Structure and Activity Changes of Proteins Caused by Adsorption on

Chapter 5

Structure and Activity Changes of Proteins Caused by Adsorption on Material Surfaces 1

2

2

1

Hiroko Sato , Takashi Tomiyama , Hiroyuki Morimoto , and Akio Nakajima 1

Research Center for Medical Polymers and Biomaterials, Kyoto University, 53 Kawaharacho, Shogoin, Kyoto 606, Japan

2

Department of Polymer Chemistry, Kyoto University, 53 Kawaharacho, Shogoin,

Downloaded by UNIV OF BATH on March 1, 2017 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch005

Kyoto 606, Japan

The activity changes of alkaline phosphatase, thrombin, and Factor X adsorbed onto glass beads coated with various polymers were studied in the absence and pre sence of pre-coating with albumin. Activity losses of the adsorbed enzymes were more pronounced than structural changes such as α-helix and β-structure of albumin desorbed from material surfaces. In general, the activity losses of enzymes adsorbed onto polyether urethane nylon (PEUN) and hydrophilic polymer surfaces was smaller than those of enzymes adsorbed to glass and hydrophobic polymer surfaces. Opposite results, however, were obtained by albumin-precoating: thrombin and Factor X adsorbed onto the albumin-precoated PEUN surface were more inactivated than those adsorbed onto the albumin-precoated glass surface. That is, albumin adsorbed selectively onto PEUN from plasma could bind more tightly to thrombin and Factor X . It was con cluded that this is one of the factors responsible for the excellent thromboresistance of PEUN. a

a

a

I t i s important t o study the a d s o r p t i o n behavior o f p r o t e i n s t h a t a r e major component o f plasma such as a l b u m i n , γ-globulin, and f i b r i nogen i n r e l a t i o n t o t h e a n t i t h r o m b o g e n i c i t y o f polymer m a t e r i a l s . F i b r i n o g e n i s i n t i m a t e l y i n v o l v e d i n f i b r i n f o r m a t i o n and p l a t e l e t a g g r e g a t i o n (1-3) on a r t i f i c i a l s u r f a c e s , and albumin m o l e c u l e s i n the n a t i v e s t a t e a r e well-known n o t t o be i n c l u d e d i n thrombus forma t i o n and p l a t e l e t a g g r e g a t i o n . T h e r e f o r e good t h r o m b o r e s i s t a n c e o f polymer s h o u l d be a c h i e v e d b 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 a l b u m i n o n t o a p o l y m e r i c m a t e r i a l s u r f a c e , such a s segmented p o l y e t h e r u r e t h a n e s ( 4 , 5 ) . I n o u r p r e v i o u s p a p e r (£.) , p o l y e t h e r u r e t h a n e n y l o n , a b b r e v i a t e d a s PEUN, was r e p o r t e d t o have a h i g h a d s o r p t i o n r a t e o f a l b u m i n , and was a l s o shown t o have good a n t i t h r o m b o g e n i c i t y . By a d s o r p t i o n o n t o an a r t i f i c i a l

s u r f a c e , albumin

molecules

0097-6156/87/0343-0076$06.00/0 © 1987 American Chemical Society

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

5. SATO E T AL.

77

Structure and Activity Changes of Proteins

undergo s t r u c t u r a l changes, which a r e presumed t o be dependent on the p r o p e r t i e s o f the m a t e r i a l s . Such s t r u c t u r a l changes maybe s t r o n g l y c o r r e l a t e d t o a c t i v i t y changes o f enzymes. Human serum a l b u m i n was found t o have an e s t e r a s e - l i k e a c t i v i t y such t h a t a s p i r i n and pn i t r o p h e n y l e s t e r s a r e h y d r o l y z e d (7.) · S i n c e t h e s p e c i f i c i t y o f t h e r e a c t i o n i s l o w , however, a l a r g e i n t e r f a c e onto which albumin i s a d sorbed i s r e q u i r e d t o d e t e c t t h e e s t e r a s e - l i k e a c t i v i t y . T h e r e f o r e we i n v e s t g a t e d t h e a c t i v i t y changes o f c o a g u l a t i o n f a c t o r s I I ( i . e., thrombin) and X , a b b r e v i a t e d as F X , and a l k a l i n e phosphatase, a b b r e v i a t e d as AP, adsorbed on v a r i o u s m a t e r i a l s u r f a c e s (PEUN, g l a s s , a h y d r o p h i l i c polymer, and a h y d r o p h o b i c polymer). a

