Applied Chemistry at Protein Interfaces - ACS Publications

1 Applied Chemistry at Protein Interfaces ROBERT E. BAIER Downloaded by EASTERN KENTUCKY UNIV on February 25, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch001

Calspan Corp., Environmental Systems Department, Buffalo, Ν. Y. 14221 Some of the areas where interfacial protein layers dominate the boundary chemistry are reviewed, and we introduce some nondestructive analytical methods which can be used simultaneously and/or sequentially to detect and character ize the microscopic amounts of matter at protein or other substrates which spontaneously acquire protein conditioning films. Examples include collagen and gelatin, synthetic polypeptides, nylons, and the biomedically important sur faces of vessel grafts, skin, tissue, and blood. The impor tance of prerequisite adsorbedfilmsof proteins during thrombus formation, cell adhesion, use of intrauterine con traceptives, development of dental adhesives, and preven tion of maritime fouling is discussed. Specifics of protein adsorption at solid/liquid and gas/liquid interfaces are compared.

Numerous ^

surface

physicochemical

analytical techniques, i n c l u d i n g

i n t e r n a l reflection I R s p e c t r o m e t r y , e l l i p s o m e t r y , a n d d e t e r m i n a t i o n

of c r i t i c a l surface t e n s i o n a n d c o n t a c t p o t e n t i a l v a l u e s , r e v e a l a c o m m o n interfacial chemistry among seemingly unrelated phenomena.

T h a t is,

m a n y interfaces are d o m i n a t e d b y proteins, either as the o r i g i n a l s u b strates at or w i t h i n w h i c h the k e y events o c c u r or as the first spontane o u s l y a c q u i r e d c o n d i t i o n i n g layers w h i c h are prerequisites f o r s u b s e q u e n t events. P r o t e i n s as substrates d o m i n a t e the surface c h e m i s t r y of c o l l a g e nous b i o m a t e r i a l s , p h o t o g r a p h i c e m u l s i o n s , s k i n , a n d h a i r . T h e i n t e r f a c i a l c o m p o s i t i o n a n d o r g a n i z a t i o n of proteins d e t e r m i n e the b a r r i e r properties of s k i n a n d its r e c e p t i v i t y to cosmetics a n d m e d i c a t i o n s .

The rapid and

often i r r e v e r s i b l e a d s o r p t i o n of p u r e p r o t e i n or g l y c o p r o t e i n constituents f r o m c o m p l e x m e d i a precedes m i c r o s c o p i c a l l y formed

detectable adhesion

( c e l l u l a r or l a r v a l ) elements i n a l l k n o w n c i r c u m s t a n c e s .

of All

surfaces p r o p o s e d for b l o o d - c o m p a t i b l e i m p l a n t m a t e r i a l s , for e x a m p l e , 1 In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

2

APPLIED CHEMISTRY AT PROTEIN

INTERFACES

a c q u i r e s u c h a c o n d i t i o n i n g film as d o a l l s t r u c t u r a l a n d e n g i n e e r i n g materials i m m e r s e d i n the sea, or p l a c e d i n tissue c u l t u r e or i n t o the o r a l o r u t e r i n e cavities. P r o t e i n films are spontaneously f o r m e d at a n d e l i m i n a t e d f r o m most g a s / l i q u i d interfaces i n n a t u r e , a n d the b u b b l e - s t r i p p i n g of s u c h layers f r o m the sea has b e e n i m p l i c a t e d i n o c é a n o g r a p h i e / m é t é orologie phenomena.

O n c e the c o m m o n features of i n t e r f a c i a l c h e m i s t r y

have been recognized,

significant c r o s s - f e r t i l i z a t i o n of the

fields

men-

tioned should be stimulated. S o m e of the best examples of the d o m i n a n c e of f u n c t i o n a l interfaces Downloaded by EASTERN KENTUCKY UNIV on February 25, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch001

b y b i o l o g i c a l m a c r o m o l e c u l e s o c c u r i n the r e s e a r c h area of " b i o a d h e s i o n . " T h i s area i n c l u d e s the use of d e n t a l restoratives; the use of

polymeric

s u r g i c a l adhesives w h i c h r e p l a c e m e c h a n i c a l l i n k s s u c h as staples a n d sutures; the d e v e l o p m e n t of p r o s t h e t i c i m p l a n t s w h i c h r e p l a c e , i m p r o v e , o r s u p p l e m e n t almost e v e r y p a r t of the h u m a n b o d y ; the m u l t i p l i c a t i o n of e x t r a c o r p o r e a l c i r c u i t s for m e d i c a l treatment i n artificial k i d n e y c e n ters, c o r o n a r y care u n i t s , a n d h o m e d i a l y s i s ; the s t u d y of b l o o d c l o t t i n g reactions as they are i n d u c e d b y contact w i t h n o n p h y s i o l o g i c

surfaces

r a n g i n g f r o m the struts of *an i m p l a n t e d heart v a l v e to t h e transected ends of a n o r m a l b l o o d vessel w h i c h i n i t i a t e w o u n d h e a l i n g ; a n d the v a r i o u s m a r i n e f o u l i n g events

w h i c h encrust ships w i t h barnacles

and

tube

w o r m s a n d w h i c h s l o w o i l - c a r r y i n g supertankers b y a l g a l a d h e s i o n at t h e h i g h l y stressed w a t e r l i n e w i t h l a r g e a c c u m u l a t i o n s of seagrass.

The

c o m m o n i n t e r f a c i a l features of these p r o b l e m s are often o v e r l o o k e d researchers

focus l a r g e l y o n the v o l u m e

phase

effects a n d the

as fluid

d y n a m i c a l effects i n v o l v e d . Analytical

Methods and

Materials

F i g u r e 1 shows the p h y s i c o c h e m i c a l surface m e t h o d s u s e d

exten-

s i v e l y i n our l a b o r a t o r y to assess the i n t e r f a c i a l structure a n d p r o p e r t i e s of p r e d o m i n a n t l y p r o t e i n substrates l i k e s k i n , c o l l a g e n , a n d l i v i n g c e l l surfaces a n d also to assess the i n i t i a l sequence of events at c l e a n s o l i d substrates u p o n t h e i r exposure to b l o o d , s a l i v a , a n d sea w a t e r . T h e m o l e c u l a r s t r u c t u r e of the film a d s o r b e d o n a substrate s u c h as g e r m a n i u m , s i l i c o n , or

various

common

I R - t r a n s m i t t i n g salts

[either

b e f o r e or after t h e i r surfaces w e r e m o d i f i e d b y s t a n d a r d t e c h n i q u e s s u c h as m o n o l a y e r f o r m a t i o n ( J , 2 ) ] , is r e a d i l y d e d u c e d b y the i n t e r n a l reflect i o n t e c h n i q u e w h i c h has b e e n d e s c r i b e d ( 3 ) .

W h e n the substrate is a

m a t e r i a l of h i g h r e f l e c t i v i t y a n d h i g h i n t r i n s i c r e f r a c t i v e i n d e x s u c h as g e r m a n i u m ( w h i c h is u s e d i n most of our experiments ), film thickness a n d refractive index m a y be techniques

(4).

determined nondestructively by

ellipsometric

A third nondestructive and noncontacting

technique,

w h i c h is easily a p p l i e d to t h i n film samples o n g e r m a n i u m or a n y c o n -

In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

1.

BAIER

3

Applied Chemistry

d u c t i v e substrate, is the d e t e r m i n a t i o n of

the contact p o t e n t i a l f r o m

v i b r a t i n g r e e d electrometer studies u s i n g e x p e r i m e n t a l a p p a r a t u s d e s i g n e d b y B e w i g a n d Z i s m a n (5,

6).

A m a j o r t e c h n i q u e u s e d i n a l l o u r studies is contact a n g l e d a t a t a k e n a c c o r d i n g to the m e t h o d s of Z i s m a n ( 7 )

to d e r i v e the c r i t i c a l surface

tension. A l l of these t e c h n i q u e s c a n be a p p l i e d s i m u l t a n e o u s l y or s e q u e n t i a l l y to the same i n t e r f a c i a l area, w i t h a s e n s i t i v i t y for a l l of the m e t h o d s great e n o u g h to detect layers less t h a n 10 A a n d r e p r e s e n t i n g less t h a n 0.1 μg of m a t e r i a l o n a substrate surface area s i m i l a r to a glass m i c r o s c o p e Downloaded by EASTERN KENTUCKY UNIV on February 25, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch001

slide.

F o r a n y i n t e r f a c i a l film, easily a d a p t e d a n d w e l l - c a l i b r a t e d t e c h

n i q u e s c a n d e t e r m i n e first the m o l e c u l a r c o m p o s i t i o n of the m a t e r i a l , t h e n its d e g r e e of o r g a n i z a t i o n ( f r o m e l l i p s o m e t r i c d e d u c t i o n s of its thickness a n d r e f r a c t i v e i n d e x ) , its degree of e l e c t r i c a l a s y m m e t r y ( f r o m

dipole

presence a n d o r i e n t a t i o n d e d u c e d via contact p o t e n t i a l d a t a ) , a n d w h i c h of the m o l e c u l a r clusters present w i t h i n the

film

finally

(identified b y

i n t e r n a l reflection I R ) m u s t b e outermost a n d i n c o n t r o l of interactions w i t h the external e n v i r o n m e n t ( u s i n g the i m p o r t a n t contact

angle-deter

m i n e d c r i t i c a l surface tension v a l u e s ) . Proteins as Substrates C o n t a c t angle m e t h o d o l o g y for p r o b i n g the surface c h e m i c a l f u n c t i o n a n d a r c h i t e c t u r e of p r o t e i n - d o m i n a t e d surfaces has b e e n the most r e v e a l ing technique.

T h e contact a n g l e (Θ) of a l i q u i d o n a s o l i d surface is

defined as that i n t e r n a l angle, g e n e r a l l y b e t w e e n 0 ° a n d 150°, m e a s u r e d t h r o u g h the l i q u i d d r o p to the tangent d r a w n to its p e r i p h e r a l b o u n d a r y . A p l o t of t h e cosine of the contact angle vs. the i n d e p e n d e n t l y d e t e r m i n e d l i q u i d / v a p o r surface tension for e a c h d i a g n o s t i c l i q u i d u s e d forms

a

l i n e a r r e l a t i o n s h i p f r o m w h i c h one c a n i n f e r a c r i t i c a l surface tension v a l u e at the cosine θ = coworkers

(7)

1 axis. T h e d i r e c t c o r r e l a t i o n w h i c h Z i s m a n a n d

have developed

between

intercepts a n d the true outermost

s u c h c r i t i c a l surface

tension

a t o m i c c o n s t i t u t i o n of l o w

energy

o r g a n i c solids is often c a l l e d a w e t t a b i l i t y s p e c t r u m .

F o r selected

low

energy surfaces the a c t u a l a t o m i c c o n s t i t u t i o n of the surface c a n b e c o r r e l a t e d o n a 1:1

basis w i t h the c r i t i c a l surface t e n s i o n e x p e r i m e n t a l l y

determined. Collagen and

Gelatin

C o n t a c t angle t e c h n i q u e s u s e d to evaluate t h i n films of w a t e r - s o l u b l e c o l l a g e n , w i t h care to a v o i d d e n a t u r i n g effects, gave a c r i t i c a l surface tension a p p r o a c h i n g 40 d y n e s / c m ( 8 ) .

A n anomalous nonwettability by

some of the l o w surface-tension, d i s p e r s i o n - f o r c e - o n l y l i q u i d s w a s e v i d e n t , a n d this w a s a t t r i b u t e d to o r g a n i z e d w a t e r a d s o r b e d at the surface


of

4

APPLIED CHEMISTRY A T PROTEIN

INTERFACES

these t h i n films. T h e r e w e r e n o s p e c i a l interactions w i t h h y d r o g e n - b o n d ing liquids.

