - d r i e d . T h e

r e s u l t i n g s a m p l e s w e r e so l u b i l i z e d q u a n t i t a t i v e l y w i t h 1 .0 S D S

s o l u t i o n a t 3 7 ~ f o r S D S - P A G E .

Crys ta l Growth Exper imen t s

T h e i n h i b i t o r y a c t i v it y o f t h e p u r i fi e d a m e l o g e n i n s , p r o t e i n s 2 a

a n d 2 b , o n H A c r y s t a l g r o w t h w a s s t u d i e d u s i n g t h e s a m e H A

p r e p a r a t i o n a s i n a d s o r p t i o n s t u d i e s , a s s e e d m a t e r i a l . A c c u -

r a t e ly w e i g h e d s a m p l e s ( 5 0 m g ) o f t h e H A w e r e e q u i l i b r a t e d w i th

p r e d e t e r m i n e d a m o u n t s o f e a c h p r o t e i n i n t h e a c e t a t e b u f f e r a t

i c e -c o l d t e m p e r a t u r e f o r 1 h o u r . T h e s u s p e n s i o n w a s t h e n c e n t r i-

f u g e d , a n d t h e s u p e r n a t a n t w a s r e m o v e d . A n a l y s e s o f t h e c al -

c i u m a n d p h o s p h o r u s c o n c e n t r a t i o n s i n t h e s u p e m a t a n t s h o w e d

t h a t t h e a m o u n t s o f t h e s e e d c r y s t a ls d i s s o l v e d d u r i n g t h is p r e -

t r e a t m e n t s t e p w e r e w i t h i n 5 o f t h e o ri g i n al l y w e i g h e d s e e d

c r y s t a ls . T h e p r e c o a t e d s e e d c r y s t a l s w e r e d i s p e r e d i n 3 m l o f 5 0

m M N a C 1 w i th a v o r t e x , a n d t h e n i n t r o d u c e d i n t o a n e x p e r i-

m e n t a l s o l u t i o n s u p e r s a t u r a t e d w i t h r e s p e c t t o h y d r o x y a p a t i t e .

T h e e x p e r i m e n t a l s o l u t i o n ( 1 0 0 m l i n v o l u m e ) w a s p r e p a r e d

f r o m s t o c k s o l u t i o n s o f C a C I2 , KH2PO4, K 2HPO4, a n d Na C1. A l l

o f t h e s o l u t i o n s w e r e p r e p a r e d u s i n g r e a g e n t - g r a d e c h e m i c a l s .

T h e d i s t i l l e d - d e i o n i z e d w a t e r u s e d w a s b o i l e d t o r e m o v e d i s -

s o l v e d C O 2 a n d s t o r e d u n d e r N 2 . T h e t w o p h o s p h a t e s o l u t io n s

w e r e m i x e d i n a p p r o p r i a t e p r o p o r t i o n s t o o b t a i n a fi n a l p H

o f 7 . 4 . T h e n o m i n a l c o n c e n t r a t i o n s o f c a l c i u m a n d p h o s p h a t e

w e r e 1 . 0 6 a n d 0 . 6 3 r a M , r e s p e c t i v e l y . A l l s o l u t i o n s c o n t a i n e d

5 0 m M N a C 1 a s a b a c k g r o u n d e l e c tr o l y t e. T h e d e g r e e o f s u p e r-

s a t u r a t i o n w i t h r e s p e c t t o h y d r o x y a p a t i t e w a s 7 .4 5 1 0 ~ i n

t e r m s o f t h e r a t io o f t h e i o n i c a c t i v i ty p r o d u c t o f t h e H A

[ ( C a2 + ) 5 (O H ) (P O 4 ) 3] t o i t s t h e r m o d y n a m i c s o l u b i l i t y p r o d u c t .

T h e v a l u e u s e d f o r t h e s o l u b i l i t y p r o d u c t c o n s t a n t a t 3 7 ~ w a s

KIjA = 7 .36 10 -60 [32].

P r i o r t o i n t r o d u c t i o n o f t h e p r e t r e a t e d s e e d s , t h e e x p e r i m e n t a l

s o l u t i o n w a s t h e r m o s t a t e d a t 3 7~ a n d u l t r a- p u r e n i t r o g e n g a s ,

p r e s a t u r a t e d w i t h 50 m M N a C I s o l u t i o n , w a s b u b b l e d t h r o u g h

t h e s o l u t i o n t o m i n i m i z e c o n t a m i n a t i o n w i t h a t m o s p h e r i c C O 2.

D u r i n g t h e e x p e r i m e n t s , t h e s o l u t i o n w a s s t i r r e d w i t h a T e f l o n -

c o a t e d m a g n e t i c s t i rr e r. T h e p H o f th e s o l u t i o n w a s c o n t i n u o u s l y

m o n i t o r e d w i t h a p H m e t e r s t a n d a r d i z e d w i t h b u f fe r s o f p H

v a l u e s 7 . 4 9 8 a n d 6 . 5 2 0 , ( N a 2 H P O 4 a n d K H 2 P O 4 ) , f r o m t h e N a -

t i o n a l B u r e a u o f S t a n d a r d s . W h e n t h e e x p e r i m e n t a l s o l u t io n

r e a c h e d t h e r m a l e q u i l i b r i u m , a s e v i d e n c e d b y a c o n s t a n t p H

r e a d i n g , t h e p r e c i p i t a t i o n r e a c t i o n w a s i n i t i a t e d b y t h e a d d i t i o n

o f t h e s l u r r y o f t h e p r e c o a t e d s e e d s ( e x p e r i m e n t a l t i m e z e ro ) .