a

a


Experimental M a t e r i a l s . B o v i n e plasma a l b u m i n ( L o t . 12F-9365) and b o v i n e plasma γ-globulin ( L o t . 116C-0147), 99 % e l e c t r o p h o r e t i c a l l y p u r e , were p u r c h a s e d from Sigma C h e m i c a l Co., and b o v i n e plasma f i b r i n o g e n ( L o t . 2 6 ) , 9 5 % c l o t t a b l e , was p u r c h a s e d from M i l e s L a b o r a t o r y I n c . The c o n c e n t r a t i o n o f t h e s e plasma p r o t e i n s was d e t e r m i n e d by spe c t r o p h o t o m e t r y , and t h e i r a b s o r p t i o n c o e f f i c i e n t s were t a k e n t o be as f o l l o w s ; Ε Y § =6.67 f o r a l b u m i n (£) ; E £ =11.2 f o r g l o b u l i n ( 2 ) ; E =15.06 f o r f i b r i n o g e n ( 1 0 ) . AP ( L o t . 21F-0270), d e r i v e d from c a l f i n t e s t i n e , was o b t a i n e d from Sigma C h e m i c a l Co. The c o n c e n t r a t i o n was d e t e r m i n e d by u s i n g the a b s o r p t i o n c o e f f i c i e n t E ^ | =7.2 (11) . The s y n t h e t i c s u b s t r a t e used f o r AP was p - n i t r o p h e n y l p h o s p h o r i c a c i d d i s o d i u m s a l t , a b b r e v i a t e d as NPP, o b t a i n e d from N a k a r a i C h e m i c a l Co., K y o t o . Bovine thrombin ( L o t . T-002) was g e n e r o u s l y s u p p l i e d by Mochida Pharma c e u t i c a l Co., Tokyo, and had a s p e c i f i c a c t i v i t y o f 1400 u n i t s / m g p r o t e i n . The a c t i v a t e d F a c t o r X ( L o t . 10F39544) was p u r c h a s e d from Sigma C h e m i c a l Co., and c o n t a i n e d 25 mg b o v i n e a l b u m i n p e r u n i t X . The f l u o r o g e n i c s u b s t r a t e s used f o r t h r o m b i n and X were B o c - V a l Pro-Arg-MCA and Boc-Ile-Glu-Arg-MCA, r e s p e c t i v e l y , where Boc and MCA 2 8

1

%

a

a

-NIH-CH 4 ~ •NBr z 6

15

^

-CH„ -

a

>-NH-C-NHCH„CH 0H 2 2 o

PEUN


78

PROTEINS AT

INTERFACES

are t e r t i a r y - b u t o x y c a r b o n y l and 7-amino-4-methyl coumarin amide, r e s p e c t i v e l y . Both s u b s t r a t e s were p u r c h a s e d from P r o t e i n R e s e a r c h F o u n d a t i o n , Osaka. Three k i n d s o f polymers were c o a t e d on g l a s s beads; PEUN (MW, 50,000) o b t a i n e d from Toyo C r o s s Co., p o l y v i n y l a l c o h o l (MW, 83,000), a b b r e v i a t e d as PVA, o b t a i n e d from K u r a r a y C h e m i c a l Co., and poly-γb e n z y l L-glutamate (MW, 281,000) {12), a b b r e v i a t e d as PBLG. The


c h e m i c a l s t r u c t u r e of PEUN i s on page 77. G l a s s beads were p u r c h a s e d from T o s h i b a B a l o t i n i Co., and were c a . 1 mm i n diameter and 21.3 cm^/g in s p e c i f i c surface area. The g l a s s beads, washed i n

a c i d and r i n s e d t h o r o u g h l y , were c o a t e d by s o l v e n t e v a p o r a t i o n from v a r i o u s polymer s o l u t i o n s as f o l l o w s : 1 mg/ml PEUN i n d i m e t h y l formamide, 1 mg/ml PVA i n d i s t i l l e d w a t e r , and 1 mg/ml PBLG i n c h l o r o form. The c o a t e d g l a s s beads were d r i e d i n vacuo f o r 15 h. The PVAc o a t e d g l a s s beads were a d d i t i o n a l l y h e a t e d a t 120°C f o r 20 min t o p r e v e n t aqueous d i s s o l u t i o n . Methods. 1) A d s o r p t i o n o f plasma p r o t e i n s and s t r u c t u r e o f desorbed plasma p r o t e i n s : B e f o r e a d d i n g a p r o t e i n s o l u t i o n , 90 g beads, packed i n a column o f 16 mm d i a m e t e r and 260 mm l e n g t h (6_, 13) , were p r e t r e a t e d by r i n s i n g w i t h a 0.05 M phosphate b u f f e r a t pH 7.4, and then a s p i r a t e d f o r 5 min. A p r o t e i n s o l u t i o n was s u b s e q u e n t l y poured onto the column w i t h a s y r i n g e . The i s o t h e r m and k i n e t i c s o f p r o t e i n a d s o r p t i o n were t h e n i n v e s t i g a t e d u s i n g a H i t a c h i Model EPS-3T o r a H i t a c h i Model 200-20 s p e c t r o p h o t o m e t e r by measuring changes i n s o l u t i o n c o n c e n t r a t i o n o f the column e f f l u e n t . The s t r u c t u r a l changes o f desorbed p r o t e i n s were measured w i t h a J a s c o J-20 CD/ORD s p e c t r o p o l a r i m e t e r a f t e r a 2 hour a d s o r p t i o n , removal o f p r o t e i n s o l u t i o n , and i n c u b a t i o n w i t h the phosphate b u f f e r . 2) Albumin a d s o r p t i o n on d r y beads and a c t i v i t y o f AP adsorbed: In the p r e s e n c e o f a l b u m i n , the amount o f water adsorbed on t h e beads was e s t i m a t e d by u s i n g 30 g d r y g l a s s beads, e i t h e r c o a t e d o r n o t coated. For the purpose o f t h e measurement o f a c t i v i t y o f adsorbed AP, where a 0 . 0 5 M T r i s H C l b u f f e r c o n t a i n i n g 0.05 MKC1 a t pH 8.1 was u s e d , 5 ml o f a 0.62 mg/ml AP s o l u t i o n was added t o 10 g beads and t h i s was i n c u b a t e d f o r 2 h a t 25°C. The amount adsorbed on the beads was e s t i m a t e d by t a k i n g the d i f f e r e n c e between t h e AP i n s o l u t i o n and the amount o f AP adsorbed on the s u r f a c e o f g l a s s c o n t a i n e r s i n the absence o f beads. A f t e r t h e removal o f t h e AP s o l u t i o n w i t h an a s p i r a t o r , 5.0 ml of 3.5 mM NPP was added t o t h e AP adsorbed on t h e g l a s s beads. The r e a c t i o n was stopped a f t e r 3 min by a d d i n g 7 ml o f a 1 M KH2PO4 s o l u t i o n a d j u s t e d w i t h NaOH t o pH 8.1. The amount o f h y d r o l v z e d p - n i t r ^ o p h e n o l was e s t i m a t e d from t h e absorbance a t 402 nm; E } =1.60x10 . 3) Amount and a c t i v i t y o f thrombin adsorbed: The p o t e n t f l u o r o g e n i c s u b s t r a t e , 7-amino-4-methyl-coumarin, AMC, r e l e a s e d from t h e s u b s t r a t e s h y d r o l y z e d by b o t h t h r o m b i n and F X ( 1 4 ) , was d e t e c t e d w i t h a H i t a c h i 650-1OS f l u o r e s c e n c e s p e c t r o p h o t o m e t e r a t 380 nm f o r e x c i t a t i o n and a t 450 nm f o r e m i s s i o n . One gram o f beads i n a v i a l was immersed i n 2 ml o f a 0.05 M T r i s H C l b u f f e r c o n t a i n i n g 0.05 MKC1 a t pH 7.4 f o r 5 min, 20 μΐ o f 0.26 u n i t s / m l t h r o m b i n was added t o t h e v i a l , and t h e thrombin s o l u t i o n was shaken s l o w l y . F i v e min a f t e r t h e a d d i t i o n , 1 ml o f t h e c m