O n t h e other h a n d , i t w a s d i s c o v e r e d that b y c a s t i n g t h i n

films of c o l l a g e n f r o m h o t r a t h e r t h a n c o o l w a t e r , a m a r k e d d e p a r t u r e in the wetting properties was exhibited b y hydrogen-bonding

liquids.

B a s e d o n p r e v i o u s p u b l i s h e d w o r k (9, 10), this b e h a v i o r is e x p l a i n e d as r a n d o m i z a t i o n o f t h e n a t i v e p r o t e i n structure w h i c h a l l o w s access of t h e hydrogen-bonding

wetting

liquids

to t h e

hydrogen-bond-susceptible

a m i d e links at t h e i n t e r f a c e w h i c h serve as a substrate f o r other i n t e r actions

(e.g., p l a t e l e t a d h e s i o n

to c o l l a g e n

fibers

in vivo

initating

Downloaded by EASTERN KENTUCKY UNIV on February 25, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch001

t h r o m b o s i s ). C o m p l e t e l y w a t e r - s w o l l e n c o l l a g e n differed m a r k e d l y f r o m t h e r e l a t i v e l y d r y [ e q u i l i b r a t e d at 5 0 % r e l a t i v e h u m d i t y ( R H ) ] p r o t e i n ( 8 ) i n its w e t t a b i l i t y b y w a t e r - i m m i s c i b l e l i q u i d s .

I n t h e w a t e r - s w o l l e n case,

the a p p a r e n t c r i t i c a l surface tension d i m i n i s h e d to ca. 30 d y n e s / c m , i n d i c a t i n g that a p r o t e i n i n t e r f a c e i n n a t u r e is n o t c o r r e c t l y m o d e l e d b y d e h y d r a t e d specimens. Protein

Analogs

S y n t h e t i c p o l y p e p t i d e s serve as m o d e l s f o r proteins i n a n u m b e r o f c i r c u m s t a n c e s , p a r t i c u l a r l y i n d e d u c i n g t h e influence of b a c k b o n e c h a i n configurations o n t h e w e t t i n g p r o p e r t i e s o f p r o t e i n - d o m i n a t e d surfaces (9,11). W h e n , f o r e x a m p l e , p o l y ( y - m e t h y l g l u t a m a t e ) w a s cast as a b u l k sheet f r o m solvents w h i c h f a v o r e d t h e e x t e n d e d c h a i n b e t a - s t r u c t u r e o f t h e p o l y m e r (as v e r i f i e d b y I R a n d v a r i o u s d i f f r a c t i o n t e c h n i q u e s ) , t h e interfacial properties

were

dominated

b y the organized

side

chains

( m e t h y l ester g r o u p s i n this c a s e ) . N o e v i d e n c e o f t h e h y d r o g e n - b o n d i n g b a c k b o n e , w h i c h w a s o n l y a f e w a t o m i c diameters f r o m t h e surface, c o u l d b e f o u n d . O n t h e other h a n d , casting t h e p o l y ( y - m e t h y l g l u t a m a t e ) i n t o b u l k films f r o m solvents w h i c h f a v o r e d t h e a l p h a h e l i c a l a r r a n g e m e n t of t h e p o l y m e r b a c k b o n e d e m o n s t r a t e d a m a r k e d increase i n t h e w e t t a b i l i t y of t h e surface b y those d i a g n o s t i c w e t t i n g l i q u i d s c h a r a c t e r i z i n g h y d r o g e n - b o n d i n g i n t e r a c t i o n s . T h e s e films also s h o w e d a n e t increase i n t h e average c r i t i c a l surface t e n s i o n or a p p a r e n t surface free energy of the p o l y m e r as a r e s u l t o f this b a c k b o n e r e o r g a n i z a t i o n .

Similarly, when

p o l y ( y - m e t h y l g l u t a m a t e ) w a s cast f r o m solvents f a v o r i n g t h e r a n d o m t a n g l e s t r u c t u r e of t h e m o l e c u l e a n d a g a i n a l l o w i n g a c c e s s i b i l i t y across t h e interface to p o l y m e r i c b a c k b o n e segments c a p a b l e of e n t e r i n g i n t o h y d r o g e n - b o n d i n g i n t e r a c t i o n s , t h e w e t t i n g results w e r e s i m i l a r to those f o r t h e a l p h a h e l i c a l f o r m a n d c o m p l e t e l y d i s s i m i l a r t o those f o r t h e extended chain intermolecularly hydrogen-bonded

b e t a structure.

A n extreme e x a m p l e o f the different a p p e a r a n c e of a m a c r o m o l e c u l a r s u r f a c e to a n i n t e r a c t i n g e x t e r n a l e n v i r o n m e n t is p r o v i d e d b y p o l y a c r y l -


1.

BAIER

a m i d e (12).

5

Applied Chemistry

I n this case, the d a t a of a l l l i q u i d s w h i c h c a n enter i n t o

hydrogen-bonding

interactions f a l l together o n a Z i s m a n - t y p e

contact

angle p l o t at a c r i t i c a l surface tension of about 50 d y n e s / c m ; other l i q u i d s i n c a p a b l e of this s p e c i a l i n t e r a c t i o n f o r m a separate straight l i n e w h i c h extrapolates to a c r i t i c a l t e n s i o n some 10 d y n e s / c m l o w e r (8, 12). p r o t e i n surfaces c a n either ( a )

Thus,

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

present a n d l o c k e d i n t o the surface ) to a v a r i e t y of c h a l l e n g i n g e n v i r o n ments, or ( b ) t h r o u g h m o l e c u l a r r e a r r a n g e m e n t , present those m o l e c u l a r


g r o u p i n g s most c o m p a t i b l e w i t h the l i q u i d adjacent to the s o l i d substrate. Nylons A m o n g m o d e l m a t e r i a l s most r e l e v a n t to n a t u r a l proteins, the e n g i n e e r i n g p o l y m e r , n y l o n 2 ( d e s c r i b e d i n b i o c h e m i c a l t e r m i n o l o g y as p o l y g l y c i n e ) is i m p o r t a n t . I n p o l y g l y c i n e (10) t h e difference b e t w e e n w e t t i n g of the surface b y h y d r o g e n - b o n d i n g l i q u i d s a n d b y n o n h y d r o g e n - b o n d i n g l i q u i d s is s t r i k i n g . O t h e r n y l o n p o l y m e r s s h o w e d the same a p p a r e n t effect ( J O ) .

The

f r e q u e n c y d i s t r i b u t i o n of the a m i d e segments i n a surface is a significant d e t e r m i n a n t of the g e n e r a l w e t t a b i l i t y of the surface as w e l l as of a n y specific e n h a n c e d w e t t a b i l i t y b y h y d r o g e n - b o n d i n g l i q u i d s .

F o r nylons,

b e c a u s e of t h e i r l a c k of m a s k i n g side c h a i n s , the m o d i f i c a t i o n s of surface properties r e s u l t i n g f r o m c a s t i n g of b u l k films f r o m v a r i o u s solvents w e r e m u c h less i m p o r t a n t t h a n i n the p o l y p e p t i d e s w i t h l e n g t h y side c h a i n s w h i c h result i n significant steric h i n d r a n c e . T h e w e t t a b i l i t y b a n d for p o l y a m i d e s of the n y l o n series s h i f t e d to s h o w l o w e r slopes a n d h i g h e r c r i t i c a l surface t e n s i o n intercepts w h e n p l o t t e d i n the s t a n d a r d Z i s m a n f o r m a t (7) i n c r e a s e d (10).

as the a m i d e g r o u p d e n s i t y

U n f o r t u n a t e l y no t h e o r e t i c a l w o r k describes t h e i m p o r -

tant factors i n f l u e n c i n g the slope of s u c h plots w h i c h reflect i n a g e n e r a l w a y the s t r e n g t h of s o l i d / l i q u i d interactions. Biome die ally Important

Collagenous

Substrates

O n e of t h e m o s t a c t i v e areas of s u r g i c a l r e s e a r c h a n d p r a c t i c e i n volves

collagen-based

sought is a b l o o d

substitutes for

natural blood

vessels.

W h a t is

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

s u i t e d for a n c h o r i n g a c c u m u l a t i n g b l o o d c o m p o n e n t s w i t h o u t s i g n i f i c a n t l y d i s t o r t i n g t h e m to cause adverse subsequent reactions ( s u c h as t h r o m b u s generation, calcification, atherosclerosis).

W e have presented m u c h sur-

face t e x t u r a l a n d surface c h e m i c a l d a t a c h a r a c t e r i z i n g t h e surfaces

of

m o d i f i e d b o v i n e b l o o d vessels d o m i n a t e d b y a c o l l a g e n m a t r i x a n d of the n a t u r a l b l o o d vessels w h i c h t h e y c a n r e p l a c e

(13).

O n e of o u r m o s t



6


Figure 1.

Simultaneous non-destructive

analytical

INTERFACES

techniques

i m p o r t a n t findings was that, w h e n the t e x t u r a l l y r o u g h collagenous grafts are exposed to fresh flowing b l o o d , t h e y are r a p i d l y o v e r l a i n w i t h a second p r o t e i n film of

fibrin,

d e p o s i t e d spontaneously.

W e also d e m o n s t r a t e d

that i n c e r t a i n c i r c u m s t a n c e s c h o l e s t e r o l stéarate a n d fatty a c i d deposits a c c u m u l a t e a l o n g the l u m e n of c o l l a g e n vessels. T h e deposits a d v e r s e l y i n c r e a s e d t h e i r surface free energy a n d u l t i m a t e l y b e c a m e the site of t h r o m b u s f o r m a t i o n a n d a n e u r y s m i c f a i l u r e of the collagenous i m p l a n t s . Skin G r o w i n g f r o m d e e p e r layers is the s t r a t u m c o r n e u m , a c o l l a p s e d l a y e r of p r o t e i n - d o m i n a t e d , k e r a t i n - f i l l e d m e m b r a n o u s sacks w h i c h p r o v i d e the interface w i t h a l l environments: air, water, and various increasingly hazardous aerosol c h e m i c a l s .

I m p o r t a n t c o n t r i b u t i o n s to the s t u d y of the

surface c h e m i s t r y of this p r o t e i n substrate w e r e m a d e first i n i n d u s t r i a l q u a r t e r s , w h e r e the actions of soaps, creams, a n d other cosmetic a p p l i c a tions w e r e sought (14).

T h e studies of m a n y laboratories c o n f i r m the

surface of s k i n to b e a m o d e r a t e l y l o w energy p o l y m e r s i m i l a r to p o l y e t h y l e n e i n m a n y i n t e r a c t i o n s . M o d i f i c a t i o n s of the m e t h o d d e s c r i b e d i n F i g u r e 1 h a v e b e e n u s e d for five years to s t u d y in situ the surface I R characteristics of l i v i n g s k i n . F i g u r e 2 shows a n i n t e r n a l reflection spect r u m o b t a i n e d s i m p l y b y t o u c h i n g the f o r e a r m to a g e r m a n i u m p r i s m i n a h o r i z o n t a l a t t a c h m e n t to o u r i n t e r n a l reflection I R

spectrophotometer



1.

BAIER

Figure 2.

Applied

7

Chemistry

Skin i n situ; spectrum recorded by touching prism in horizontal attachment

forearm to

germanium

( c o n s t r u c t e d to o u r specifications b y H a r r i c k Scientific C o r p . , O s s i n i n g , Ν. Y . ). T h e d e p t h of the s k i n thus s a m p l e d is o n l y a f r a c t i o n of a m i c r o n . T h i s e x t e r n a l surface of the b o d y is o v e r w h e l m i n g l y d o m i n a t e d b y p r o t e i n components.