T h e s o l u t io n p H w a s c o n t i n u o u s l y p l o t te d i n a s t r i p - c h a r t r e -

c o r d e r a n d i t s v a lu e r e c o r d e d e v e r y m i n u t e b y a d i gi t al p r i n t e r

( M o d e l 9 5 1 , O r i o n R e s e a r c h , M A ) . A l i q u o t s o f t h e w e l l - s t i r r ed

s o l u t i o n w e r e w i t h d r a w n w i t h a sy r i n g e p r i o r t o t h e s e e d i n g ( 3

m l ) a n d d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t ( 1 m l ) a t sp e c i f i e d

t i m e d i n t e r v a l s . A f i l t e r i n g d e v i c e w a s a t t a c h e d t o t h e s y r i n g e ,

a n d t h e s u s p e n s i o n w a s p a s s e d t h r o u g h a p o l y c a r b o n a t e f i l t e r

w i t h a m a x i m u m p o r e s i ze o f 0 .3 3 ~ m ( M i l li p o r e C o r p . M A ) . T h e

c a l c iu m c o n c e n t r a t i o n o f t h e f i lt r a te w a s d e t e r m i n e d b y a t o m i c

a b s o r p t i o n s p e c t r o p h o t o m e t r y , a n d t h e p h o s p h o r u s c o n c e n t r a -

t i o n w a s d e t e r m i n e d c o l o r i m e t r i c a l l y a s a p h o s p h o - m o l y b d a t e

c o m p l e x [ 33 ]. T h e e x p e r i m e n t a l c a l c iu m a n d p h o s p h a t e s o l u t io n

3

p o l y n o m i a l o f t h e f o r m Y = ~ ] A i t , i n w h i c h Y s t a n d s f o r e i t h e r

i=o

c a l c i u m o r p h o s p h o r u s c o n c e n t r a t i o n . F o r m o s t o f t h e p r e s e n t

w o r k , a t h i r d - d e g r e e p o l y n o m i a l ( n = 3 ) p r o v i d e d a d e q u a t e f it -

t i n g t o t h e e x p e r i m e n t a l d a t a , w i t h i n d i c e s o f d e t e rm i n a t i o n ( r2)

o f 0 . 9 6 o r h i g h e r . T h e i n i t i a l c r y s t a l g r o w t h r a t e s , R o , a t t h e i n i -

t i a l d e g r e e o f s u p e r s a t u r a t i o n , w e r e o b t a i n e d b y d i f f e r e n t i a t i o n

o f t h e c o r r e s p o n d i n g polynomial with r e s p e c t t o t i m e a n d c a l c u -

l a t i n g t h e v a l u e o f t h e d e r i v a t i v e f o r t = 0 . T h e i n h i b i t i o n o n t h e

c r y s t a l g r o w t h o f h y d r o x y a p a t i t e d u e t o p r o t e i n s 2 a a n d 2 b w a s

q u a n t i t a t e d i n t e r m s o f t h e r e s u l t i n g r e d u c t i o n i n i n i ti a l p r e c i p i t a -

t i o n r a te , ( R c - R i ) / R c 1 0 0, w h e r e R c a n d R i w e r e t h e i n i t i a l

p r e c i p i t a t i o n r a t e s d e t e r m i n e d u s i n g u n t r e a t e d a n d p r e t r e a t e d

s e e d s w i t h t h e s e p r o t e i n s , r e s p e c t i v e l y .


4/9

2 84 T . A o b a e t a l .: A d s o r p t i o n o f P o r c i n e -A m e l o g e n i n s o n t o H y d r o x y a p a t i t e

esu l t s

F i g u r e 1 s h o w s S D S e l e c t r o p h o r e s i s p a t t e r n s o f t h e

e n a m e l p r o t e i n s o b t a i n e d f r o m t h e o u t e r l a y e r a n d

f r o m t h e i n n e r p o r t i o n o f c h e e s e l i k e e n a m e l ( t r a c k s

1 a n d 2 , r e s p e c t i v e l y ) , a s w e l l a s t h e p a t t e r n s o f th e

p r o t e i n s r e m a i n i n g i n t h e s u p e r n a t a n t ( t r a c k s 3 a n d

4 ) a n d a d s o r b e d o n t o t h e H A c r y s t a l s a f t e r e q u i l i -

b r a t i o n a t p H 6 . 0 ( t r a c k s 5 a n d 6 ) . T h e c o n t e n t o f

p r o t e i n 2 a w a s m u c h h i g h e r i n t h e s a m p l e s o b t a i n e d

f r o m t h e o u t e r l a y e r t h a n i n t h o s e f r o m t h e i n n e r

e n a me l t i s s u e . A s i g n i f i c a n t f i n d i n g w a s t h a t , f o l -

l o w i n g t h e e q u i l i b r a t i o n w i t h t h e H A c r y s t a l s ( 2 5

m g H A i n 2 m l o f t h e a d s o r b a t e s o l u ti o n ) , p r o t e i n

2 a w a s c o n c e n t r a t e d o n t he c r y s t a l s e v e n a f t e r

w a s h i n g t h r e e t i m e s a s d e s c r i b e d a b o v e , w h e r e a s

p r o t e i n 2 b r e ma i n e d i n t h e e q u i l i b r a t i n g s o l u t i o n .

