Applied Chemistry at Protein Interfaces

7 Wetting Properties of Collagen and

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Gelatin Surfaces R. E. BAIER1 and W. A. ZISMAN U.S. Naval Research Laboratory, Washington, D.C. 20375 Collagenous proteins dominate the organic matrix of connective tissue and teeth, so their wetting properties are important for surgical and dental adhesives. Gelatin, a randomized collagen product, is used extensively in interfacial applications. Collagen maintains a bound layer of water under all practical conditions which prevents complete spreading of low surface tension, organic liquids. Higher surface tension liquids wet collagen surfaces as they would simpler organic materials with critical surface tensions of about 40 dynes/cm. Gelatin exhibits even stronger interfacial interactions with hydrogen-bonding liquids as a distinct class. The combination of a high critical surface tension and a separate contact angle pattern for hydrogen-bonding liquids provides evidence of accessible amide groups at the surfaces of polyamide materials.

l t h o u g h proteins are e x t r a o r d i n a r i l y c o m p l e x , h i g h m o l e c u l a r w e i g h t p o l y m e r s , a c o m m o n feature is t h e i r p o l y a m i d e b a c k b o n e c h a i n . T h e surface p r o p e r t i e s of specific p r o t e i n s a n d t h e i r p o t e n t i a l r a n g e of i n t e r f a c i a l b e h a v i o r are i m p o r t a n t c o m m e r c i a l l y a n d are also of b i o l o g i c a l interest. W o r k at the N a v a l R e s e a r c h L a b o r a t o r y d u r i n g t h e last 30 years has d e m o n s t r a t e d the v a l u e of contact angle measurements i n assessing the surface characteristics of s i m p l e r p o l y m e r films; therefore, this series of experiments was i n i t i a t e d i n o r d e r to l a y a f o u n d a t i o n for e v e n t u a l r e l i a b l e i n t e r p r e t a t i o n of contact angle d a t a f o r p r o t e i n surfaces. O u r earlier reports (1, 2) c o n s i d e r e d t h e n a t u r e o f the c o n t r i b u t i o n to w e t t i n g b e h a v i o r of the c o m m o n p o l y a m i d e b a c k b o n e chains. It was 1

Present address: Calspan Corp., P.O. Box 235, Buffalo, N.Y. 14221. 155 In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

156

APPLIED CHEMISTRY AT PROTEIN

INTERFACES

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

wet

differently b y h y d r o g e n - b o n d i n g a n d 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

(2).

I n v e s t i g a t i o n of p o l y a m i d e s of the n y l o n f a m i l y (2, 3)

established that

s u c h w e t t a b i l i t y differences reflect a s p e c i a l i n t e r a c t i o n b e t w e e n 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 the surface-accessible a m i d e l i n k s of t h e p o l y m e r c h a i n . T h e d i a g n o s t i c v a l u e of this feature w a s e x p l o r e d w i t h t h e m o r e c o m p l e x p o l y p e p t i d e p o l y ( m e t h y l g l u t a m a t e ) ( P M G ) (1, 4);

it was dis

c o v e r e d that the a c c e s s i b i l i t y of the h y d r o g e n - b o n d i n g p o l y a m i d e b a c k Downloaded by UNIV OF MARYLAND COLL PARK on October 15, 2014 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch007

b o n e at the surface o f the s o l i d p o l y m e r w a s a f u n c t i o n of the m o l e c u l a r c o n f i g u r a t i o n as w e l l as the c h e m i c a l c o n s t i t u t i o n of the m a t e r i a l .