A s i m i l a r s p e c t r u m c h a r a c t e r i z e d o l d s k i n in situ o n the

t h u m b of a n e x p e r i m e n t a l subject s h o w i n g p r o t e i n d o m i n a t i o n a g a i n w i t h a s m a l l c o n t r i b u t i o n of o r g a n i c ester. C o n t r a s t i n g w i t h this is t h e s i t u a t i o n i l l u s t r a t e d s p e c t r a l l y i n F i g u r e 3, w h e r e the fresh s k i n of the o p p o s i t e t h u m b of the same subject, as generated b e n e a t h a b l i s t e r c a p , shows a n i n t e r m e d i a t e s k i n c h e m i s t r y w h e r e i n the r e l a t i v e a b u n d a n c e of f a t t y esters

Figure 3. MA.I.R. infrared spectrum blister cap (1 cm X 3 cm; right thumb atmosphere; fingerprint ridges not well and ester bands

of fresh skin area beneath peeled away i n situ, 2 days after first exposure to developed). Note ratio of hydrocarbon to amide bands.


8


INTERFACES

is greater. F i g u r e s 4 a n d 5 illustrate t h e difference i n s k i n surface c h e m istry r e c o r d e d before a n d just after the a p p l i c a t i o n of a t y p i c a l c o m m e r c i a l m o i s t u r e c r e a m a c c o r d i n g to the supplier's i n s t r u c t i o n s . T h i s e x a m p l e h i g h l i g h t s the p o t e n t i a l d i a g n o s t i c p o w e r of this m e t h o d , w h i l e c o n f i r m i n g d o m i n a n c e of the i n t e r f a c i a l c h e m i s t r y b y p r o t e i n components t i v e to o t h e r m a t e r i a l s . C o n t r o l experiments s h o w e d that the

recep-

observed

changes b e t w e e n F i g u r e s 4 a n d 5 w e r e n o t s i m p l y the result of a s k i n


coating.

Figure 4.

M.A.LR. infrared spectrum of virgin skin i n situ (female underside, immediately after soap and water wash, towel dry)

forearm,

T h u s , the outermost l a y e r of l i v i n g h u m a n s k i n is d o m i n a t e d b y p r o teins; n e w s k i n g e n e r a t e d u n d e r the o l d is s i m i l a r l y d o m i n a t e d b y proteins w i t h the a d d i t i o n of a significant f r a c t i o n of l i p i d .

T o u n d e r s t a n d the

b a r r i e r a n d other p r o t e c t i v e p r o p e r t i e s of the s k i n one m u s t l o o k at the i n t e r f a c i a l c h e m i s t r y of p r o t e i n layers.

F i g u r e 6 shows t h a t e v e n

the

e x u d a t e f r o m a s k i n w o u n d is d o m i n a t e d b y p r o t e i n components.

As

d i s c u s s e d later, the p r o t e i n s i n this case are almost e x c l u s i v e l y g l y c o p r o teins, as i n d i c a t e d i n F i g u r e 6 b y the r e l a t i v e l y strong a b s o r p t i o n b a n d at ca. 1050 c m " . I n m o d e r n p r o t e i n i n t e r f a c i a l c h e m i c a l l i t e r a t u r e , g l y c o 1

proteins d o m i n a t e the d i s c u s s i o n . Tissue and Blood O n e of the most s t r i k i n g differences b e t w e e n p r o t e i n - d o m i n a t e d s u b strates ( e.g., s k i n , tissue masses, a n d b l o o d ) a n d other s o l i d , s e m i - s o l i d , or l i q u i d surfaces is i n t h e i r w e t t a b i l i t y a n d adhesiveness w i t h

other

m a t e r i a l s . W o r k o n t h e d e v e l o p m e n t of s u r g i c a l adhesives b a s e d u p o n the p o l y ( a - c y a n o a c r y l a t e s )

u s e d successfully i n hemostasis for m a s s i v e



1.

BAIER

Figure 5. underside,

Applied

9

Chemistry

MA.LR. infrared spectrum of modified skin i n situ (female forearm, immediately after application of commercial "moisture cream' according to suppliers instructions)

w o u n d s , c l i n i c a l r e p a i r of s k i n i n c i s i o n s , a n d o r a l s u r g e r y (15, 16)

pro-

v i d e s excellent examples of this d i f f e r e n t i a l w e t t a b i l i t y a n d a d h e s i o n . F o r e x a m p l e , the h o m o l o g o u s series of a l k y l c y a n o a c r y l a t e p o l y m e r s f r o m m e t h y l t h r o u g h h e p t y l shows a n inverse o r d e r of w e t t i n g a n d a d h e s i o n o n b l o o d a n d tissue f r o m the o r d e r s h o w n o n p u r e w a t e r o r p r o t e i n - f r e e fluids.

This and ancillary evidence

(16)

shows t h a t interfaces of

wet

b i o l o g i c a l masses are d o m i n a t e d b y p r o t e i n m o l e c u l e s w h i c h c a n enter

Figure 6.

Proteinaceous

exudate from wounded (by stripping of cornified skin surface


layer)

10

APPLIED

CHEMISTRY AT PROTEIN

INTERFACES

i n t o m a n y s p e c i a l i n t e r f a c i a l c h e m i c a l reactions not p o s s i b l e i n the a b sence of p r o t e i n . P r o t e i n i n t e r f a c i a l layers also c o n t r o l t r a d i t i o n a l surface

physico-

c h e m i c a l p r o p e r t i e s of f r i c t i o n , w e a r , a n d l u b r i c a t i o n i n the joints of a r t i c u l a t e d bones—e.g., i n h i p joints. L i t t l e is k n o w n about the r u b b i n g surfaces a n d the

filling

fluids

of the n a t u r a l b a l l a n d socket connections

except that t h e y are p r e d o m i n a t e l y c a r t i l a g i n o u s , g l y c o p r o t e i n , a n d p r o teoglycan materials. A n o t h e r i m p o r t a n t e x a m p l e of the d i f f e r e n t i a l adhesiveness

which


p r o t e i n - d o m i n a t e d surfaces c a n d i s p l a y , is i n the d e v e l o p m e n t of a r t i f i c i a l s k i n e s p e c i a l l y for w o u n d dressings a n d for t e m p o r a r y covers of extensive burns. C . W . H a l l and co-workers

(17)

s h o w e d that r e l a t i v e tissue a d -

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

various

m a t e r i a l s f o l l o w s the o r d e r p r e d i c t e d b y the c r i t i c a l surface tensions o f construction material. M o h a n d a s a n d c o - w o r k e r s (18), c o n f i r m i n g p r e v i o u s findings of W e i s s a n d B l u m e n s o n (19),

have also s h o w n t h a t cells i n a n e n v i r o n m e n t free

of a d s o r b a b l e proteins ( w h i c h r a p i d l y m o d i f y the surface p r o p e r t i e s of p o l y m e r i c or i n o r g a n i c substrates) w i l l e x h i b i t a s i m i l a r d i r e c t r e l a t i o n s h i p b e t w e e n t h e i r a d h e s i o n a n d the c r i t i c a l surface tension of the surface they contacted.

D i f f e r e n t i a l a d h e s i o n of r e d b l o o d cells was m e a s u r e d b y

d e t e r m i n i n g the f r a c t i o n of cells r e t a i n e d o n a surface after t h e a p p l i c a t i o n of w e l l - c a l i b r a t e d shear stresses (18). the r e d cells

(themselves

I n protein-free

experiments,

d o m i n a t e d i n adhesive interactions b y t h e i r

p r o t e i n m e m b r a n e s ) h a d greatest a d h e s i o n to glass, i n t e r m e d i a t e a d h e s i o n to p o l y e t h y l e n e a n d s i l i c o n i z e d glass, a n d least a d h e s i o n to T e f l o n . T h i s a r t i f i c i a l b i o a d h e s i o n does not c h a r a c t e r i z e the n a t u r a l s i t u a t i o n , w h e r e spontaneous p r o t e i n a d s o r p t i o n precedes cell-surface contact. M o h a n d a s a n d co-workers

r e c o g n i z e d this p r o b l e m a n d h a v e

(18)

ex-

t e n d e d t h e i r studies to p r o t e i n - c o a t e d surfaces as w e l l . Proteins at Substrates Thrombus.

I n over 20,000 substitute heart valves i m p l a n t e d d u r i n g

the mid-1960's, t h r o m b o e m b o l i s m

( s h e d d i n g of s m a l l masses of p l a t e l e t

aggregates f r o m t h e i r l o c i o n the a r t i f i c i a l surfaces of t h e i m p l a n t e d prostheses) o c c u r r e d i n about one of five patients despite attempts to m a i n t a i n a n t i c o a g u l a t i o n (20).

T h e events of c e l l a d h e s i o n a n d b r e a k -

d o w n of s u c h a d h e s i o n after i t has p r o p a g a t e d e n o u g h so that l o c a l shear forces c a n o v e r c o m e i t is a significant c o m p l i c a t i o n of heart v a l v e r e p l a c e m e n t a n d s i m i l a r i n s e r t i o n of n o n p h y s i o l o g i c a l m a t e r i a l into the c a r d i o v a s c u l a r system. dominantly

T h e first events i n v o l v e a d s o r p t i o n of p r o t e i n s , p r e -

fibrinogen,

as m o d i f y i n g or c o n d i t i o n i n g films o n the i m p l a n t s


1.

BAIER

Applied

11

Chemistry

T h e n platelets, w h i c h h a d b e e n a r r i v i n g b u t not a d h e r i n g

(21, 22, 23).

p r i o r to the b u i l d u p of a c e r t a i n thickness of the p r o t e i n l a y e r , adhere to f o r m a s a t u r a t e d l a y e r (24,

25, 26).

D e p e n d i n g o n the n a t u r e of the

o r i g i n a l substrate as t r a n s d u c e d t h r o u g h the n o n e q u i l i b r i u m l a y e r

of

p r o t e i n present at the t i m e of i n d u c t i o n of platelet a d h e s i o n , either p l a t e let a g g r e g a t i o n into the l u m e n occurs, or the o r i g i n a l platelets are shed. I n the f o r m e r , m o r e c o m m o n

case, the a g g r e g a t i n g mass grows

down-

stream i n a w a k e p a t t e r n , finally i n d u c i n g f o r m a t i o n of a n interaggregate fibrin

and red cell mesh, w i t h complete

flow

block by thrombus

and


subsequent e m b o l i . A c t i v a t i o n of the c o a g u l a t i o n factors X I I , X I I I , etc. is a n i m p o r t a n t i n d e p e n d e n t

event w h i c h also c a n b e g i n b y

contact; i t enhances the f o r m a t i o n of flowing

stream.

fibrin

O n l y t w o , s e l d o m seen routes to f a v o r a b l e

outcomes h a v e b e e n o b s e r v e d .

surface

i n the v o l u m e phase of the biomedical

I n the first, the o r i g i n a l l a y e r of adherent

platelets does not b e c o m e s t i c k y to a r r i v i n g s i b l i n g s . T h r o u g h a p o o r l y understood

secondary

a d h e s i o n of w h i t e cells

(predominantly

neutro-

p h i l s ) , the o r i g i n a l platelets are r e m o v e d to leave a r e s i d u a l p r o t e i n

film

i n d y n a m i c e q u i l i b r i u m w i t h the b l o o d stream. N o f u r t h e r c e l l u l a r d e p o sition is n o t e d .