T h i s c o n c e n t r a t i o n o f p r o t e i n 2 a o n th e s u r f a c e o f

t h e H A c r y s t a l s w a s a l s o e v i d e n t w i t h e n a m e l

s a m p l e s f r o m t h e i n n e r la y e r o f t h e c h e e s e l i k e

e n a m e l , e v e n t h o u g h t h e c o n c e n t r a t i o n o f t h is p r o -

t e i n in s u c h s a m p l e s w a s m u c h l o w e r th a n i n t h o s e

f r o m t h e s u r f a c e l a y e r s . T h e s e r e s u l t s s u g g e s t t h a t

t h e n a t u r e o f t h e r e m a i n i n g p r o t e i n 2 a , w i t h r e s p e c t

t o i t s a d s o r p t i o n b e h a v i o r o n t o t h e H A c r y s t a l s ,

w a s n o t a l t e r e d d u r i n g t h e p o s t s e c r e t o r y p r o g r e s -

s i v e ma t u r a t i o n ; i t i s e v i d e n t , h o w e v e r , t h a t i t s a d -

s o r p t i o n a f f i n i t y i s g r e a t l y d i m i n i s h e d w h e n t h e

p r o t e i n w a s d e g r a d e d t h r o u g h t h e c l e a v a g e o f t h e

c - t e r m i n a l s e g m e n t i n t o p r o t e i n 2 b a n d o t h e r

s m a l l e r m o l e c u l e s . I n a d d i t i o n , h i g h e r m o l e c u l a r

w e i g h t p r o t e i n s , r a n g i n g b e t w e e n 6 0 K a n d 9 0 K

d a l t o n , a l s o a d s o r b e d p r e f e r e n t ia l l y o n t o t h e a d s o r -

b e n t c r y s t a l s t o g e t h e r w i t h p r o t e i n 2 a .

T h e r e s u l t s s h o w n i n F i g u r e 1 s t r o n g l y in d i c a t e d

t h a t t h e v a r i o u s p r o t e i n m o l e c u l e s p r e s e n t i n t h e

c h e e s e l i k e e n a m e l s h o w e d q u i t e d i f f e r e n t a d s o r p -

t i o n a f fi n it ie s o n t o t h e H A c r y s t a l s . T h e a d s o r p t i o n

s e l e c t i v i t y w a s f u r t h e r i n v e s t i g a t e d t h r o u g h e q u i l i -

b r a t i o n s o f t h e p r e p a r e d e n a m e l p r o t e i n s ( m i x t u re s

o f a m e l o g e n i n s a n d e n a m e l i n s ) u s in g t h e s a m e t o t a l

c o n c e n t r a t i o n o f t h e a d s o r b a t e ( 2 m l o f 0 . 1 5 s o lu -

t i o n ) w i t h in c r e a s i n g a m o u n t s o f t h e a d s o r b e n t

( f r o m 3 - 1 5 0 m g ) . O n e w o u l d e x p e c t t h a t i n t h e s e

e x p e r i m e n t s , t h e a d s o r b a t e s s h o w i n g th e h i g he s t

a f f i n i t y f o r t h e a d s o r b e n t w o u l d a d s o r b p r e f e r e n -

t i a l ly u n d e r c o n d i t i o n s o f l i m i t e d a v a i l a b l e s u r f a c e

o f H A . F i g u r e 2 s h o w s t h e S D S - P A G E p a t t e rn s o f

t h e p r o t e i n s r e m a i n i n g i n t h e s o l u t i o n ( t r a c k s 2 - 7 )

a n d a d s o r b e d o n t o t h e c r y s t a l s ( t r a c k s 8 - 1 3 ) a f t e r

e q u i l i b ra t i o n i n t h e 5 0 m M a c e t a t e b u f f e r a t t h e i c e-

c o l d te m p e r a t u r e . A c o m p a r i s o n o f t h e s e p a t t e rn s

c l e a r l y d e mo n s t r a t e s t h a t p r o t e i n 2 a ( 2 5 K d a l t o n )

a s w e l l a s t h e h i g h e r m o l e c u l a r - w e i g h t c o m p o n e n t s

( 6 0 - 9 0 K d a l t o n s ) a d s o r b e d p r e f e r e n t i a l l y e v e n o n

F i g . 1. S o d i u m d o d e c y l s u l f a t e g el e l e c t r o p h o r e t o g r a m o f

e n a m e l p r o t e i n s s e p a r a t e d f r o m p o r c i n e p e r m a n e n t i n c i s o r s i n

t h e s e c r e t o r y s t a g e . 1 ) W h o l e e n a m e l p r o t e in s o b t a i n e d f r o m t h e

o u t e r y o u n g ) ti s s u e o f t h e i m m a t u r e t e e t h . N o t e t h e h i g h c o n -

t e n t o f p r o t e i n 2 a . 2 ) W h o l e e n a m e l m a t r i x o b t a i n e d f r o m t h e

i n n e r o l d ) t i s s u e . 3 a n d 4 ) P r o t e i n s r e m a i n i n g i n t h e s u p e r n a t a n t

a f t e r e q u i l i b r a ti n g w i t h 2 5 m g o f H A c r y s t a l s i n t h e a c e t a t e

b u f f e r p H 6 . 0) a t i c e - c o l d t e m p e r a t u r e . T r a c k s 3 a n d 4 s h o w t h e

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

a d s o r b a t e s , r e s p e c t i v e l y . 5 a n d 6 ) P r o t e i n s a d s o r b e d o n t o H A

c r y s t a l s . T r a c k s 5 a n d 6 c o r r e s p o n d t o t r a c k s 3 a n d 4 , r e s p e c -

t i v e ly . N o t e t h e s e l e c t i v e a d s o r p t i o n o f p r o t e i n 2 a o n t o H A , a s

c o m p a r e d t o p r o t e i n 2 b . 7 ) M o l e c u l a r w e i g h t s t a n d a rd s .

t h e s m a l l e st a m o u n t ( 3 .2 m g ) o f t h e a d s o r b e n t ( s e e

t r a c k 8 ) . W i t h in c r e a s i n g s u r f a c e a r e a s o f a d s o r -

b e n t , t h e s m a l l e s t m o l e c u l a r - w e i g h t c o m p o n e n t s