a


5.

SATO

ET

79


AL.


s u p e r n a t a n t was added t o 1 ml o f a 20 μΜ s u b s t r a t e s o l u t i o n i n a q u a r t z c e l l t o e s t i m a t e t h e amount o f thrombin a d s o r b e d on t h e mat e r i a l s u r f a c e s by s u b t r a c t i n g from t h e amount o f thrombin u s e d . Then, a n o t h e r 1 ml o f 20 μΜ s u b s t r a t e s o l u t i o n was added t o t h e r e m a i n i n g s o l u t i o n and beads, and 1 ml o f t h e s u p e r n a t a n t l i q u i d was withdrawn a f t e r 2 min. The amount o f AMC formed by thrombin i n t h e r e m a i n i n g s o l u t i o n and a d s o r b e d on t h e beads was e s t i m a t e d a f t e r 2 min by b a c k - e x t r a p o l a t i o n on t h e r e c o r d e r . (The 1 ml withdrawn s o l u t i o n was mixed w i t h 1 ml o f 10 μΜ s u b s t r a t e s o l u t i o n , which was used f o r t h e adjustment o f t h e base l i n e o f f l u o r e s c e n c e . ) F i n a l l y , t h e a c t i v i t y o f t h e thrombin a d s o r b e d was deduced by s u b t r a c t i n g t h e amount o f AMC formed by thrombin i n t h e f i r s t - w i t h d r a w n s u p e r n a t a n t from t h e amount o f AMC formed by thrombin i n t h e r e m a i n i n g s o l u t i o n and on t h e beads. In t h e c a s e o f t h e e x p e r i m e n t s f o r a l b u m i n - p r e c o a t e d m a t e r i a l s u r f a c e s , a 1 mg/ml albumin s o l u t i o n i n a 0.05 M T r i s H C l b u f f e r and 0.05 M KC1 a t pH 7.4 was used i n s t e a d o f t h e same b u f f e r i n t h e absence o f a l b u m i n . 4) R e l a t i v e a c t i v i t y o f F X a d s o r b e d : As F X r e a c t i v i t y w i t h t h e s y n t h e t i c s u b s t r a t e was lower t h a n t h a t o f t h r o m b i n , 40 μΐ o f 0.4 u n i t s / m l F X s o l u t i o n was added t o 2 ml o f a 0.05 M phosphate b u f f e r c o n t a i n i n g 0.05 M KC1 a t pH 7.4. The F X a d s o r p t i o n on g l a s s beads was i n v e s t i g a t e d u s i n g a l m o s t t h e same methods a s t h o s e adopted f o r thrombin, e x c e p t t h a t s p e c i f i c a c t i v i t i e s o f F X i n t h e r e m a i n i n g s o l u t i o n and on beads were e v a l u a t e d as v a l u e s r e l a t i v e t o a F X solution. a

a

a

a

a

a

R e s u l t s and D i s c u s s i o n B l o o d plasma i s a c o n c e n t r a t e d p r o t e i n s o l u t i o n , and t h e a d s o r p t i o n b e h a v i o r o n t o m a t e r i a l s u r f a c e s may be e s t i m a t e d from t h e amount and the t y p e o f a d s o r p t i o n f o r major plasma p r o t e i n s i n d i l u t e s o l u t i o n , where t h e a d s o r p t i o n t h e o r y f o r a monomolecular l a y e r i s a p p l i e d . The a d s o r p t i o n i s o t h e r m s f o r a l b u m i n , γ-globulin, and f i b r i n o g e n a t a c o n c e n t r a t i o n lower t h a n c a . 1 mg/ml obeyed t h e Langmuir-type adsorption formula. That i s ,