T h e second

favorable

circumstance

occurs

when, in

i m p l a n t s of sufficiently l a r g e d i a m e t e r or i n regions of sufficiently h i g h rates of flow, the o r i g i n a l l a y e r of platelet t h r o m b u s ( w i t h or w i t h o u t fibrin fibrin

strands a n d t r a p p e d e r y t h r o c y t e s )

remodels

to f o r m a

smooth

l a y e r or to s u p p o r t c e l l u l a r i n g r o w t h ( p r o b a b l y of e n d o t h e l i a l cells

s u c h as those o r i g i n a l l y l i n i n g b l o o d vessels ) to p r o v i d e a passive p s e u d o intima. Some g e n e r a l observations o n the a d h e s i o n of b l o o d platelets c a n be m a d e b a s e d o n experiments p e r f o r m e d i n a v a r i e t y of c a r e f u l l y d e signed

flow

deposited,

chambers even

scopically and reflection

(24,

though filmed.

spectroscopy,

27).

arriving

A l a g of 3 0 - 6 0 i n abundance,

sec before platelets was

observed

micro-

A n c i l l a r y studies b y e l e c t r o n m i c r o s c o p y , i n t e r n a l ellipsometry, staining, antibody,

and

contact

angle techniques p r o v i d e d e v i d e n c e that no c e l l a d h e s i o n occurs

from

n a t u r a l b l o o d w i t h o u t the presence of this i n t e r v e n i n g l a y e r of p r o t e i n w h i c h is s e l e c t i v e l y a n d u n i f o r m l y d e p o s i t e d o n a l l n o n p h y s i o l o g i c strates (21, 25, 28, 29).

sub-

It has b e e n p r o p o s e d r e c e n t l y that the i n t e r a c t i o n

of platelets themselves w i t h this d e p o s i t e d p r o t e i n film is m e d i a t e d b y a n e x t r a c e l l u l a r p r o t e i n l a y e r of c o n t r a c t i l e p r o t e i n o n the c e l l surface ( 30, 31 ).

F i g u r e 7 is a h i g h l y m a g n i f i e d , e l e c t r o n m i c r o s c o p i c v i e w of

the e d g e of a single b l o o d p l a t e l e t w h e r e i t contacts (epoxy).

a foreign solid

T h i s v i e w illustrates b o t h the p r e r e q u i s i t e a d s o r b e d

film

and

the p l a t e l e t surface f u z z w h i c h m a y be i n v o l v e d i n this adhesive i n t e r action.


12

CHEMISTRY

A T PROTEIN

INTERFACES


APPLIED

Figure

7.

The edge of a platelet adhering to an epoxy slab, as coated with a protein conditioning film

N o e x p l a n a t i o n exists f o r t h e o b s e r v a t i o n r o u t i n e l y c o n f i r m e d that, although spontaneously

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

interfaces

has a u n i f o r m i n i t i a l a p p e a r a n c e a n d c h e m i s t r y , t h e a r r i v i n g b l o o d p l a t e lets d o n o t a d h e r e u n i f o r m l y t o t h a t l a y e r . T h e a d h e r e n t platelets a l w a y s l e a v e a p p a r e n t l y u n o c c u p i e d space b e t w e e n themselves a n d t h e i r nearest


1.

BAIER

Applied

13

Chemistry

n e i g h b o r s , g e n e r a l l y g i v i n g a s a t u r a t i o n p o p u l a t i o n d e n s i t y of 70 a n d 90 platelets p e r 1000 μ

2

between

A c t u a l cell adhesion on preformed

(26).

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

(18).

T h e r a p i d i t y of the p r o t e i n a d s o r p t i o n has b e e n d e m o n s t r a t e d else where.

I n as little as 5 sec, for e x a m p l e , the film thickness w a s a l r e a d y

of the o r d e r of 50 A (21).

I t is n o w a c c e p t e d

that a d s o r b e d

protein

accumulates over the same t i m e p e r i o d to a b o u t the same thickness o n a l l f o r e i g n s o l i d surfaces i n b l o o d .

E l e c t r o n m i c r o g r a p h s of the platelets


o r i g i n a l l y a d h e r e n t to surfaces h a v i n g different o r i g i n a l characters

(32)

p r o v i d e some i n s i g h t o n the o r i g i n of a c o n t i n u e d d i f f e r e n t i a l e n d p o i n t existing a m o n g t h r o m b o r e s i s t a n t or t h r o m b o g e n i c

materials. O n

those

surfaces w h i c h are t h r o m b o r e s i s t a n t after l o n g t e r m i m p l a n t a t i o n s , t h e o r i g i n a l l y a d h e r e n t platelets r e m a i n m o r e m o r p h o l o g i c a l l y i n t a c t t h a n those w h i c h o r i g i n a l l y a d h e r e d to the ( e m p i r i c a l l y f o u n d ) materials.

thrombogenic

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

or d i s c - l i k e shape a n d t h r o w out v e r y f e w p s e u d o p o d s across the surface are no l o n g e r o b s e r v e d o n the surface after flow times as short as 2 hrs i n m a n y cases ( 3 3 ) , e v e n t h o u g h t h e y w e r e present f r o m times of a b o u t 1 m i n (24)

to 10 m i n (26).

T h u s , i n some u n k n o w n w a y , differences i n

the a d s o r b e d p r o t e i n , e v e n the same a d s o r b e d p r o t e i n , m u s t b e m a r k e d e n o u g h to p r o v i d e this s t r i k i n g l y different response i n a d h e r i n g b l o o d platelets. C h a n g e s i n the zeta p o t e n t i a l a n d other e l e c t r o k i n e t i c p r o p e r ties across a d s o r b e d p r o t e i n layers are p r o b a b l y not great e n o u g h

to

e x p l a i n the r e l a t i v e c o m p a t i b i l i t y , or l a c k of c o m p a t i b i l i t y , of a v a r i e t y of p r o p o s e d i m p l a n t m a t e r i a l s (35,

36).

L e o V r o m a n a n d c o - w o r k e r s , w h o present a d d i t i o n a l d a t a i n this v o l u m e , h a v e m a d e m a j o r c o n t r i b u t i o n s to o u r u n d e r s t a n d i n g of the f u n d a mentals of this a d s o r p t i o n process.

T h e y h a v e s h o w n that events at the

s u b s t r a t e / b l o o d i n t e r f a c e are not static after the first l a y e r of p r o t e i n is a d s o r b e d , b u t that the p r o t e i n l a y e r is c o n t i n u o u s l y r e m o d e l e d , w i t h , or c o n v e r t e d b y other surface-active c o m p o n e n t s

reacted

i n intact plasma

(37). T h e l a y e r w h i c h continues to exist i n a p p a r e n t e q u i l i b r i u m w i t h t h e b l o o d after l o n g t e r m i m p l a n t a t i o n s of o t h e r w i s e i n e r t s o l i d materials r e m a i n s m y s t e r i o u s ; i t is not r e c o g n i z e d b y a n y of t h e specific component

antibodies t r i e d to date.

blood

Often, it remains thick enough

that w h e n a n a l y z e d b y i n t e r n a l reflection spectroscopy

so

(sensitive to, at

the most, a f e w m i c r o n s of a substrate surface s a m p l e ) , the u n d e r l y i n g p o l y m e r i c substrate cannot be d e t e c t e d at a l l . A n i n t e r e s t i n g e x c e p t i o n to this finding has c o m e f r o m o u r recent r e s e a r c h w i t h i n o r g a n i c s u b strates w h i c h h a d b e e n s c r u p u l o u s l y c l e a n e d b y g l o w d i s c h a r g e treat ment.

W h e n b o r o s i l i c a t e glass t u b i n g was t r e a t e d b y this process a n d


14

APPLIED

CHEMISTRY AT PROTEIN

INTERFACES

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

location

(the

canine

thoracic

inferior vena

cava).

A

p a s s i v a t i n g l a y e r of p r o t e i n , w h i c h w a s r e m a r k a b l y p u r e b y s p e c t r o s c o p i c c r i t e r i a a n d a b u n d a n t after 2 hrs of b l o o d contact, c o u l d b e b a r e l y d e m onstrated b y I R after 480 days

(38).

I m p r o v e d a n a l y t i c a l t e c h n i q u e s are n e e d e d to detect the i m p o r t a n t c o n f i g u r a t i o n a l a n d c h e m i c a l differences spontaneously

from complex

among adsorbed

films

solutions o n v a r i o u s substrates.

formed Internal

reflection I R does not r e v e a l significant differences i n the a d s o r b e d p r o Downloaded by EASTERN KENTUCKY UNIV on February 25, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch001

t e i n films w h i c h a c c u m u l a t e o n v a r i o u s l y treated S t e l l i t e 21 devices

(a

c o b a l t - c h r o m i u m a l l o y u s e d to m a k e s y n t h e t i c h e a r t v a l v e struts a n d s e a t s ) ; i n some instances these devices are t h r o m b o g e n i c , a n d i n others they

are

apparently

thromboresistant

(39).

Yet,

scanning

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

electron

differences

i n t h e films i m m e d i a t e l y o n contact w i t h t h e m . T e x t i l e G r a f t s . F l e x i b l e k n i t t e d a n d w o v e n tubes ( u s u a l l y of D a c r o n and Teflon

fibers)

was d e m o n s t r a t e d

are often i m p l a n t e d as s y n t h e t i c b l o o d c o n d u i t s . as e a r l y as 1958 that these p l a s t i c

fiber

It

grafts a l l

b e c o m e coated, o n b o t h outer a n d i n n e r surfaces, w i t h fibrous tissues as s o o n as 30 days after i m p l a n t a t i o n (40). surface-energy

I n a d d i t i o n the n o m i n a l l y h i g h e r

fabrics, constructed from polyamide

m o r e p r o n e to a c c u m u l a t e t h i c k layers of s u c h

a n d polyester,

fibrous

are

tissue t h a n are

the l o w e r surface-energy grafts of T e f l o n . D r a w i n g u p o n s u c h scattered observations,

along w i t h

our o w n

d a t a o n the surface

-apparently thromboresistant i m p l a n t s

(41),

we

properties

proposed

of

a tentative

c o r r e l a t i o n of the r e l a t i v e surface energies of solids w i t h t h e i r b i o l o g i c a l interactions (13, 24, 42).

T h e most significant feature of our hypothesis

is a m i n i m u m i n r e l a t i v e b i o l o g i c a l i n t e r a c t i o n a l o n g the scale f r o m v e r y l o w surface-energy m a t e r i a l s ( t y p i f i e d b y the surface-energy

plastics

(typified by

fluorocarbons

) to the h i g h e r

the v a r i o u s polyesters

and

poly-

a m i d e s ). E v i d e n c e (42, 43) suggests that the z o n e of m a x i m u m b i o c o m p a t i b i l i t y — a s j u d g e d b y m i n i m u m depositions of debris o n i m p l a n t s or m i n i m u m d i s t o r t i o n of cells a d h e r e n t t h r o u g h a n i n t e r m e d i a t e l a y e r of p r o t e i n to the s u r f a c e s — f a l l s i n the c r i t i c a l surface r e g i o n b e t w e e n 20 a n d 30 d y n e s / c m .

O n the basis of w e l l d e v e l o p e d correlations b e t w e e n p o l y -

m e r surface c o n s t i t u t i o n a n d c r i t i c a l surface tension ( 7 ) , s u c h a range m u s t b e essentially d o m i n a t e d b y the - C H t e r m i n a l groups as side chains 3

(as i n p o l y d i m e t h y l s i l o x a n e ) acids, a m i d e s ,

and long

organic backbones.

or t e r m i n a l a t o m i c clusters (as i n fatty

chain aliphatic alcohols)

on more

T h e a p p a r e n t c r i t i c a l surface tension of

complex organized

w a t e r ( d e t e r m i n e d f r o m w e t t i n g experiments i n v o l v i n g s i m p l e l i q u i d s w h i c h c a n i n t e r a c t o n l y b y d i s p e r s i o n forces a n d not b y p o l a r interactions o r h y d r o g e n - b o n d i n g i n t e r a c t i o n s ) falls also i n this z o n e (45, 46);


this

1.