( 5 - 6 K d a l t o n s ) f o u n d i n th e v i c i n it y o f t h e e l u t io n

f r o n t , c o m m e n c e d t o a d s o r b o n t o t h e c r y s t a l s , f o l -

l o w e d b y t h e a d s o r p t i o n o f 1 4 - 1 8 K d a l t o n p r o -

t e i n s . T h e a d s o r p t i o n o f p r o t e i n 2 b ( 2 0 K d a l t o n ) a s

w e l l a s t h e p r o t e in s o f 2 1 - 2 3 K d a l t o n s w e r e d e -

t e c t e d o n l y i n t h e e x p e r i m e n t s u s i n g t h e g r e a t e r

a m o u n t s o f a d s o r b e n t , b u t s t il l t h e l a r g e s t p o r t i o n s

o f t h e s e p r o t e i n s r e m a i n e d i n t h e s u p e r n a t a n t . I t

w a s a l s o a p p a r e n t t h a t s o m e o f t h e s m a l l e r m o l e c -

u l a r w e i g h t p r o t e i n s ( 1 0 - 1 3 K d a l t o n s ) s h o w e d n o

s ig n i fi c a nt a d s o r p t i o n o n t o t h e c r y s t a l s , e v e n w h e n

t h e e n a m e l p r o t e i n s w e r e e q u i l i b r a t e d w i t h t h e

l a rg e s t a m o u n t s o f H A .

T h e f u n c t i o n a l r e l a t io n s h i p b e t w e e n t h e t o ta l

a m o u n t o f pr o t e in s a d s o r b e d o n t o t h e H A c r y s ta l s

a n d t h e a v a i l a b l e s u r f a c e a r e a o f th e a d s o r b e n t i s

s h o w n i n F i g u r e 3 . T h e t o t a l a d s o r b a t e u s e d w a s 3

m g i n e a c h e x p e r i m e n t . T h e a m o u n t a d s o r b e d i n -

c r e a s e d w i t h i n c r e a s i n g s u r f a c e a r e a o f t h e a d s o r -


5/9

T. Aob a e t a l .: Adsorpt io n of Porcine-Amelogen ins onto Hydroxy apat i te 285

Fig. 2 . SDS elect rophoretogram showing the se lec t ive adsorp-

t ion of enamel prote ins separa ted f rom the outer t i ssue of the

immatur e porc ine tee th . 1) Or iginal enamel prote ins used as ad-

sorbate . 2-7) Prote ins remaining in the supernatan t af ter equi li -

bra t ing wi th increas ing amou nts of HA crysta ls f rom lef t to

right, 3.2 mg, 7.3 mg, 19.1 mg, 34.4 mg, 108.2 mg, and 148.5 rag)

in the aceta te buffer pH 6.0) a t ice-cold temperature . 8-13)

Prote ins adso rbed on to HA crysta ls . The resul ts shown in t racks

8 through 13 correspond to those in t racks 2 through 7, respec-

t ively . 14) The same molec ular weight s tandards as show n in

Figure 1.

bent , bu t substan t ia l amou nts of p ro te ins remained

in t h e su p e rn a t an t ev en a f t e r eq u i l i b ra t i n g w i th

large amou nts of c rysta l s . In fac t , the increase in

ad so rb ed p ro t e in s b y a ch an g e o f su r face a rea fro m

0.3 m 2 to 0.7 m 2 was negligible. Prote in 2a wa s the

major cont r ibu tor to the in i tia l s teep increase in ad-

s o r p t i o n ; i n c o n t r a s t , p r o t e i n 2 b w a s th e m a j o r

species among the pro te ins remain ing in the so lu-

t ion.

N ex t w e a t t emp ted s imi l a r ex p e r imen t s a t d i f -

fe ren t p H co n d i ti o n s an d i n t h e p re sen se o f u rea o r

g u an id in e -H Cl ; t h e p u rp o se o f t h e se ex p e r imen t s

w as t o a sce r t a in w h e th e r t h e se l ec t iv e ad so rp t io n o f

p ro t e in 2 a may b e a f fec t ed b y ch an g es i n p H , a s

expe cted fro m the h igh conten t o f h is t id ine res idues

in the amelogenins , and whether the observed ad-

sorp t ion o f pro te in 2a was brought a bout by the in -

t e r m o l e c u l a r a n d / o r i n t r a m o l e c u l a r h y d r o g e n

bondings or hydrophobic in terac t ions , which lead

to aggregat ion . Figures 4 and 5 show the resu l t s o f

adsorp t ion s tud ies a t pH 7 .4 in the presence of 3 M

urea , and 2 and 4 M guanid ine-HC1, respect ive ly .

These resu l t s were ob ta ined using a design para l le l

to tha t used in connect ion wi th Figure 3 . The re-

su l t s show n in Figures 4 and 5 confi rm ed again tha t

pro te in 2a , as wel l as the h igher molecular compo-

nents , adsorb onto the HA crysta l s p referen t ia l ly as

co mp ared t o p ro t e in 2 b an d t h e o th e r w eak ad so r -

bate species . I t was apparen t , though, tha t the to ta l

amo u n t s o f p ro t e in 2 a ad so rb ed o n t h e c ry s t a l s

~3

0

0

f

I 4 8

tot l surf ce re of

HA m=xlO

Fig. 3 . A plot of the to ta l amounts of enamel prote ins adsorbed

onto HA crysta ls as a funct ion of the avai lable surface area of

the adsorbent . Note that the in i t ia l s teep increase in adsorpt ion

was due mainly to prote in 2a see Fig . 2).

Fig . 4 . SDS elect rophoretogram showing the se lec t ive adsorp-

t i on o f enam e l p ro t e in on to H A in t he p r e sence o f 3 M u rea .