1 δ

1

= =

ρ

Qp

+

1

1

KQp

" c

where Q i n d i c a t e s t h e amount o f p r o t e i n a d s o r b e d a t t h e p r o t e i n c o n c e n t r a t i o n , c , a t e q u i l i b r i u m , Κ i s a c o n s t a n t , and Qp i s t h e maximum amount o f p r o t e i n a d s o r b e d on t h e m a t e r i a l s u r f a c e as a monomolecular l a y e r . The Qp v a l u e s o f a l b u m i n , γ-globulin, and f i b r i n o g e n a r e l i s t e d i n T a b l e I . The r a t e c o n s t a n t s k o f t h e Langmuirt y p e a d s o r p t i o n s , e x p r e s s e d by E q u a t i o n 2, a r e o b t a i n e d u s i n g t h e Qp v a l u e s l i s t e d i n T a b l e I and from e x p e r i m e n t s on t h e a d s o r p t i o n k i n e t i c s o f p r o t e i n s o n t o t h e m a t e r i a l s u r f a c e s . When t h e amount Qp o f p r o t e i n i s a d s o r b e d on a m a t e r i a l s u r f a c e d u r i n g t h e e l a p s e d t i m e , t : p

L

= k

e?

-

T

· t

QP


(2)

PROTEINS AT INTERFACES

80 Table

I.

S a t u r a t e d Amounts Q o f Plasma P r o t e i n s A d s o r b e d on M a t e r i a l S u r f a c e s * p

PEUN Glass PVA PBLG

(mg/m ) Fibrinogen

γ-Globulin

Albumin 10.3 9.5 1.9 4.9

14.3 13.7 11.6 11.6

17.9 19.2 6.6 13.7

Source: Reproduced w i t h p e r m i s s i o n Society of Polymer Science.


2

Qp

Materials

f r o m R e f . 6.

Copyright

1 9 8 4 The

The r a t e c o n s t a n t s i n E q u a t i o n 2 i n c l u d e t h e s o l u t i o n c o n c e n t r a t i o n of t h e p r o t e i n which i s assumed t o be c o n s t a n t i n t h e i n i t i a l s t a g e s of a d s o r p t i o n . S i n c e t h e c o n c e n t r a t i o n s o f plasma p r o t e i n s albumin: γ-globulin:fibrinogen a r e i n t h e r a t i o 42.0 : 22.4 : 5.6, t h e r a t e c o n s t a n t s from E q u a t i o n 2 were w e i g h t e d a c c o r d i n g l y t o g i v e t h e v a l u e s shown i n T a b l e I I . Thus, t h e a p p a r e n t r a t e c o n s t a n t , k j ^ p i t f o r p r o t e i n a d s o r p t i o n c a n be e s t i m a t e d i n t h e i n i t i a l a d s o r p t i o n p r o c e s s when v a r i o u s m a t e r i a l s u r f a c e s a r e p u t i n c o n t a c t w i t h plasma. As i s o b v i o u s from T a b l e I I , t h e r a t e c o n s t a n t o f a l b u m i n i n the i n i t i a l a d s o r p t i o n p r o c e s s i n plasma, k p i , on a PEUN s u r f a c e i s v e r y l a r g e compared w i t h o t h e r s u r f a c e s . The h i g h r a t e c o n s t a n t f o r albumin may c o n t r i b u t e t o t h e e x c e l l e n t t h r o m b o r e s i s t a n c e o f PEUN, even though t h e amount o f albumin a d s o r b e d o n t o PEUN was a l m o s t t h e same as onto g l a s s . L #

The phenomenon o f t h e r a p i d a d s o r p t i o n o f a l b u m i n o n t o a PEUN s u r f a c e may be a s s o c i a t e d w i t h h y d r o p h o b i c and h y d r o p h i l i c i n t e r a c t i o n s o f t h e PEUN s u r f a c e w i t h some sequences o f r e l a t i v e l y hydropho b i c amino a c i d r e s i d u e s i n t h e i n t e r i o r o f a l b u m i n . An albumin mole c u l e i s composed o f three-subdomains (15). T h e r e a r e two gaps between the subdomains. One i s a h y d r o p h o b i c p o c k e t w i t h an a f f i n i t y c o n s t a n t , K-, = l . l x l 0 ^ M f o r s t e a r i c a c i d ; t h e o t h e r i s an i n t e r m e d i a t e _ 1

fc>

1

hydrophobic pocket with K =l.5x10 M f o r b i l i r u b i n (16). Perhaps the s t r u c t u r e o f a d s o r b e d a l b u m i n i n c o n t a c t w i t h a PEUN s u r f a c e i s composed o f h y d r o p h o b i c and h y d r o p h i l i c r e g i o n s c o r r e s p o n d i n g o r complementary t o t h o s e o f t h e PEUN s u r f a c e , even though t h e e x t e r i o r of n a t i v e albumin i s r i c h i n h y d r o p h i l i c amino a c i d s i d e c h a i n s . When l a r g e amounts o f c o a t e d o r u n c o a t e d beads were added t o a p r o t e i n s o l u t i o n o f volume V and c o n c e n t r a t i o n c , the c o n c e n t r a t i o n a

Q

Table

II.