BAIER

15

Applied Chemistry

suggests that v e r y h i g h l y h y d r a t e d masses s u c h as h y d r o g e l s m i g h t also f u n c t i o n b y this m o d e r a t e surface-energy m e c h a n i s m w h e n t h e y r e m a i n thromboresistant. Cell Adhesion. E x c e p t for m o d e l experiments w i t h r e d cells i n a r t i ficial

p r o t e i n - f r e e e n v i r o n m e n t s , there is n o c o u n t e r e x a m p l e to the g e n -

e r a l i z a t i o n t h a t c e l l a d h e s i o n does not o c c u r to a n y s o l i d surface w i t h o u t an i n t e r v e n i n g t h i n l a y e r of a d s o r b e d or p r e v i o u s l y d e p o s i t e d p r o t e i n d o m i n a t e d matter.

A n excellent d e m o n s t r a t i o n of t h e r e q u i r e m e n t f o r

s u c h a d s o r b e d layers w a s p r o v i d e d b y A . C . T a y l o r (47)

i n his studies of


a d h e s i o n b e t w e e n g i n g i v a l e p i t h e l i a l cells a n d h a r d e n a m e l surfaces at the d e n t a l m a r g i n a n d b e t w e e n a l l l i v i n g cells w h i c h h e s t u d i e d a n d a r t i f i c i a l substrates i n c u l t u r e . M o r e r e c e n t l y , this finding has b e e n c o n firmed

i n examples as m u c h i n contrast as the a d h e s i o n of s t a n d a r d c e l l

lines to u n m o d i f i e d glass surfaces a n d to c o m p l e t e l y s i l i c o n e - m a s k e d glass surfaces (48).

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

a s i l i c o n i z i n g m a t e r i a l w h i c h not o n l y c h a n g e d the surface free

energy

of the s o l i d b u t also r e v e r s e d its n o r m a l e l e c t r i c a l c h a r g e ( f r o m n e g a t i v e to p o s i t i v e ) ; yet p r o t e i n a d s o r p t i o n p r e c e d e d c e l l a d h e s i o n b o t h b e f o r e a n d after the glass was o v e r c o a t e d . I n this v o l u m e B a i e r a n d W e i s s demonstrate t h e r e a l i t y of the s p o n taneous a d s o r p t i o n of r e a s o n a b l y p u r e g l y c o p r o t e i n films f r o m

common

c e l l c u l t u r e m e d i a (as g e n e r a l l y s u p p l e m e n t e d w i t h c a l f s e r u m )

prior

to c e l l a t t a c h m e n t a n d g r o w t h o n substrate surfaces. I n a d d i t i o n to s u r face c h e m i c a l a n d charge influences o n c e l l a d h e s i o n at the s o l i d / s o l u t i o n interface, there is a d e p e n d e n c e o n the r e l a t i v e sizes a n d geometries the cells a n d t h e i r p o t e n t i a l substrates (48).

of

I n some cases, w h e r e the

substrates are q u i t e l a r g e w i t h respect to the cells, the cells w i l l s i m p l y g r o w over, a l o n g , or u n d e r s u c h f o r e i g n m a t e r i a l . are v e r y s m a l l a n d a k i n to phagocytosis

fibrous,

or o t h e r w i s e

finely

W h e n t h e substrates

p a r t i c u l a t e d , a process

occurs—i.e., the cells cluster o n a n d a r o u n d the

f o r e i g n s o l i d surfaces. P a u l W e i s s c a t a g o r i z e d these types of reactions as i n d i c a t i v e of a tactile c h e m i c a l response (49). (50, 5 1 ) a n d G o o d ( 5 2 )

V a n Oss a n d c o - w o r k e r s

h a v e r e c e n t l y t a k e n some of these factors i n t o

a c c o u n t i n t h e i r s t u d y of m e c h a n i s m s of phagocytosis

as i t u n d e r l i e s

p a r t i c l e e n g u l f m e n t i n h e a l t h a n d disease. A n i m p o r t a n t feature of the i n c u b a t i o n of f o r e i g n s o l i d surfaces i n natural biological media, especially i n media

containing

adsorbable

m a c r o m o l e c u l a r c o m p o n e n t s s u c h as s e r u m g l y c o p r o t e i n s , is that h i g h e r surface free-energy materials ( s u c h as g l o w - d i s c h a r g e c l e a n e d glass ) a n d m o d e r a t e l y l o w surface free-energy m a t e r i a l s ( s u c h as d i c h l o r o d i m e t h y l s i l a n e - m o d i f i e d glass) c o n t i n u e to e x h i b i t d i f f e r e n t i a l s u r f a c e p r o p e r t i e s t h r o u g h a n a d s o r b e d p r o t e i n b l a n k e t of e q u a l thickness. T h e s e p r o p e r t i e s are e x h i b i t e d i n b o t h contact a n g l e ( w e t t i n g a n d s p r e a d i n g )


and cell

16

APPLIED CHEMISTRY

AT PROTEIN

INTERFACES

a d h e s i o n experiments w h i c h are easily d o n e i n the l a b o r a t o r y (47). c h a r a c t e r i z e d a n d w e l l b e h a v e d glass c o a t i n g c o m p o u n d s s u r f a c e free energy are n e e d e d

Well

of v e r y

low

to v e r i f y t h a t p r o t e i n a d s o r p t i o n

will

o c c u r o n that substrate i n the same w a y i t does o n the h i g h a n d m o d e r a t e surface free-energy substrates. C e l l s a d h e r e m o r e t i g h t l y a n d i n greater abundance

to

smooth

smooth moderate

higher

surface

free-energy

( b e t w e e n 20 a n d 30 d y n e s / c m )

t a c h e d to the h i g h surface-free-energy

substrates

than

to

substrates; cells at-

substrates, t h r o u g h a n i n t e r v e n i n g

l a y e r of spontaneously a d s o r b e d p r o t e i n , s h o w m u c h greater f r e q u e n c y of Downloaded by EASTERN KENTUCKY UNIV on February 25, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch001

irregular perimeters

(i.e., m a n y p r o t r u s i o n s , p s e u d o p o d s , a n d

projections f r o m the b o r d e r a t t a c h i n g to the s u r f a c e ) .

fibrous

C e l l s o n the b i o -

c o m p a t i b l e surfaces r e m a i n r o u n d e d a n d p o o r l y adherent.

E q u a l l y sig-

n i f i c a n t is that, i n the case of c e l l contact w i t h p r o t e i n - c o a t e d

moderately

l o w - e n e r g y surfaces, t h e cells prefer to aggregate ( o v e r a p e r i o d of t i m e ) w i t h one a n o t h e r i n tissue-like masses r a t h e r t h a n m a i n t a i n i n d e p e n d e n t a n d separate a d h e s i o n w i t h a substrate (47).

W e have previously dis-

c u s s e d a z o n e of m i n i m a l c e l l s p r e a d i n g a n d a t t a c h m e n t o n s u c h surfaces ( 41 ) a n d r e v i e w e d these processes f r o m the p o i n t of v i e w of c e l l m o t i l i t y a n d m i g r a t i o n as necessarily takes p l a c e d u r i n g w o u n d h e a l i n g

(53).

F u t u r e s t u d y m u s t s h o w h o w , at the l e a d i n g e d g e of m i g r a t i n g cells, the a d h e s i o n b e t w e e n c e l l surface structures a n d the u n d e r l y i n g i m m o b i l i z e d substrate surface structures c a n b e so r a p i d l y m a d e a n d b r o k e n as to a l l o w t h e o b s e r v e d r o t a t i n g - t a n k t r e a d - l i k e m o t i o n of the c e l l surface a n d the f o r w a r d progress of the c e l l b o d y to occur. Intrauterine Devices.

L e i n i n g e r (54)

has r e v i e w e d the u t i l i t y of

v a r i o u s p o l y m e r s as i m p l a n t s . O b j e c t s w h i c h are n e i t h e r a tissue i m p l a n t n o r a c a r d i o v a s c u l a r i m p l a n t b u t w h i c h m a y h a v e features of b o t h , are c o n t r a c e p t i v e f o r e i g n bodies.

Since this subject has not b e e n

discussed

often e n o u g h i n the surface c h e m i c a l l i t e r a t u r e , interactions of

devices

p l a c e d i n the uterine c a v i t y r e m a i n p o o r l y u n d e r s t o o d .

devices

These

m i n i m i z e i n some u n k n o w n m a n n e r , chances for c o n c e p t i o n a n d p r e g n a n c y . A b r i e f a r t i c l e w h i c h discusses t h e specifics of spontaneous i n t e r f a c i a l m o d i f i c a t i o n of these contraceptives is i n c l u d e d i n this v o l u m e

(57).

T h e m a j o r finding is that a l l s u c h inserts a c c u m u l a t e a r e m a r k a b l y u n i f o r m c o a t i n g of g l y c o p r o t e i n b y spontaneous a d s o r p t i o n f r o m the c e r v i c a l mucous

fluid.

It has b e e n s p e c u l a t e d t h a t this l a y e r of a d s o r b e d i n t e r -

f a c i a l m a t e r i a l m o d e s t l y activates a n t i b o d y a n d r e j e c t i o n m e c h a n i s m s i n the s u r r o u n d i n g tissue, t h e r e b y p r e v e n t i n g c a p a c i t a t i o n of s p e r m o b l i g e d to s w i m t h r o u g h this s u b t l y c h e m i c a l l y m o d i f i e d z o n e Dental Adhesion.

A

problem

of

(41).

increasing importance

w h i c h is

a t t r a c t i n g i n c r e a s i n g scientific w o r k e r s , is the e s t a b l i s h m e n t i n t h e m o i s t saline, e n z y m a t i c a l l y a c t i v e , heat-and-cold-stressed,

and

mechanically

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


the

1.

BAIER

Applied

17

Chemistry

n a t u r a l surfaces a n d s y n t h e t i c p r o s t h e t i c m a t e r i a l s (50, 57, 5 8 ) .

This

h a p p e n s spontaneously i n most h e a l t h y a n d m a n y diseased persons w h e n d e n t a l p l a q u e accumulates a n d transforms i n t o d e n t a l c a l c u l u s . A s w e h a v e s h o w n elsewhere

(59),

a n d as Q u i n t a n a r e v i e w s i n this v o l u m e

( 6 0 ) , s o l i d surfaces i n t h e o r a l c a v i t y e v e n w h e n s c r u p u l o u s l y p r e c l e a n e d do not r e m a i n free of a d s o r b e d m a c r o m o l e c u l a r c o m p o n e n t s for m o r e than a few

seconds.

T h e m a t e r i a l w h i c h is s p o n t a n e o u s l y

adsorbed

is a specific g l y c o p r o t e i n c o m p o n e n t of s a l i v a a n d not a heterogeneous r a n d o m selection f r o m a l l surface-active

c o m p o n e n t s present.

In

our


laboratories, w e are d e t e r m i n i n g the surface c h e m i s t r y of h u m a n teeth as t h e y n o r m a l l y rest i n a h e a l t h y h u m a n m o u t h , b y m a k i n g contact angle measurements ( 5 9 ) .