1-4) Prote ins adsorbed onto HA af ter equi l ibra t ing wi th in-

creas ing amou nts of the crys ta ls f rom left to r ight , 3.2 mg, 8.1

mg, 17.0 mg, an d 30.6 mg) in the 0.05 M Tris buffer pH 7.4) at

room t em pera tu r e . 5 -8 ) P ro t e ins r em a in ing i n t he supe rna t an t.

The resul ts shown in t racks 5 through 8 correspond to those in

t racks 1 through 4, respect ively .

wer e red uced s ign if ican tly in the prese nce of these

dissocia t ive so lvents , suggest ing tha t i t s o r ig ina l

conformat ion might have p layed an importan t ro le

in the i r adsorp t ion onto the crysta l s . However , the

h ig h c o n c e n t r a t i o n s o f t h e s t r u c t u r e - b r e a k i n g

agents ch ange dras t ica l ly the prope rt ies o f the so l -

v en t i n w h ich t h e ad so rb a t e s a re d i s so lv ed an d ,

t h e reb y , t h e ac t i v i t ie s o f t h e ad so rb in g sp ec i e s .

Mo re u n amb ig u o u s i n fo rma t io n o n t h i s ma t t e r w as

o b t a in ed f ro m ad so rp ti o n ex p e r imen t s a t p H 6 .0 5 0

m M a m m o n i u m a c e t a t e ) , p H 7 .8 5 0 m M a m -

mon ium bicarbona te) , and pH 10.8 50 mM sodium

carb o n a t e ) ; t h e p e r t i n en t SD S p a t t e rn s a re sh o w n

in Figure 6 . I t i s apparen t tha t the marked adsorp-

t ion se lec t iv i ty of pro te in 2a d isp layed a t pH 6 .0


6/9

286 T. Aob a e t a l . : Adsorp t ion of Porcine-Amelogen ins onto Hydrox yapat i te

l 3 4

i!i84 !il

; ;F i :

Fig. 5 . SDS elect rophoretogram showing the se lec t ive adsorp-

t ion of enamel prote ins onto HA in the presence of 2 and 4 M

guanidine-HC1. 1-3) Prote ins adso rbed onto the crys ta ls af ter

equi l ibra t ing wi th increas ing amounts of the adsorb ent f rom lef t

to right, 3.3 mg, 7.9 mg, and 29.7 mg) in the 0.05 M Tris buffer

pH 7.4) a t room temperatur e . The equi l ibra t ion was carr ied out

in the pres ence of 4 M guanidine-HC1. 4) Prote ins adsorbed

onto 7 .3 mg of the crys ta ls in the presence of 2 M guanidine-

HC1. 5-8) Prote ins remaining in the supernatant . The resul ts

shown in t racks 5 through 8 corresponds to those in t racks 1

through 4.

( t rack 2) i s substan t ia l ly reduced a t pH 7 .8 ( t rack

3 ); fu r t hermore , ana ly ses o f the adso rbed phases

(not g iven here) showed a substan t ia l reduct ion in

to t a l p ro t e in adso rbed a t pH 7 .8 by compar i son

wi th the adsorp t ion a t pH 6 , and ex t remely low ad-

sorption at pH 10.8 (track 4).

The resu l t s of the adsorp t ion s tudies descr ib ed

abov e were obta ined using a ll o f the enam el pro-

t e i n s , i n c l u d i n g t h e a m e l o g e n i n s ( o r i g i n a l a n d

d e g r a d e d p r o d u c t s ) a n d t h e e n a m e l i n s . I n s u c h

a~dso rp t i on s t ud i e s , one may a rgue t ha t t he ob -

s e r v e d c o n c e n t r a t i o n o f p r o t e i n 2 a o n t h e H A

crysta l s was due not to d i rec t in terac t ion be tween

prote in 2a and the crysta l s , bu t to in terac t ion wi th

the coexis t ing enamel ins , which have been gener-

a l ly considered to adsorb s t rongly onto HA [7 , 13 ,

34] . To e luc ida te th is mat ter , adsorp t ion experi -

men t s w ere conduc t ed w i th t he i so la t ed frac t ions o f

2a and 2b . Figure 7 shows the SDS-PAGE pat terns

of pro te ins 2a and 2b af ter equi libra ting a t pH 6 .0

w i th fou r a l iquo t s o f H A (2 .5 -25 mg) so a s t o have

increasing surface areas . The HA was equi l ibra ted

w i th 2 ml o f t he ace t a t e bu f fe r con t ain ing 0 .1

Fig. 6 . Compar ison of the se lec t ive adsorpt ion of enamel pro-

te ins onto HA at d i f ferent pH condi t ions . The adsorbate mate-

r ia ls sho wn in t rack 1 were sep ara ted f rom the ou ter t i ssue of

po rc ine pe rm anen t i nc i so rs i n t he s ec r e to ry s t age . The am oun t

of HA used was 17.0 mg. Tracks 2 through 4 show prote ins ad-

sorbe d onto HA af ter equi l ibra t ing a t pHs 6 .0 , 7 .8 , and 10.8,

respect ively . Note the sensi t iv i ty of adsorpt ion behav ior of pro-

te in 2a due to chang es in pH.

(w /v ) o f t he p ro t e in i n ques t i on . The pa t t e rn s i n

Figure 7 showe d tha t la rger amou nts o f pro te in 2a

were adso rbed on the crysta l s wi th increasing avai l-

ab l e su r face a rea s o f H A . O n the o the r hand , mos t

of pro te in 2b remained in the so lu t ion , a l though a

sl igh t increase in adsorp t ion wi th increasing surface

area was a l so d iscern ib le . Figure 8 p lo ts the to ta l

amoun t s o f t he pu r i f ied p ro t e in s adso rbed on t he

crysta l s a t p H 6 .0 and 7 .8 as a funct ion of availab le

surface area of c rysta l s . I t is apparent tha t la rger

a m o u n t s o f p r o t e i n 2 a w e r e a d s o r b e d o n t h e

crysta l s a t pH 6 .0 , as compared to pro te in 2b , bu t

th is d i fference d isappeared a t pH 7 .8 . In addi t ion ,

the adsorp t ion of 2b was insensi t ive to the change

in pH in the experimenta l range .