C a l c u l a t e d Rate C o n s t a n t s i n t h e I n i t i a l A d s o r p t i o n P r o c e s s i n Plasma* kL,pl

Materials Albumin PEUN Glass PVA PBLG

(

m i n

*)

γ-Globulin

7.27 4.49 2.10 0.92

Source: Reproduced w i t h p e r m i s s i o n S o c i e t y of Polymer Science.

Fibrinogen

0.72 1.77 1.08 1.41 f r o m R e f . 6.

0.15 0.45 0.13 0.13 Copyright

1984 The


5.

Structure and Activity Changes of Pro teins

SATO ET AL.

81

o f the p r o t e i n s o l u t i o n was o b s e r v e d g e n e r a l l y t o i n c r e a s e owing t o the a d s o r p t i o n o f water o n t o the beads. I f t h e amount o f adsorbed water i s r e p r e s e n t e d by Q , the f o l l o w i n g i n e q u a l i t y i s o b t a i n e d : w

V(c - c ) D

Qw

=

c

2p

( 3 )

= °

If Q o f E q u a t i o n 3 i s p l o t t e d a g a i n s t c, i t i s found t h a t Q de c r e a s e s h y p e r b o l i c a l l y w i t h i n c r e a s i n g c . On the o t h e r hand, Qp obeys the Langmuir-type a d s o r p t i o n f o r m u l a , e x p r e s s e d by E q u a t i o n 1. In a monomolecular l a y e r o f a d s o r b e d p r o t e i n , i t may be assumed t h a t the t o t a l number, N, o f a d s o r p t i v e s i t e s on a m a t e r i a l s u r f a c e i s c o n s t a n t , as i n d i c a t e d i n E q u a t i o n 4, and t h a t t h e a d s o r b e d p r o t e i n molecules contribute η water-binding sites.


w

w

Ν = Q

w

+ nQ

=Q™= nQp = c o n s t a n t

p

(4)

where Q^ i s the amount o f a d s o r b e d water a t s u r f a c e s a t u r a t i o n . Thus, the r e l a t i o n s among t h e amounts o f water and p r o t e i n a d s o r b e d on material s u r f a c e s may be r e p r e s e n t e d as f o l l o w s . Q

P

=

Qp

1

, _ 1 + ac

CD

Qw

(5)

1 + ac

Q,

where a i s a c o n s t a n t , η becomes c o n s t a n t , i f no s t r u c t u r a l changes o c c u r i n the p r o t e i n . In F i g u r e 1 (a) and ( b ) , t h e amounts o f water and albumin ad s o r b e d , r e s p e c t i v e l y , on PEUN and g l a s s a r e p l o t t e d a g a i n s t the c o n c e n t r a t i o n s o f a l b u m i n s o l u t i o n s a t e q u i l i b r i u m . Our experimental 00 values of Q appear t o a c c o u n t f o r the amount o f water from c a p i l l a r y a c t i o n among t h e beads as w e l l as t h e amount o f water a d s o r b e d on g l a s s beads. The h y d r a t i o n o f a l b u m i n m o l e c u l e s has been e s t i m a t e d t o be 0.0004 g BÔ/mg a l b u m i n by i s o p i e s t i c measurements o f water v a p o r ( 1 7 ) . A l t h o u g h Q^lb v a l u e s on a PEUN s u r f a c e a r e l a r g e r than on a g l a s s s u r f a c e i n t h e lower c o n c e n t r a t i o n r e g i o n (below 0.4 mg/ml), QAlb Qw l b o t h PEUN and g l a s s s u r f a c e s a r e a l m o s t the same i n the c o n c e n r a t i o n range from 0.7 t o 1.0 mg/ml. The Qîb and Q v a l u e s , and t h e i r r a t i o , Q / Q ^ i b / o b t a i n e d f o r a 1 mg/ml a l b u m i n s o l u t i o n , a r e summarized i n T a b l e I I I . The s t r u c t u r a l changes o f a l b u m i n d e s o r b e d from m a t e r i a l s u r f a c e s a r e a l s o shown i n T a b l e I I I (6). The Q v a l u e f o r a PVA s u r f a c e seems t o o s m a l l , p e r h a p s because the s m a l l amounts a d s o r b e d a r e s u b j e c t t o exp e r i m e n t a l e r r o r . The water c o n t e n t s o f t h e a d s o r b e d l a y e r f o r PEUN and g l a s s s u r f a c e s a r e a l m o s t t h e same, w h i l e t h e water c o n t e n t o f a h y d r o p h o b i c s u r f a c e i s s m a l l . The degree o f d i s r u p t i o n o f t h e n a t i v e and r e g u l a r s t r u c t u r e s such as α-helix and 3 - s t r u c t u r e i n a l b u m i n a d s o r b e d on PEUN and g l a s s s u r f a c e s a r e t h e same w i t h i n e x p e r i m e n t a l e r r o r , w h i l e a l b u m i n d e s o r b e d from t h e PVA s u r f a c e may cause a m i l d e r d i s r u p t i o n . S i n c e t h e s t r u c t u r a l changes s h o u l d be c l o s e l y r e l a t e d t o the enzymatic a c t i v i t y , more pronounced e f f e c t s may be e x p e c t e d w

o

r

v

a

u

e

s

o

n

w

w

w




0

0.6

0.2 C

Alb

1 .0 (

m

g

/

m

l

)