O n c e the m a i n features of this i n t e r f a c i a l c h e m i s t r y

a n d the d o m i n a n c e of most interfaces b y a d s o r b e d h y d r a t e d proteinaceous layers b e c o m e s m o r e g e n e r a l l y u n d e r s t o o d , the d e v e l o p m e n t of excellent d e n t a l adhesives w i l l f o l l o w . S u c h adhesives, s u p p o r t e d b y s p e c i a l d e n t a l treatment, w i l l a l l o w greater a d h e s i o n b e t w e e n b i o l o g i c a l materials a n d h a r d substrates, as i n i m p l a n t s at the d e n t a l m a r g i n . i m p r o v e d surface c h e m i c a l k n o w l e d g e

I n other cases,

s h o u l d a l l o w treatments to

be

d e v e l o p e d w h i c h w i l l m i n i m i z e a d h e s i o n b e t w e e n c e l l u l a r elements, s u c h as b a c t e r i a l flora, a n d s o l i d surfaces i n the m o u t h so that d e n t a l p l a q u e , cavity initiation, and calculus formation w i l l be m i n i m i z e d . Biological Fouling.

B a c t e r i a l a d h e s i o n is a p r i m a r y event i n the

e a r l y phases of d e n t a l p l a q u e f o r m a t i o n .

A less p o p u l a r l y u n d e r s t o o d

example

of b a c t e r i a l a d h e s i o n is that w h i c h occurs o n f o r e i g n

surfaces

i n n a t u r a l waters.

K e v i n Marshall i n Australia and

solid

William

C o r p e i n the U n i t e d States h a v e l e d the s t u d y of p r i m a r y b a c t e r i a l f o r m a t i o n o n f o r e i g n s o l i d surfaces (61, 62).

T h e y have

film

demonstrated,

i n d e p e n d e n t l y a n d i n c o l l a b o r a t i o n w i t h this author, the i n t e r f a c i a l m o d i fication

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

b y a d s o r b e d g l y c o p r o t e i n layers. It is not yet c e r t a i n that these e x t r a c e l l u l a r layers o r i g i n a t e f r o m d i s s o l v e d c o m p o n e n t s i n n a t u r a l seawater or i n s u s p e n d i n g m e d i a i n l a b o r a t o r y experiments. films

These conditioning

of a d s o r b e d p r o t e i n m a y result f r o m a c t i v e p a r t i c i p a t i o n of

the

b a c t e r i a i n e x t r u d i n g s u c h m a t e r i a l or f r o m the d i s i n t e g r a t i o n of some of the b a c t e r i a to p r o v i d e the a d s o r b a b l e c o m p o n e n t s i n a

nonspecific

m a n n e r . M a r s h a l l has also s h o w n that the p r o p a g a t i o n of these b a c t e r i a i n chains a n d clusters p r o c e e d s f r o m the surface t h r o u g h the i n t e r m i n g l i n g or a d h e s i o n of

fibrillar

e x t r a c e l l u l a r p o l y m e r i c m a t e r i a l s i m i l a r to t h a t

i n v o l v e d i n the o r i g i n a l adhesive event the i n v o l v e m e n t of

fiber-forming

(63).

fibrinogen

T h i s o b s e r v a t i o n recalls

a n d the m u t u a l a g g r e g a t i o n

of b l o o d platelets d u r i n g the i n i t i a l events of t h r o m b u s f o r m a t i o n s c r i b e d e a r l i e r (21,

29).


de-

18

APPLIED CHEMISTRY A T PROTEIN

INTERFACES

E v e n i n t h e absence of m i c r o s c o p i c a l l y o b s e r v a b l e b a c t e r i a l a d h e s i v e events, a l l substrates i n n a t u r a l w a t e r , a n d e s p e c i a l l y i n s u b t r o p i c a l areas w h e r e m a r i t i m e f o u l i n g a b o u n d s , are spontaneously c o a t e d w i t h a d s o r b e d p r o t e i n films ( 6 5 ) . I n fact, a l l o w i n g f o r differences i n r e l a t i v e t i m e scales w h i c h reflect differences i n r e l a t i v e concentrations of a d s o r b a b l e

com-

ponents, the sequence of events at s o l i d surfaces i m m e r s e d i n c o m m o n f r e s h or seawater ( c o n t a i n i n g l i v i n g o r g a n i s m s )

is essentially i d e n t i c a l

to t h a t i n t h e n a t u r a l m a r i t i m e e n v i r o n m e n t . T h e sequence b e g i n s w i t h r a p i d , n e a r l y m o n o m o l e c u l a r l a y e r c o v e r a g e of specifically a d s o r b e d g l y Downloaded by EASTERN KENTUCKY UNIV on February 25, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch001

coproteins f o l l o w e d b y l a y e r t h i c k e n i n g a n d finally a d h e s i o n a n d g r o w t h of discrete organisms ( 6 5 ) . W i t h samples p r e p a r e d b y M . C o o k i n E n g l a n d , w e h a v e

demon-

strated t h a t s u c h a d s o r b e d films o n s o l i d substrates are also i n v o l v e d i n the a d h e s i o n of mussel byssus discs to s u c h surfaces ( 6 6 ) . A s e c o n d a r y a d h e s i v e p r o d u c t , a n a l y t i c a l l y s i m i l a r to g l y c o p r o t e i n s , is that o n the u n d e r s i d e of the mussel byssus discs ( 6 7 ) . I n the case of the a b i l i t y of b a r n a c l e c y p r i d s to a d h e r e u n d e r adverse circumstances to substrates as d i v e r s e as T e f l o n , steel, a n d h i g h l y toxic p a i n t surfaces, t h e o r i g i n a l m o d i f i c a t i o n of the s o l i d substrate i n t e r f a c e is p r o v i d e d b y

adsorbed

p r o t e i n - d o m i n a t e d layers. C r i s p (68) has s h o w n c o n v i n c i n g l y t h a t b a r n a c l e c y p r i d s i n t h e i r adhèrent stage w i l l choose p r o t e i n - c o a t e d substrates f o r s e t t l i n g rather t h a n f r e s h l y i n s e r t e d or freshly c l e a n e d surfaces. I n this case, as w i t h the mussel byssus d i s c a n d w i t h a d h e r e n t b a c t e r i a , the extruded cement

(itself a g l y c o p r o t e i n )

m u s t b e e s t a b l i s h i n g its great

a d h e s i o n n o t d i r e c t l y w i t h t h e substrate b u t w i t h t h e a l r e a d y - p r e s e n t g l y c o p r o t e i n film f o r m e d p r i o r to c y p r i d settling. W e suggested earlier that a l l b i o l o g i c a l f o u l i n g begins b y a n a d s o r p t i v e event d o m i n a t e d b y a c c u m u l a t i o n of g l y c o p r o t e i n m a t e r i a l a n d that s e c o n d a r y a d h e s i o n of the f o r m e d c e l l u l a r or l a r v a l organisms is t h r o u g h a p r o t e o g l y c a n

type

m a t e r i a l ( 6 7 ) . W e suppose that the s e c o n d a r y c e m e n t interacts, c a r b o hydrate-group-to-carbohydrate-group, chains w i t h s i m i l a r o l i g o s a c c h a r i d e

t h r o u g h its exposed groups

glyco

side

of t h e o r i g i n a l l y a d h e r e n t

l a y e r i n m u c h the same m a n n e r as p o l y s a c c h a r i d e chains m e r g e i n w e t c e l l u l o s i c p u l p s to g i v e p a p e r p r o d u c t s t h e i r strength. B a r n a c l e s a c t u a l l y g r o w n o n t h e faces of i n t e r n a l reflection p r i s m s h a v e a c e m e n t p r e d o m i n a t e l y of t h e g l y c o p r o t e i n class. I n most d e t a c h a b l e b i o l o g i c a l l i n k s , s u c h as f o r m e d b y t h e mussel byssus discs, l i m p i d s , snails, a n d f r e s h - w a t e r r e m o v a b l e b a c t e r i a , the a d h e s i v e is g e n e r a l l y of the p o l y s a c c h a r i d e or p r o t e o g l y c a n class. B a s e d u p o n i m m e r s i o n studies s t i l l i n progress, the z o n e of m i n i m a l b i o l o g i c a l a d h e s i o n s i g n a l e d b y the c r i t i c a l surface t e n s i o n r a n g e b e t w e e n

20 a n d 30 d y n e s / c m

i n tissue

i m p l a n t a t i o n , c e l l c u l t u r e , a n d b l o o d c o m p a t i b i l i t y experiments w i l l also b e t h e p r o p e r f u n c t i o n a l z o n e for m i n i m u m b i o l o g i c a l f o u l i n g ( 6 4 ) .


1.

BAIER

Applied

Specifics of Protein

19

Chemistry Adsorption

Substrates of d i f f e r i n g i n i t i a l surface p r o p e r t i e s , w h e n i m m e r s e d i n media containing adsorbable macromolecules

s u c h as proteins, a t t a i n at

e q u i l i b r i u m essentially the same a m o u n t of the same a d s o r b e d m a t e r i a l (69).

A l t h o u g h the e q u i l i b r i u m c o n d i t i o n s

at the surfaces

substrates are, i n the absence of other components l i v i n g cells, s i m i l a r , these surfaces

of

various

and especially

of

differ r e m a r k a b l y w h i l e a t t a c h i n g ,

a d s o r b i n g , or o t h e r w i s e c o n t a c t i n g c e l l u l a r elements before e q u i l i b r i u m Downloaded by EASTERN KENTUCKY UNIV on February 25, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch001

thicknesses or constant c h e m i c a l c o n d i t i o n s

have been

obtained.

To

u n d e r s t a n d b i o a d h e s i v e events one m u s t k n o w t h e specific c o n f i g u r a t i o n a n d other details of o r g a n i z a t i o n of the films d u r i n g t h e i r c o n v e r s i o n f r o m i n i t i a l l y a t t a c h e d molecules desorption

e q u i l i b r i u m . Some

especially

the differences

to the establishment of

aspects

adsorption-

of the a d s o r p t i o n process,

w h i c h continue

to

exist even

under

l i b r i u m c o n d i t i o n s , are d e s c r i b e d i n other p u b l i c a t i o n s (69,

Adsorbed

and equi-

70).

Films

U s i n g the p r o t e i n b e t a - l a c t o g l o b u l i n as a m o d e l , G e o r g e L o e b has s h o w n that the r a t i o of essentially n a t i v e to c o n f i g u r a t i o n a l l y m o d i f i e d p r o t e i n i n s p o n t a n e o u s l y a d s o r b e d films varies d i r e c t l y w i t h the a m o u n t of m a t e r i a l a d s o r b e d (71).

A t the lowest a m o u n t a d s o r b e d

(presumably

the first m o n o l a y e r c o v e r a g e ) , the r a t i o of n a t i v e to a l t e r e d m o l e c u l a r c o n f i g u r a t i o n is about 0.5. O n l y as the film achieves a d s o r p t i o n e q u i l i b r i u m at a s i g n i f i c a n t l y greater thickness does this r a t i o b e c o m e about 0.8 ( f o r films a d s o r b e d o n n o m i n a l l y h i g h surface e n e r g y m a t e r i a l s ) .

Loebs

p a r a m e t e r of s t r u c t u r a l a l t e r a t i o n was the I R - d e t e c t a b l e shift i n h y d r o gen-bonding

arrangements w h i c h differentiate the p r e d o m i n a t e l y

beta-

s t r u c t u r e d ( i n t e r m o l e c u l a r l y h y d r o g e n - b o n d e d e x t e n d e d c h a i n ) f r o m the alpha-helical

configuration

bonded amino acid units).

(stabilized by

intramolecularly

hydrogen-

O n l y w h e n the substrate a v a i l a b l e f o r a d -

s o r p t i o n of p r o t e i n m a c r o m o l e c u l e s f r o m s o l u t i o n was i n the m o d e r a t e l y low-surface-free-energy i n the a d s o r b e d

film

class d i d the ratio of n a t i v e to d e n a t u r e d m a t e r i a l a p p r o a c h one.