S i n c e i t w a s d e m o n s t r a t e d i n t h e a d s o r p t i o n

studies that there was a significant difference in ad-

so rp t i on be tw een p ro t e in s 2a and 2b , i t b ecame o f

in t e re s t t o i nves t i ga t e w he the r d i f fe rences i n ad -

sorp t ion behavior were ref lec ted in the inh ib i tory

act iv i ty of these pro te ins as seede d apat i te c rysta l

growth . Figure 9 shows changes in pH of the super-

sa tu ra t ed so lu t i on a s a func t i on o f t he t ime fol -

lowing the seeding wi th precoated crysta l s a t t ime

zero . Calc ium and phosp hate an alyses of so lu t ion

samples taken a t var ious in tervals confi rmed tha t

t he dec reas ing pH va lues w ere p ropo r t i ona l t o t he


7/9

7 4

Fig. 7. Comp ar ison of adsorpt ion on to HA betwee n par t ia l ly pu-

r i f ied prote ins 2a and 2b. 1-4) Pro te in 2a adsorb ed onto HA

after equi l ibra t ing wi th increas ing amoun ts of the crys ta ls f rom

left to fight, 2.5 rag, 5.5 mg, 7.5 rag, and 25 mg) in the acetate

buffer pH 6.0) a t ice-cold temperature . 5-8) Prote in 2b ad-

so rbed on to H A unde r t he s am e cond i t ions a s de sc r ibed above .

9-12) a nd 13-16) Prote ins 2a and 2b remaining in the superna-

tant , respect ively .

Z2

7.O

6 8

T. Aob a e t a l .: Adsorp t ion of Porcine-Amelogen ins onto Hydrox yapat i te 287

I i i

I 2

T i m e h r

Fig, 9. Change s in pH of the supersa tura ted solut ion as a func-

t ion of t ime on addi t ion of hydroxyapat i te seed. A) 50 mg of HA

crys t a ls w e re used a s s eeds w i thou t p r e t r ea tm en t w i th enam e l

prote ins . B and C) The same amo unt of HA seeds was pre-

t rea ted wi th prote in ~b see text and Table 1). D and E) The

same amount of HA seeds was pre t rea ted wi th prote in 2a .

_o

o

o

o

E

=o

O

0

2 a p r o t e i n 2 b p r o t e i n

78

= i

I I

total surfa ce area of HA crystals rnZxlO

~ ~ 1 oH 6 .0

P H I 8

2

Fig. 8 . Plots of the amounts of prote in 2a or 2b adsorbed onto

HA at pHs 6 .0 and 7 .8 as a funct ion of the to ta l avai lable surface

areas of the adsorben t . N ote the higher adsorpt ion of prote in 2a

at pH 6.0 and the sensi t iv i ty of i t s adsorpt ion due to changes

in pH.

amo un t o f p rec ip i t a t ed apa t i t e . Tab le 1 g ives the

amoun t s o f t he p ro te in s u sed and adso rbed on t he

c rys t a l s i n t he p recoa t i ng t rea tmen t and the re -

su l ting inhib i t ion of the H A c rysta l g rowth . Thes e

resu l t s ind ica te tha t the pre t rea tm ent of the seed

crysta l s wi th increasing conc ent ra t ions of pro te in

2a brought abou t very s igni f icant c rysta l g rowth in-

h ib i t ion wh erea s the inh ib i tory ac t iv i ty of 2b was

negligible. From the data in Table 1 i t is apparent

tha t the inh ib i tory ac t iv i ty i s c lose ly re la ted to the

ex t en t o f p ro t e in adso rbed on to t he H A seeds . In

fac t i t was qual i ta t ive ly veri f ied tha t substan t ia l

amou n t s o f p ro t e in 2a w ere a ssoc i a t ed w i th t he

seed crysta l s a f te r growth for 3 hours in the super-

sa tura ted so lu t ion; no a t tempt was made to quant i -

ta re this resu l t becau se of the apprec iab le mas s of

seeds t ha t had been rem oved f rom the sy s t em in the

suspens ion samp les t aken du r ing t he g row th pe -

riod.

iscussion

The resu l t s o f the present invest iga t ion show very

clear ly tha t the or ig ina lly secre ted amelogenin 25

K molecular weight and r ich in pro l ine and h is t i -

d ine adso rbs se l ec t i ve ly on to H A c rys t a l s a t pH

values of 6 .0 and 7 .4 even in the presenc e of d isso-

c ia t ive so lvents such as 3 M urea and 4 M guani -

d ine-HCl . Co nsequent ly i t i s reasonab le to assum e

tha t t he pa ren t ame logen in mo lecu l e sec re t ed in

rive prote in 2a ma y in terac t wi th the surfaces of

exis t ing enamel crysta l s and tha t i t has a poten t ia l

ro le in amelogen esis . Adm it ted ly on the basis of

the pres ent resu l t s no speci fic and comple te ame-

logene t i c mechan i sm can be advanced i n w h ich

prote in 2a wou ld fu lf il l a wel l -def ined funct ion . On

the o ther hand the ra ther h igh adsorp t ion aff in i ty

o f p ro t e in 2a and i ts i nh ib i t o ry a c t i v i t y on t he

seeded c ry s t a l g row th o f H A a re now d i f f i cu l t t o

ignore in re la t ion to an ac t ive ro le in amelogenesis .

The so lubi l i ty of amelogenins i s ra ther low but suf-

ficient [29] to yield s olutions in the ord er o f 10 -6 M.