( a)

0

0.2

0.6 C

Alb

1.0 (mg/ml)

( b )

F i g u r e 1. Amounts o f albumin Q^x^ and c a l c u l a t e d amounts o f water Q a d s o r b e d on PEUN ( a ) and g l a s s ( b ) s u r f a c e s a r e p l o t t e d a g a i n s t c o n c e n t r a t i o n s c ^ - ^ o f albumin s o l u t i o n s a t equilibrium; A corresponds t o Q J « w

1 K


5.

83

Structure and Activity Changes of Pro teins

SATO ET AL.

from the v a r i o u s p r o p e r t i e s o f the m a t e r i a l s u r f a c e s r a t h e r t h a n from the e f f e c t s o f the water c o n t e n t o f the a d s o r b e d l a y e r . AP, c l a s s i f i e d as a c e l l u l a r enzyme, e x i s t s a l s o i n plasma (18) , and has a h i g h e r r e a c t i v i t y w i t h p - n i t r o p h e n y l phosphate t h a n a l b u min. (The M i c h a e l i s - M e n t e n c o n s t a n t , K = 1 . 1 3 x l 0 ~ M f o r AP and NPP i n a 0.05 M phosphate b u f f e r c o n t a i n i n g 0.05 M KC1 a t pH 8.1.) Table IV shows amounts, the s p e c i f i c , and the r e l a t i v e s p e c i f i c a c t i v i t i e s o f AP a d s o r b e d on m a t e r i a l s u r f a c e s . T a b l e V shows the amounts and 5

m


Table I I I .

Amounts o f Albumin (Qî^) and Water (Q ) Adsorbed on M a t e r i a l S u r f a c e s i n a 1 mg/ml Albumin S o l u t i o n (a), and S t r u c t u r a l Changes o f Desorbed Albumin w

Qw Materials

PEUN Glass PVA PBLG native

2

(mg/m )

(ml/g

VÂlb (ml/mg)

beads)

7.4

0.62

1.31

0.73

0.56

7.1

0.58

1.28

0.67

0.50

1.8

0.11

0.97

0.85

0.63

4.6

0.25

0.85

—

albumin

1

(a) T o t a l a r e a o f beads u s e d i s 0 . 0 6 3 9 m . Source: Reproduced w i t h p e r m i s s i o n from Ref. S o c i e t y of Polymer S c i e n c e . T a b l e IV.

Q *AP (mg/m )

Specific

PEUN Glass PVA PBLG solution

T a b l e V.

Activity

(ymoles/mg/min)

2

(a) The

(16%)

1984

The

Relative Speci fic Activity 0.76

2.8

3.72

1.6

2.88

0.59

—

3.76

0.77

1.8

3.40

0.70

—

4.88

1.00

Thrombin Q-J-J Relative Spe, o cific Activity (yg/m ) z x

Solution

1

Copyright

Thrombin ( i n the Absence o f Albumin) and 0 . 2 0 mg/ml Albumin) Adsorbed on M a t e r i a l

Materials

PEUN Glass PVA PBLG

6.

— (67%)

Amounts and A c t i v i t y Changes o f A l k a l i n e Phosphatase Adsorbed on M a t e r i a l S u r f a c e s f o r 2 h from a 0 . 6 2 mg/ml AP s o l u t i o n

Materials

AP

R e l a t i v e Contents* i n Desorbed Albumin f o r α-Helix; 3 - S t r u c t u r e

FX (in Surfaces

FXa Relative Spe cific Activity

0.92

0.30

0.963

0.92

0.21

0.921

0.29

0.990

0.17

0.911

1.00

1.00

0.82 1.02

(a)

— s i g n i f i c a n t f i g u r e s are

a

(a)

(a) (a)

(a)

substantially 2 figures.