L o e b also s h o w e d

i n a series of

experiments i n w h i c h the c o n c e n t r a t i o n of p r o t e i n i n the o r i g i n a l s o l u t i o n was s y s t e m a t i c a l l y m o d i f i e d , t h a t the p r o p o r t i o n of n a t i v e m a t e r i a l ( c o n figurationally

u n d i s t o r t e d at the l e v e l of secondary structure d e t e c t e d

by

I R ) w a s l o w e r r e l a t i v e to the l o w e r o r i g i n a l c o n c e n t r a t i o n of p r o t e i n i n solution.

I n s i m i l a r systems s t u d i e d b y e l l i p s o m e t r y , e q u i l i b r i u m

film

thicknesses are n o t a t t a i n e d u n t i l ca. 1500 sec of a d s o r p t i o n ; this i l l u s trates the s u b s t a n t i a l difference

i n t i m e scales b e t w e e n

a t t a i n m e n t of

e q u i l i b r i u m p r o t e i n a d s o r p t i o n a n d the t i m e ( 3 0 - 4 8 0 sec) at w h i c h cells


20


u s u a l l y b e c o m e adhesive to the t h i c k e n i n g the N a t i o n a l B u r e a u of S t a n d a r d s ( 6 9 )

films.

INTERFACES

R e c e n t results f r o m

i l l u s t r a t e the f u r t h e r s e n s i t i v i t y

of p r o t e i n a d s o r p t i o n to the specific surface free e n e r g y of the s o l i d s u b strate. S u c h w o r k has s h o w n that a l t h o u g h the mass of m a t e r i a l a d s o r b e d o n v a r i o u s surfaces m i g h t r e m a i n essentially constant u n d e r e q u i l i b r i u m c o n d i t i o n s , the p r o t e i n extension out i n t o the s o l u t i o n phase c a n b e s u b s t a n t i a l l y different.

O n a v a r i e t y of surfaces, proteins of s p e c i a l interest

i n t h r o m b u s f o r m a t i o n a n d b l o o d c l o t t i n g h a v e t h e i r greatest extension f r o m those surfaces of l o w e r free e n e r g y ( 6 9 ) .

S u c h studies h a v e s u g -


gested the i n t e r p r e t a t i o n , d i s c u s s e d i n d e t a i l b y N y i l a s (72),

that l o w

i n t e r a c t i o n energies across s o l i d / p r o t e i n s o l u t i o n b o u n d a r i e s w i l l ensure short residence times of t h e a d s o r b i n g m a c r o m o l e c u l e s .

Proper balance

of the i n t e r f a c i a l c h e m i s t r y w i t h the c h e m i s t r y of the a d s o r b i n g species w h i c h tenacious,

irre-

versible protein adsorption ( a n d configuration modification) induce

c a n p r o b a b l y m i n i m i z e t h e adverse

(73).

Gas/Liquid

influences

Interfaces

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

at s o l i d / s o l u -

t i o n interfaces are often difficult to p e r f o r m a n d e v e n m o r e difficult to interpret properly.

Fortunately w e have reported a n d reviewed

else-

w h e r e (11, 22) that b o t h a d s o r p t i o n a n d d e l i b e r a t e s p r e a d i n g of proteins and synthetic polypeptides

(as m o d e l p r o t e i n s ) at a i r / l i q u i d interfaces

p r o v i d e films w h o s e structures are u s u a l l y i n d i s t i n g u i s h a b l e f r o m those f o r m e d b y a d s o r p t i o n to solids. r e v i e w e d b y M a l c o l m (74)

It has also b e e n s h o w n , as r e c e n t l y

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

g a s / l i q u i d i n t e r f a c i a l p o l y m e r i c films c a n b e grossly m a n i p u l a t e d , c r u m pled, a n d collapsed into

fibrous

f u n d a m e n t a l s t r u c t u r e (75, 76).

b u n d l e s w i t h o u t m o d i f i c a t i o n of t h e i r L o e b has s h o w n that for p r e d o m i n a n t l y

a l p h a - h e l i c a l proteins, a d s o r p t i o n at the s o l i d / l i q u i d b o u n d a r y a n d m o r e p a r t i c u l a r l y s p r e a d i n g at the a i r / l i q u i d i n t e r f a c e does not s i g n i f i c a n t l y degrade their original secondary

s t r u c t u r e (77).

O n the other h a n d ,

p r e d o m i n a n t l y b e t a - c h a i n m a t e r i a l s , s u c h as b e t a - l a c t o g l o b u l i n , do d u r i n g a d s o r p t i o n or a i r / w a t e r i n t e r f a c i a l film f o r m a t i o n c h a n g e to a h e l i c a l f o r m (71).

more

T h i s w a s p r e v i o u s l y s h o w n , b y m u l t i p l e attenuated

i n t e r n a l reflection s p e c t r o s c o p y t e c h n i q u e s i d e n t i c a l to those d e s c r i b e d here, to o c c u r w i t h t h e s i m p l e r p o l y p e p t i d e

poly(y-methylglutamate)

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

was

beta

film

(extended

chain)

became about 5 0 %

and

in which

c o i l e d (76).

its a i r / w a t e r i n t e r f a c i a l

I n b e t a - l a c t o g l o b u l i n , the f r a c t i o n of

n a t i v e m a t e r i a l r e m a i n i n g i n a m o n o l a y e r d e p e n d e d on the surface p r e s sure u n d e r w h i c h the film w a s t r a n s f e r r e d to i n t e r n a l reflection p r i s m s f o r u l t i m a t e analysis. T h e h i g h e r pressures f a v o r e d m o r e n a t i v e m a t e r i a l


1.

BAIER

Applied

i n the t r a n s f e r r e d

21

Chemistry film.

W i t h this exception, i t c a n b e g e n e r a l i z e d t h a t

the strong p r e f e r e n c e is for c o i l e d r a t h e r t h a n e x t e n d e d chains of surfacelocalized macromolecules. U s i n g d i r e c t extensions

of

film

transfer t e c h n i q u e s

originally de-

s c r i b e d b y L a n g m u i r a n d B l o d g e t t ( 1 ), w e h a v e a p p l i e d the surface

film

r e t r i e v a l m e t h o d d e s c r i b e d to films a c c u m u l a t e d at n a t u r a l g a s / l i q u i d i n t e r f a c i a l b o u n d a r i e s s u c h as those b e t w e e n the sea a n d the atmosphere a b o v e lakes a n d oceans ( 78, 79, 80, 81 ). G i v e n p r i s m s w h i c h h a d b e e n


s c r u p u l o u s l y c l e a n e d i n the l a b o r a t o r y p r i o r to p a c k a g i n g , so that t h e i r surface properties w e r e p r e d o m i n a n t l y h y d r o p h i l i c at the t i m e of

field

i m m e r s i o n , i t was d e m o n s t r a t e d t h a t even i n a r e a s o n a b l y c h o p p y m a r i time environment where

precise

i m m e r s i o n a n d w i t h d r a w a l was

not

possible, o n l y a single thickness of the a m b i e n t resident film w a s t r a n s f e r r e d for analysis ( b y the c o m b i n a t i o n of t e c h n i q u e s d e s c r i b e d e a r l i e r here).

It w a s also s h o w n that, b y first c o n d i t i o n i n g the p r i s m w i t h a

hydrophobic

c o a t i n g ( s u c h as t h a t p r o v i d e d b y s i l i c o n i z a t i o n or b y a

d r i e d m o n o l a y e r of stearic a c i d or stéarate s a l t ) , m u l t i p l e i m m e r s i o n s a n d w i t h d r a w a l s of a p r i s m t h r o u g h a s u r f a c e - f i l m - c o v e r e d l i q u i d w o u l d r e s u l t i n the easy transfer of p r o p o r t i o n a t e l y t h i c k e r ( m u l t i p l e a m b i e n t layers ) films for easier analysis. T h i s t e c h n i q u e i n v o l v e s r e m o v i n g a p r i s m i n a s p e c i a l s m a l l h o l d e r f r o m its p l a s t i c p a c k a g e , a t t a c h i n g i t to a snap h o o k o n a fishing l i n e a n d l o w e r i n g i t t h r o u g h the n a t u r a l a i r / w a t e r i n t e r f a c e ( or o u t f a l l of i n d u s t r i a l wastes, l a y e r of f o a m , o i l s l i c k ) of interest, s l o w l y w i t h d r a w i n g the p r i s m , g i v i n g it a b r i e f a i r d r y i n g , a n d r e p a c k a g i n g for later analysis. T h e first l a r g e scale a p p l i c a t i o n of the t e c h n i q u e w a s i n C h a u t a u q u a L a k e i n N e w Y o r k state, w h i c h w a s r e p e t i t i v e l y s a m p l e d over the entire 1969 r e c r e a t i o n a l season to establish spectroscopic ters to p e r m i t c h a r a c t e r i z a t i o n of its surface q u a l i t y (78).

parame-

M o r e recently,

the t e c h n i q u e has b e e n e x t e n d e d to the major oceans a n d seas of the earth.

I n g e n e r a l , i n a l l n o n p o l l u t e d locations or i n p o l l u t e d locations

w h i c h w e r e a l l o w e d a f e w days for n a t u r a l c l e a n s i n g to o c c u r at the interface, n a t u r a l a i r / w a t e r b o u n d a r i e s are d o m i n a t e d b y a n d p r o t e o g l y c a n t y p e films (80, W i t h r a r e exceptions,

glycoprotein

81).

s u c h as a l o n g the n o r t h w a l l of the

Gulf

S t r e a m w h e r e i n t e r f a c i a l films are sometimes d o m i n a t e d b y l i p i d

com-

ponents, contact angle d a t a o n the t r a n s f e r r e d d r i e d films h a v e i n d i c a t e d c r i t i c a l surface tensions b e t w e e n 30 a n d 40 d y n e s / c m , t h e r e b y c o n f i r m i n g the presence of o x y g e n a t e d a n d p r e s u m a b l y also n i t r o g e n a t e d as d o m i n a n t c o m p o n e n t s

components

i n s u c h films. T h e s t a b i l i z i n g film at the g a s /

l i q u i d b o u n d a r i e s so p r o l i f i c i n l o n g - l a s t i n g sea foams is p r e d o m i n a n t l y g l y c o p r o t e i n a n d p r o t e o g l y c a n m a t e r i a l h a v i n g its o r i g i n i n sea-surface films c o n t r i b u t e d p r i m a r i l y b y p l a n k t o n b l o o m s (81, 82, 83,

84).


22


INTERFACES

W i t h respect to g a s / l i q u i d interfaces c r e a t e d i n the b u l k of solutions, b u b b l e s r i s i n g t h r o u g h solutions c o n t a i n i n g m a c r o m o l e c u l e s of b i o l o g i c a l o r i g i n , a n d e s p e c i a l l y proteins, w i l l n o t o n l y spontaneously c o l l e c t these p o l y m e r s at the n e w g a s / l i q u i d interfaces of the r i s i n g b u b b l e b u t w i l l concentrate t h e m i n t o i n s o l u b l e fibrous debris w h i c h is s p u n off the d i s a p p e a r i n g t r a i l i n g e d g e of a m o v i n g b u b b l e (85).

B u b b l e s t r a v e l i n g to

the w a t e r surface f r o m the s o l u t i o n also c a r r y , w i t h t h e i r g a s / l i q u i d b o u n d a r i e s , at least a p o r t i o n of t h e i r a d s o r b e d b u r d e n to t h a t surface. D u r i n g b u b b l e b r e a k i n g , this m a t e r i a l is ejected a l o n g w i t h film f r a g Downloaded by EASTERN KENTUCKY UNIV on February 25, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch001

ments f r o m the a m b i e n t g a s / l i q u i d i n t e r f a c i a l film a l r e a d y r e s i d i n g there. B l a n c h a r d , i n this v o l u m e , gives a n excellent r e v i e w of the i m p o r t a n c e of s u c h processes o n a w o r l d w i d e scale a n d of t h e i r i m p l i c a t i o n s i n oceanography and meteorology.