The

p o l y a m i d e b a c k b o n e was effectively m a s k e d f r o m t h e interface b y the a r r a y of m e t h y l ester side chains t h r u s t u p p e r m o s t

w h e n P M G was

f o r c e d b y solvent changes i n t o the e x t e n d e d c h a i n , b e t a c o n f o r m a t i o n . W h e n P M G w a s cast i n t o films w i t h a c o i l e d a l p h a h e l i c a l structure or a r a n d o m t a n g l e of c h a i n s , the contact angles s i g n a l e d the a v a i l a b i l i t y of the p o l y a m i d e b a c k b o n e to Η - b o n d i n g at the s o l i d - g a s interface. T h e s e investigations w e r e a i d e d s u b s t a n t i a l l y b y use of the m u l t i p l e a t t e n u a t e d i n t e r n a l reflection ( M A I R ) spectroscopic t e c h n i q u e to r e c o r d surface specific, i n f r a r e d ( I R )

spectra i n o r d e r to d e t e r m i n e structure

a n d m o n i t o r p r e p a r a t i o n a n d c l e a n i n g methods. I n this r e p o r t , these concepts are a p p l i e d to r e a l p r o t e i n s : to c o l l a g e n , a n i m p o r t a n t s t r u c t u r a l m a t e r i a l i n tendons, bones, teeth, a n d s k i n , a n d to g e l a t i n , the d e n a t u r e d p r o d u c t of c o l l a g e n t h a t is so i m p o r t a n t industrially.

T h e s e m a t e r i a l s are c o m p l e x b e c a u s e of t h e i r 18 different,

c o m p o n e n t a m i n o a c i d side c h a i n s ; i n a d d i t i o n , t h e y present e x p e r i m e n t a l difficulties because of t h e i r w a t e r s o l u b i l i t y — t h e y cannot b e

washed

(e.g., w i t h a n aqueous d e t e r g e n t ) to assure surface cleanliness. F u r t h e r m o r e , t h e y are often of u n k n o w n p u r i t y .

T h e y d o h a v e the

common

p o l y a m i d e b a c k b o n e , a n d it is possible to t r a n s f o r m the m o l e c u l a r c o n figuration.

T h e d a t a a r e i n d i c a t i v e of the p o t e n t i a l u t i l i t y of contact a n g l e

measurements o f i m p o r t a n t , n a t u r a l m a t e r i a l s .

N o c l a i m is m a d e

for

a d e q u a t e a t t e n t i o n to the c o m p l e x b i o c h e m i s t r y of these m a t e r i a l s . O u r t e n t a t i v e conclusions are as f o l l o w s .

Purified collagen prepara

tions present no e v i d e n c e for the a v a i l a b i l i t y of Η - b o n d i n g p o l y a m i d e l i n k s at the surface of s o l i d films, b u t i n c r e a s i n g l y d e n a t u r e d (i.e.

ran

d o m i z e d ) gelatins d o reflect the stronger i n t e r a c t i o n of the p o l y m e r w i t h the Η - b o n d i n g w e t t i n g l i q u i d s . I n a d d i t i o n , c o l l a g e n a n d g e l a t i n e v i d e n c e anomalous,

nonspreading behavior w i t h

low

surface

tension,

organic

l i q u i d s o n t h e i r surfaces; this is a t t r i b u t e d to the presence of s t r o n g l y a d s o r b e d w a t e r at t h e i r surfaces. T h i s a t t r i b u t i o n is b a s e d o n experiments w i t h water-swollen material a n d w i t h synthetic polyacrylamide, a n d on w o r k (5, 6) o n other i n t r i n s i c a l l y h i g h surface energy m a t e r i a l s l i k e glass and- metals,.

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

7.

B A i E R A N D zisMAN

Collagen

and

Collagen

and Gelatin

157

Surfaces

Gelatin

W i l k i e s u m m a r i z e d the i m p o r t a n t i n t e r f a c i a l p r o p e r t i e s of g e l a t i n ( 7 ) , a n d S t e n z e l a n d co-workers p r e p a r e d a s i m i l a r r e v i e w of the features of c o l l a g e n w h i c h u n d e r l i e that protein's f u n d a m e n t a l i m p o r t a n c e as a biomaterial

(8,9).