84


r e l a t i v e s p e c i f i c a c t i v i t i e s o f thrombin and F X a d s o r b e d on mate r i a l s u r f a c e s . Under e x p e r i m e n t a l c o n d i t i o n s the same as t h o s e i n T a b l e V, K = 7 . 8 1 x l 0 ~ M f o r thrombin and K = 7 . 6 9 x l 0 ~ M f o r F X . AP seems a r e l a t i v e l y s t a b l e enzyme ( 1 9 ) , compared w i t h t h r o m b i n , because o f the s m a l l e r a c t i v i t y l o s s o f a d s o r b e d AP. The enzymes used have d i f f e r e n t r e a c t i v i t i e s w i t h t h e same c o r r e s p o n d i n g s u b s t r a t e s . A l s o , albumin i s p r e s e n t i n the F X system, which seems t o i n f l u e n c e the F X a d s o r p t i o n on s u r f a c e s . However, i t may be c o n c l u d e d t h a t o f t h e t h r e e enzymes s t u d i e d thrombin l o s e s t h e most a c t i v i t y on a d s o r p t i o n . Thrombin a d s o r b e d on cuprophane o r PVC {20) , and on p o l y e t h y l e n e {2V) l o s t a c t i v i t y c o m p l e t e l y . The s t r u c t u r e o f t h r o m b i n , t h e r e f o r e , i s l a b i l e c a u s i n g p r o f o u n d i n a c t i v a t i o n o f a d s o r b e d t h r o m b i n . The d e c r e a s e o f enzyme a c t i v i t y due t o a d s o r p t i o n on m a t e r i a l s u r f a c e s depends n o t o n l y on the p r o p e r t i e s o f t h e enzymes, on t h e i r a f f i n i t y and r e a c t i v i t y t o the s u b s t r a t e , on the e f f e c t s o f added a l b u m i n , and — e s p e c i a l l y f o r thrombin — on t h e s p e c i f i c a c t i v i t y b u t a l s o on the p r o p e r t i e s of the m a t e r i a l s u r f a c e s . I t i s o b v i o u s from T a b l e s IV and V t h a t the a c t i v i t i e s o f enzymes a d s o r b e d on PEUN and PVA s u r f a c e s a r e h i g h e r than on g l a s s and on PBLG. Thrombin o f a h i g h s p e c i f i c a c t i v i t y l o s e s a c t i v i t y r a p i d l y i n a b u f f e r s o l u t i o n w i t h o u t a l b u m i n , b u t remains a c t i v e i n an albumin s o l u t i o n ( 1 8 ) . F o r example, the time dependence o f thrombin a c t i v i t y i n s o l u t i o n s i s p l o t t e d i n F i g u r e 2, where a c t i v i t y changes of thrombin a r e compared i n t h e absence o f albumin and i n t h e p r e sence o f 1 mg/ml a l b u m i n . In the absence o f a l b u m i n , the e f f e c t s o f a d s o r p t i o n t o a g l a s s c o n t a i n e r as w e l l as s e l f - h y d r o l y s i s o f thromb i n seem t o be d e t e c t e d w i t h i n t h e i n i t i a l 30 min, and t h e n t h e e f f e c t s o f s e l f - h y d r o l y s i s p r e v a i l . T h e r e f o r e the d a t a on the r e l a t i v e a c t i v i t i e s o f thrombin i n T a b l e V a r e c o r r e c t e d on t h e b a s i s o f t h e d a t a o b t a i n e d from F i g u r e 2. In T a b l e VI, the e f f e c t s o f a l b u m i n - p r e c o a t e d s u r f a c e s on t h e enzyme a d s o r p t i o n a r e compared f o r PEUN and g l a s s s u r f a c e s , because Ç>Alb and Q v a l u e s a r e s i m i l a r w i t h b o t h s u r f a c e s , as i s seen i n T a b l e I I I . A c t i v i t i e s o f b o t h thrombin and F X a d s o r b e d on t h e PEUN s u r f a c e a r e h i g h e r than on t h e g l a s s s u r f a c e i n t h e c a s e o f t h e b a r e m a t e r i a l s u r f a c e s , as i s shown i n T a b l e V. However, the r e l a t i v e s p e c i f i c a c t i v i t i e s o f b o t h thrombin and F X a d s o r b e d on an a l b u m i n p r e c o a t e d PEUN s u r f a c e a r e s i g n i f i c a n t l y lower t h a n on an a l b u m i n p r e c o a t e d g l a s s s u r f a c e . T h a t i s , albumin m o l e c u l e s a d s o r b e d on a PEUN s u r f a c e may b i n d t o thrombin and F X w i t h h i g h e r a f f i n i t y &

6

5

m

m

a

a


a

w

a

a

a

Table VI.

Materials

Thrombin

2lla 2

(yg/m ) PEUN Glass Solution

and F X A d s o r p t i o n on A l b u m i n - p r e c o a t e d PEUN and G l a s s Beads a

Thrombin Relative Specific Activity

F

â Relative Specific Activity

1.1 1.1

0.44 0.54

0.73 0.80

-

1.00

1.00



SATO ET AL.


F i g u r e 2. Time dependence o f thrombin a c t i v i t y i n t h e presence o f 1 mg/ml albumin ( Ο ) and i n t h e absence o f albumin ( # ) i n a 0.05 M T r i s H C l b u f f e r c o n t a i n i n g 0.05 M KC1 a t pH 7.4. Con c e n t r a t i o n o f t h e s u b s t r a t e = 20 μΜ.