I n a more modest but i n d i v i d u a l l y more

l e t h a l e x a m p l e , the p r e f e r r e d m e t h o d of b l o o d o x y g e n a t i o n d u r i n g o p e n heart s u r g e r y is to a l l o w a c o l u m n of gas b u b b l e s to rise t h r o u g h t h e blood.

T h i s creates a v i g o r o u s f o a m i n g w h i c h r e q u i r e s s e c o n d a r y

foam

b r e a k i n g a n d filtering of p a r t i c u l a t e d e b r i s b e f o r e the b l o o d is r e i n j e c t e d into the patient. I n b u b b l e s r i s i n g t h r o u g h the o c e a n , t h e i n s o l u b l e p r o t e i n s h e d m a y b e c o n t r i b u t i n g to the o r g a n i c d e t r i t i s necessary as f o o d f o r l o w e r - d w e l l i n g organisms b e l o w the p h o t i c z o n e ; i n b l o o d oxygenators, the proteins lost i r r e v e r s i b l y f r o m the v o l u m e phase are u s u a l l y c r u c i a l to the h e a l t h of the p a t i e n t .

L o s s of s u c h c r u c i a l m a t e r i a l , e s p e c i a l l y

a n t i b o d i e s , c o u l d b e l a r g e l y r e s p o n s i b l e for the f r e q u e n t deaths

from

s i m p l e i n f e c t i o n , p n e u m o n i a , a n d other diseases of patients w h o

have

h a d successful o p e n h e a r t surgery.

Cell-Cell

Interactions

L i v i n g cells c a n u n d e r g o changes i n t h e i r surface p r o p e r t i e s , a n d these p r o p e r t i e s d i c t a t e t h e r e l a t i v e adhesiveness of cells to t h e i r n e i g h bors (86).

T h i s surface c h e m i c a l interference to a d h e s i o n t h e n correlates

w i t h decreased strength of c e l l - t o - c e l l joints a n d the i n c r e a s e d m o b i l i t y a n d invasiveness c h a r a c t e r i s t i c of m a l i g n a n t cells i n tumors a n d other forms of cancer. I n this r e g a r d , i t is w o r t h w h i l e to consider m e c h a n i s m s w h e r e the a d h e s i o n is b e t w e e n t w o s i m i l a r or d i s s i m i l a r cells; f o r e i g n s o l i d substrates, as d i s c u s s e d earlier, are not i n v o l v e d here.

It is l i k e l y

that c e l l - t o - c e l l adhesions are also m e d i a t e d b y a d s o r b e d m a c r o m o l e c u l a r components,

however.

A l m o s t c e r t a i n l y , g l y c o p r o t e i n or

proteoglycan

materials a c c o u n t for the g a p of a b o u t 100 A s h o w n b y e l e c t r o n m i c r o s c o p y to exist b e t w e e n closely a p p o s e d c e l l surface m e m b r a n e s . task for chemists to d e c i p h e r the specific

It is a serious

constitution, configuration,

structure, a n d f u n c t i o n of s u c h g l y c o p r o t e i n s a n d p r o t e o g l y c a n s at interfaces.

Since the g l y c o p r o t e i n materials c a n c o n t a i n

present

anywhere


1.

BAIER

Applied

Chemistry

23

f r o m 1 / 1 0 o f 1 % to a b o u t 1 5 % c a r b o h y d r a t e i n side chains, a n d since the m o l e c u l a r w e i g h t s of these materials are generally a r o u n d 1 m i l l i o n , a n d since p r o t e o g l y c a n s ( u n t i l r e c e n t l y u s u a l l y c a l l e d m u c o p o l y s a c c h a rides i n the b i o c h e m i c a l literature ) c a n have every other a m i n o a c i d a l o n g the p r o t e i n b a c k b o n e s u b s t i t u t e d w i t h short sugar chains a n d also c a n range i n m o l e c u l a r w e i g h t f r o m ca. 20,000 to > 1 m i l l i o n , i t is a f o r m i d a b l e c h a l l e n g e to d e c i p h e r the specifics of i n t e r f a c i a l c h e m i s t r y a n d o r g a n i z a


t i o n r e q u i r e d for f u r t h e r progress.

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27. Petschek, H., Adamis, D., Kantrowitz, A. R., Trans. Amer. Soc. Artif. Intern Organs (1968) 14, 256. 28. Vroman, L., Thromb. Diath. Haemorrh. (1964) 10, 455. 29. Vroman, L., Adams, A. L., J. Biomed. Mater. Res. (1969) 3, 43. 30. Booyse, F. M., Rafelson, Jr., M. E., Ser. Haematol. (1971) 4, 152. 31. Booyse, F. M., Sternberger, L. Α., Zschocke, D., Rafelson, Jr., M. E., J. Histochem. Cytochem. (1971) 19, 540. 32. Schoen, F. J., Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1971) 30, 1647. 33. Gott, V. L., Baier, R E., "Materials Compatible with Blood," Vol. 2, Ann. Rep. Contract PH 43-68-84 (1973) available Nat. Tech. Inf. Serv., Springfield, Va. 34. Moyer, L. S., Gorin, M. H., J. Biol. Chem. (1940) 133, 605. 35. Mirkovitch, V., Beck, R. E., Andrus, P. G., Leininger, R.I.,J. Surg. Res. (1964) 4, 395. 36. Vroman, L., Adams, A. L., Klings, M., Fischer, G., ADVAN. CHEM. SER. (1975) 145, 255. 37. Baier, R. E., DePalma, V. Α., Furuse, Α., Gott, V. L., Lucas, T., Sawyer, P. N., Srinivasan, S., Stanszweski, B., unpublished data. 38. Baier, R. E., Dutton, R. C., Gott., V. L., Advan. Exp. Med. Biol. (1970) 7, 235. 39. Harrison, J. H., Amer. J. Surg. (1958) 95, 3. 40. Baier, R. E., Gott, V. L., Furuse, Α., Trans. Amer. Soc. Artif. Intern. Org (1970) 16, 50. 41. Baier, R. E., "Adhesion in Biological Systems," R. S. Manly, Ed., Chap. 2, Academic, New York, 1970. 42. Ward, C. Α., Stanga, D., Collier, R. P., Zingg, W., Trans. Amer. Soc. Artif. Intern. Organs (1974) 20. 43. Kolobow, T., Stool, E. W., Weathersby, P. K., Pierce, J., Hayano, F., Trans. Amer. Soc. Artif. Intern. Organs (1974) 20. 44. Johnson, Jr., R. E., Dettre, R. H., J. Colloid Interface Sci. (1966) 21, 610. 45. Shafrin, E. G., Zisman, W. A., J. Phys. Chem. (1967) 71, 1309. 46. Taylor, A. C., "Adhesion in Biological Systems," R. S. Manly, Ed., Aca demic, New York, 1970. 47. Wilkins, J. F., "Adhesion of Ehrich Ascites Tumor Cells to Surface-Modified Glass," Ph.D. Dissertation, State University of New York at Buffalo, 1974. 48. Maroudas, N., J. Theor. Biol., in press. 49. Weiss, P., Harvey Lect. (1959) 55, 13. 50. Neumann, A. W., van Oss, C. J., Szekely, J., Kolloid Ζ. Z. Polym. (1973) 251, 415. 51. van Oss, C. J., Gillman, C. F., Neumann, A. W., Immunol. Chem. (1974) 3, 77. 52. van Oss, C. J., Good, R. J., Neumann, A. W., J. Electroanal. Chem. (1972) 37, 387. 53. Baier, R. E., "Epidermal Wound Healing," H. I. Maibach and D. T. Rovee, Eds., Chap. 2, Year Book Medical, Chicago, 1972. 54. Leininger, R. I., Crit. Rev. Bioeng. (1972) 1, 333. 55. Baier, R. E., Lippes, J., ADVAN. CHEM. SER. (1975) 145, 308. 56. Glantz, P., Odontol. Revy (1969) Suppl. 17. 57. Glantz, P., Hansson, L., Odontol. Revy (1972) 23, 205. 58. Lasslo, Α., Quintana, R. P., Eds., "Surface Chemistry and Dental Integu ments," C. C. Thomas, Springfield, 1973. 59. Baier, R. E., "Surface Chemistry and Dental Integuments," A. Lasslo and R. P. Quintana, Eds., Chap. 5, C. C. Thomas, Springfield, 1973. 60. Quintana, R. P., ADVAN. CHEM. SER. (1975) 145, 290. 61. Marshall, K. C., Stout, R., Mitchell, R., J. Gen. Microbiol. (1971) 68, 337. In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.


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62. Corpe, W. Α., "Adhesion in Biological Systems," R. S. Manly, Ed., Chap. 4, Academic, New York, 1970. 63. Marshall, K. C., Proc. 3rd Intern. Cong. Marine Corrosion and Fouling, p. 625, Northwestern University Press, Evanston, 1973. 64. Goupil, D. W., DePalma, V. Α., Baier, R. E., Proc. Marine Technol. Soc., 9th Ann. Conf., Washington, D.C., 1973, p. 445. 65. Baier, R. E., Proc. 3rd Intern. Cong. Marine Corrosion and Fouling, p. 633, Northwestern University Press, Evanston, 1973. 66. Cook, M., Baier, R. E., Sargent, H., Proc. 21st Mtg. Brit. Div. IADR, 1973, Abst. 116. 67. Cook, M., "Adhesion in Biological Systems," R. S. Manly, Ed., Chap. 8, Academic, New York, 1970. 68. Crisp, D. J., Ryland, J. S., Proc. Roy. Soc. London (1963) 158B, 364. 69. Fenstermaker, C. Α., Grant, W. H., Morrissey, B. W., Smith, L. E., Stromberg, R. R., "Interaction of Plasma Proteins with Surfaces," Nat. Bur. Stand. Rep. (1974) 74-470. 70. Lee, R. G., Adamson, C., Kim, S. W., Thromb. Res. (1974) 4. 71. Loeb, G. I., J. Colloid Interface Sci. (1969) 31, 572. 72. Nyilas, E., Proc. 23rd Ann. Conf. Eng. Med. Biol. (1970) 12, 147. 73. Andrade, J. D., Med. Instrum. (1973) 7 ,110. 74. Malcolm, B. R., Progr. Surface Membrane Sci. (1973) 7, 183. 75. Loeb, G. I., J. Colloid Interface Sci. (1968) 26, 236. 76. Loeb, G. I., Baier, R. E., J. Colloid Interface Sci. (1968) 27, 38. 77. Loeb, G. I., Amer. Chem. Soc., Div. Polym. Chem., Prepr. 11, 1313 (Chi cago, September, 1970). 78. Baier, R. E., Proc. 13th Conf. Great Lakes Res., Ann Arbor, Michigan (1970) p. 114. 79. Baier, R. E., J. Geophys. Res. (1972) 77, 5062. 80. Baier, R. E., Goupil, D. W., Bull. Union Oceanogr. France (1973), A 1. 81. Baier, R. E., Goupil, D. W., Perlmutter, S., King, R. W., J. Rech. Atmos., in press. 82. Abe, T., Rec. Oceanogr. Works Jap. (1954) 1, 18. 83. Abe, T., J. Oceanogr. Soc. Jap. (1962) 20, 242. 84. Baier, R. E., Aust. Nat. Hist. (1972) 17, 162. 85. Goldacre, R. D., J. Anim. Ecol. (1949) 18, 36. 86. Baier, R. E., Shafrin, E. G., Zisman, W. Α., Science (1968) 162, 1360. RECEIVED

June 28, 1974.


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