At such concent ra t ions o ther c rysta l g rowth inhib-

i tors [9 35 36] of biological origin are repo rted to

eff ic ien t ly check the growth of apat i t ic c rysta l s .


8/9

2 8 8 T . A o b a e t a l. : A d s o r p t i o n o f P o r c i n e - A m e l o g e n i n s o n t o H y d r o x y a p a t i t e

T a b l e 1 . I n h i b i t i o n o f h y d r o x y a p a t i t e c r y s t a l g r o w t h d u e t o a m e l o g e n i n ( 2 a a n d 2 b ) p r o t e in s

T o t al a m o u n t s o f A m o u n t s a d s o r b e d i n h i b i t i o n o f

K i n e t i c p r o t e i n s u s e d o n t o c r y s t a l s a i n i t i a l p r e c i p i t a t i o n

e x p e r i m e n t P r o t e i n u s e d ( m g ) ( m g ) r a t e

A N o n e - - - - C o n t r o l

B A m e l o g e n i n 2 b 0 . 42 0 .0 3 < 5

C A m e l o g e n i n 2 b 0 . 6 4 0 . 0 4 = 5

D A m e l o g e n i n 2 a 0 . 3 3 0 . 2 6 2 8

E A m e l o g e n i n 2 a 0 . 6 6 0 . 4 7 3 3

a H A s e e d c r y s t a l s ( 6 . 3 9 m 2 /g ; 5 0 m g / 1 0 0 m l ) w e r e p r e i n c u b a t e d i n 2 m l o f 50 m M a c e t a t e b u f f e r ( p H 6 . 0 ) c o n t a i n i n g t h e p r e d e t e r m i n e d

a m o u n t s o f 2 a o r 2 b p r o t e i n s

A ve r y s ign i f i c a n t f i nd ing i s t he obse r ve d d i f f e r -

e n c e s i n a d s o r p t i o n a f fi n it ie s o n t o H A o f p r o t e in s

2 a a n d 2 b a n d t h e r e b y t h e i r d i f f e r e n t i n h i b i to r y a c -

t i v it y o f H A c r y s t a l g r o w t h . S u c h a b e h a v i o r i s

p r o b a b l y r e l a t e d t o t h e i r p r i m a r y s t r u c t u r e s . T h e

N - t e r m i n a l s e q u e n c e is v ir t u a ll y t h e s a m e 5 4 r e s -

idue s ) i n bo th p r o t e ins [ 22 , 26] ; c onse que n t ly , t he

h i g h e r c o n t e n t s r e p o r t e d [ 2 2 ] f o r p r o t e i n 2 a i n

a s p a r t i c a n d g l u t a m i c a c i d s , l y s i n e , a n d a r g i n i n e

m u s t r e f l e c t d i f fe r e n c e s in t h e c - t e r m i n a l s e q u e n c e s

o f t h e t w o p r o t e i n s w h i c h , p r e s u m a b l y , p l a y a n im -

p o r t a n t r o l e i n t h e a d s o r p t i o n p r o c e s s . A l s o , i t

s e e m s p o s s i b l e t h a t p r o t e i n 2 b i s a d e g r a d e d

p r o d u c t o f p r o t e i n 2 a o r i g i n a t i n g f r o m c l e a v a g e s )

o f it s c - t e r m i n a l s e g m e n t . T h i s p o s s i b i l it y c o u l d

t h e n b e r e l a t e d t o a p o t e n t i a l r o l e o f a m e l o g e n i n s i n

t h e a c t i v e c o n t r o l o f e n a m e l c r y s t a l g r o w t h i n t h e

s e c r e t o r y s t ag e . P r o t e i n 2 a , t h r o u g h a d s o r p t i o n a n d

b l o c k a g e o f g r o w t h s i t e s , c o u l d e s s e n t i a ll y s t o p t h e

gr o wth o f t he i n i t ia l l y p r e c ip i t a t e d c r ys t a l l i t e s ; e n -

z y m a t i c d e g r a d a t i o n o f 2 a w o u l d r e s u l t i n p r o t e i n

2 b a n d o t h e r p e p t i d e s w i t h a l o w e r a d s o r p t i o n a f -

f i n i t y a nd h ighe r so lub i l i t y , i . e . , a l owe r i nh ib i to r y

a c t i v i t y o f c r y s t a l g r o w t h . I f t h e d e g r a d a t i o n o c -

c u r r e d i n t h e a d s o r b e d s t a t e , a g r a d u a l d e s o r p t i o n

o f t h e p r o d u c t s c o u l d b e a n t i c i p a t e d w i t h a c o n c o m -

i t a n t e n h a n c e m e n t in t h e r a t e o f c r y s t a l g r o w t h .

T h e m o s t p l a u s i b l e r e a s o n w h y t h e s e l e c t i v e a d -

s o r p t i o n o f t h e a m e l o g e n i n s h a s b e e n n e g l e c t e d in

t h e p r e v i o u s s t u d i e s i s t h e d i f f i c u lt y i n o b t a i n in g t h e

o r i g in a l l y s e c r e t e d p r o t e i n , i f t h e e n a m e l p r o t e i n s

a r e c o l l e c t e d f r o m t h e w h o l e s o f t t i s su e . A s m e n -

t i o n e d a b o v e , t h e o r i g i n a l a m e l o g e n i n s ) is d e g r a d -

e d , m o s t p r o b a b l y t h r o u g h e n z y m a t i c ac t iv i t y ,

w i t h p r o g r e s s i v e m a t u r a t i o n a n d , t h e r e f o r e , t h e r e is

l e s s poss ib i l i t y o f ob t a in ing the o r ig ina l a m e loge n in

e v e n f r o m t h e s o f t e n a m e l t i s s u e s i n t h e s e c r e t o r y