86


c o n s t a n t s t h a n albumin m o l e c u l e s adsorbed on a g l a s s s u r f a c e . T h i s f a c t a l s o may be r e l a t e d t o t h e e x c e l l e n t i n v i v o t h r o m b o r e s i s t a n c e o f PEUN (6) , f o r which albumin was adsorbed most r a p i d l y among t h e major component plasma p r o t e i n s , a l t h o u g h competing a d s o r p t i o n and exchange (23) by plasma p r o t e i n s o n t o t h e a l b u m i n - c o v e r e d PEUN s u r f a c e under c o n d i t i o n s o f f l o w , and a t t a c k by b l o o d c e l l s c a n be presumed t o o c c u r i n v i v o . Because albumin c a n b i n d t h r o m b i n , albumin m o l e c u l e s s h o u l d d i s t u r b t h e r e a c t i o n o f t h r o m b i n and t h e s y n t h e t i c s u b s t r a t e . The i n h i b i t i o n constant, , o f albumin was s t u d i e d i n t h e system o f t h r o m b i n and t h e f l u o r o g e n i c s u b s t r a t e i n a 0.05 M T r i s H C l b u f f e r c o n t a i n i n g 0.05 M KC1 a t pH 7.4; we found t h a t K i = 1 . 7 9 x l 0 ~ M. From the v a l u e , t h e i n h i b i t o r y e f f e c t o f albumin i s n e g l i g i b l y small i n t h e h y d r o l y s i s r e a c t i o n by t h r o m b i n . That i s , t h e e f f e c t o f a l b u min i s t h e p r o t e c t i o n o f t h r o m b i n from s e l f - h y d r o l y s i s r a t h e r t h a n i n h i b i t i o n o f t h r o m b i n . However, albumin i n b u f f e r supposed t o have a h i g h e r a f f i n i t y f o r t h r o m b i n and F X t h a n albumin adsorbed and t h u s d e n a t u r e d . F i n a l l y a s c h e m a t i c model f o r t h r o m b i n adsorbed on a l b u m i n - p r e c o a t e d PEUN and g l a s s s u r f a c e s i s shown i n F i g u r e 3. Thrombin adsorbed on t h e a l b u m i n - p r e c o a t e d g l a s s s u r f a c e i s more a c t i v e toward t h e s u b s t r a t e t h a n t h r o m b i n on t h e a l b u m i n - p r e c o a t e d PEUN s u r f a c e .


4

a

F i g u r e 3. A schematic model f o r thrombin a d s o r b e d on a l b u m i n p r e c o a t e d PEUN and g l a s s beads. Thrombin m o l e c u l e s a r e shown t o be more a c t i v e on t h e a l b u m i n - p r e c o a t e d g l a s s s u r f a c e .

Literature Cited 1. 2. 3. 4.

Chuang, H. Y. K.; Sharpton, T. R.; Mohammad, S. F. Thromb. Haemostas. 1981, 46, 304. London, J.; McManama, G.; Kushner, L . ; Merrill, E.; Salzman, E. Thromb. Haemostas. 1985, 54, 3. Hawiger, J.; Timmons, S.; Kloczewiak, M.; Strong, D. D.; Doolittle, R. F. Proc. Natl. Acad. Sci. USA 1982, 79, 2068-71. Lyman, D. J.; Brash, J. L.; Chaikin, S. W.; Klein, K. G.; Carini, M. Trans. Am. Soc. Artif. Intern. Org. 1968, 14, 25055.


5. SATO ET AL.

5. 6. 7. 8. 9.


10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.


87

Lyman, D. J.; Metcalf, L. C.; Albo, D., Jr.; Richards, K. F.; Lamb, J. Trans. Am. Soc. Artif. Intern. Org. 1974, 20, 474-79. Sato, H.; Morimoto, H.; Nakajima, A.; Noishiki, Y. Polymer J. 1984, 16, 1-8. Kurono, Y.; Maki, T.; Yotsuyanagi, T.; Ikeda, K. Chem. Pharm. Bull. 1979, 27, 2781-86. Zurawski, V. R., Jr.; Kohr, W. J.; Foster, J . F. Biochemistry 1975, 14, 5579-86. Lowry, O. H.; Rosebrough, J.; Farr, J.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-75. Mihalyi, E. Biochemistry 1968, 7, 208-23. Malamy, M.; Horecker, B. L. Methods Enzym. 1966, 9, 639-42. Noishiki, Y.; Y. Nakahara, Y.; Sato, H.; Nakajima, A. Artif. Org. 1980, 9, 678-82. Soderquist, M. E.; Walton, A. G. J. Colloid Interface Sci. 1980, 75, 386-97. Morita, T.; Kato, H.; Iwanaga, S.; Takada, K.; Kimura, T.; Sakakibara, S. J . Biochem. 1977, 82, 1495-98. Brown, J. R.; Shockley, P.; Behrens, P. Q. In The Chemistry and Physiology of the Human Plasma Proteins; Bing, D. Η., Ed.; Pergamon: New York, 1978; pp 29-37. Anderson, L. O. In Plasma Proteins; Blombäck, B.; Hanson, L. Å., Ed., John Wiley & Sons: New York, 1979; pp 47-53. Bull, H. B.; Breese, K. Arch.Biochem. Biophys. 1968, 128, 48896. Salthouse, T. N. J. Biomed. Mat. Res. 1976, 10, 197-229. Mizutani, Τ. J. Pharm. Sci. 1980, 69, 279-82. Chuang, Η. Y. K.; Mohammad, S. F.; Sharma, N. C.; Mason, R. G. J. Biomed. Mat. Res. 1980, 14, 467-76. Larsson, R.; Olsson, P.; Lindahl, U. Thromb. Res. 1980, 19, 43 -54. Anderson, M. M.; Gaffney, P. J.; Seghatchian, M. J. Thromb. Res. 1980, 20, 109-22. Brash, J . L.; Uniyal, S.; Samak, Q. Trans. Am. Soc. Artif. Intern. Org. 1974, 20, 69-76.

RECEIVED January 28, 1987


Structure and Activity Changes of Proteins Caused by Adsorption on

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