s t a g e . F o r e x a m p l e , i n th e c a s e o f b o v i n e a m e l o -

ge n in , T a ka g i e t a l . [ 17] r e p or t e d a n a m ino a c id se -

q u e n c e o f 1 7 0 r e s i d u e s , w h i l e t h e r e s u l ts o f S n e a d

e t a l. [ 1 6 ] s u g g e s t e d t h e p r e s e n c e o f e x t r a 8 - 1 0 r e s -

i d u e s i n t h e c - t e r m i n a l e n d . I n f a c t , C r e n s h o w v e r i-

f i e d s u c h a c - t e r m i n a l s e g m e n t i n th e p a r e n t b o v i n e

a m e l o g e n i n 2 7 - 2 8 K d a l to n ) p e r s o n a l c o m m u n i -

c a t i o n ) . I n t h e p i g e n a m e l , t h e c o n c e n t r a t i o n o f

p r o t e i n 2 a d e c r e a s e d s u b s t a n t i a l l y f r o m t h e o u t e r

1 0 0 - 1 5 0 Ix m i n d e p t h f r o m t h e f o r m i n g s u r f a c e ) t o

i n n e r l a y e r . C o n c e r n i n g t h e p r o s p e c t e d d e g r a d a t i o n

p r o c e s s o f t h e s e c r e t e d a m e l o g e n i n , S t ra w i c h a n d

G l i m c h e r [ 2 5 ] r e c e n t l y d e m o n s t r a t e d t h a t t h e 2 5 K

m o l e c u l a r w e i g h t s p e c ie s w a s s e c r e t e d f r o m t h e

c e l ls a n d w a s f i rs t d e t e c t e d i n t h e e x t r a c e l l u l a r o r -

g a n i c m a t r i x ; t h o s e i n v e s t i g a t o r s a l s o s h o w e d t h a t

a n 11 K s p e c i e s a n d s u b s e q u e n t l y a n o t h e r o f a p -

p r o x i m a t e l y 6 . 5 K d a l t o n , w e r e d e t e c t e d i n t h e o r -

g a n i c m a t r i x c o n c o m i t a n t w i t h a r e l a t i v e d e c r e a s e

i n t h e 2 5 K c o m p o n e n t . F u r t h e r m o r e , S h i m i z u e t

a l. [ 3 7 ] p u r i f ie d t h e p r o t e o l y t i c e n z y m e s ) , w h i c h

c a n d e g r a d e p r o t e i n 2 b i n t o 1 1 K a n d 9 k d a l t o n

m o l e c u l e s . O n e o f t h e m i s s i n g k e y r e a c t i o n s i n th i s

p o s t s e c r e t o r y d e g r a d a t io n o f t h e a m e l o g e n i n s is t h e

m e c h a n i s m b y w h i c h t h e c - t e r m i n a l s e g m e n t is s e p -

a r a t e d f r o m t h e p a r e n t p r o t e i n , 2 a , a t a c o n s i d e r -

a b l y f a s t r a t e in vivo f o l lo w i n g t h e s e c r e t i o n o f t h e

p r o t e i n b y a m e l o b l a s t s . F r o m t h e p r e s e n t s t u d y , i t

a p p e a r s l i k el y t h a t t h e p a r t ia l d e g r a d a t i o n o f t h e

p a r e n t a m e l o g e n i n m a y b e b r o u g h t a b o u t o n t h e

c r y s t a l s u r f a c e s , t h r o u g h e n z y m a t i c o r c a t a l y t i c a c -

t ivi t ies .

I n c o n c l u s i o n , t h e r e s u l t s o f t h e p r e s e n t s t u d y i n -

d i c a t e t h a t t h e o r ig i n a ll y s e c r e t e d a m e l o g e n i n 2 5 K

d a l t o n ) , a s w e l l a s t h e 6 0 - 9 0 K d a l t o n s m o l e c u l e s ,

c a n b e s e l ec t iv e l y a d s o r b e d o n t o h y d r o x y a p a t i t e

a nd tha t t h i s a dsor p t ion i s r e l a t e d t o i t s i nh ib i to r y

a c t i v i t y o n t h e c r y s t a l g r o w t h o f h y d r o x y a p a t i t e . I n

c o n t r a s t , t h e p a r ti a l ly d e g r a d a t e d p r o d u c t 2 0 K

d a l t o n ) , n e i t h e r a d s o r b s s u b s t a n t i a l l y o n t o t h e

c r y s t a l s n o r i n h i b i ts t h e c r y s t a l g r o w t h o f a p a t i te .

In vivo

i t s e e m s r e a s o n a b l e t o a s s u m e t h a t p o s t s e -

c r e t o r y d e g r a d a t i o n o f a m e l o g e n i n s w o u l d p r o c e e d

o n t h e c r y s t a l s u r f a c e , t h e r e f o r e e n a m e l m i n e r a li z a -

t i o n i n t h e e a r l y d e v e l o p m e n t a l s t a g e s c o u l d b e r e g -

u l a t e d b y t h e s e c r e t i o n o f i n h ib i t o rs o f c r y s t a l

g r o w t h , a n d t h e i r i n a c t i v a t i o n t h r o u g h p a r t i a l d e g -

r a d a t i o n o f t h e s e c r e t e d a m e l o g e n i n s . T h e a d s o r p -

t i o n a f f i n i ty o n t o h y d r o x y a p a t i t e o f in d i v i d u a l a m e -


9/9

T. Aoba et al.: Adsorption of Porcine-Amelogenins onto Hydroxyapati te 289

logenins and enamelins and the effects of amelo-

genin-enamelin in teract ions on the enamel

mineralization should be elucidated in future work.

Acknowledgments

This investigation was supported in part by

USPHS Grant DE 07623 and DE 3187 from the National Insti-

tute of Dental Research.

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1987.

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