C o l l a g e n is the most a b u n d a n t p r o t e i n i n a n i m a l s . I n fact, o n e - t h i r d of a l l the p r o t e i n i n m a m m a l s is c o l l a g e n . C o l l a g e n ' s p h y s i c a l properties Downloaded by UNIV OF MARYLAND COLL PARK on October 15, 2014 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch007

of inertness a n d s t r e n g t h enable i t to f u n c t i o n as a p r o t e c t i v e a n d s u p p o r t i n g structure i n s k i n , t e n d o n , a n d c a r t i l a g e .

C o l l a g e n is also the

o r g a n i c c o m p o n e n t of teeth a n d bone. C o l l a g e n , l i k e a l l proteins, is m a d e u p of α-amino acids c o n n e c t e d i n p e p t i d e sequence. T h e structure of c o l l a g e n , as d e t e r m i n e d b y its x - r a y d i f f r a c t i o n p a t t e r n , consists of three p o l y p e p t i d e chains e a c h of w h i c h has a h e l i c a l structure. T h e three helices c o i l a r o u n d one another i n a g r a d u a l , r i g h t - h a n d e d h e l i x to f o r m m o l e c u l a r c o l l a g e n k n o w n as t r o p o c o l l a g e n . A t r o p o c o l l a g e n m o l e c u l e is a b o u t 14 A i n d i a m e t e r a n d 2800 A l o n g w i t h a m o l e c u l a r w e i g h t of a b o u t 300,000 (10).

C o l l a g e n represents a p a r

t i c u l a r a g g r e g a t i o n state of c o n s t i t u e n t t r o p o c o l l a g e n m o l e c u l e s .

Non-

h e l i c a l a p p e n d a g e s d e t e r m i n e the m o l e c u l a r interactions a n d i m m u n o l o g i c properties of c o l l a g e n

(11).

T h e a m i n o a c i d content of c o l l a g e n is o n e - t h i r d g l y c i n e , o n e - t h i r d c y c l i c , a n d one-fifth d i b a s i c or d i c a r b o x y l i c a m i n o a c i d s ; a s m a l l p r o p o r t i o n of o l i g o s a c c h a r i d e groups is also present.

T h i s u n u s u a l b l e n d no

d o u b t accounts for the e x t r a o r d i n a r y p h y s i c a l properties of c o l l a g e n .

The

g l y c i n e content is u n u s u a l l y h i g h , a n d that of the a r o m a t i c a n d s u l f u r c o n t a i n i n g a m i n o acids is l o w .

C o l l a g e n is u n u s u a l a m o n g proteins i n

h a v i n g a h i g h content of the a m i n o acids p r o l i n e a n d h y d r o x y p r o l i n e . I n fact, c o l l a g e n has the highest c o n c e n t r a t i o n of p r o l i n e of a n y k n o w n p r o t e i n , a n d it is the o n l y p r o t e i n w h i c h contains b o t h h y d r o x y p r o l i n e and hydroxyglycine.

T h e compositions of v a r i o u s c o l l a g e n a n d g e l a t i n

specimens are s u m m a r i z e d i n T a b l e I

(12-33).

C o l l a g e n is classified as a fibrous or s c l e r o p r o t e i n . T h e fibrous s t r u c t u r e of s o l i d c o l l a g e n c a n be d i v i d e d into v e r y s m a l l fibrils w i t h a p e r i o d i c i t y of a b o u t 640 A d e p e n d i n g o n the degree of h y d r a t i o n (24).

Native

c o l l a g e n fiber is b u i l t u p b y a h i g h l y o r d e r e d process of l i n e a r a n d l a t e r a l a g g r e g a t i o n o f the t h i n , h i g h l y e l o n g a t e d t r o p o c o l l a g e n m o l e c u l e s .

Three

types of fibrous c o l l a g e n constitute the p r o t e i n c o m p o n e n t s of c o n n e c t i v e tissue. C o l l a g e n is i n s o l u b l e i n w a t e r , b u t 0 . 5 - 3 % of c o n n e c t i v e tissue c o l l a g e n is either n e u t r a l salt s o l u b l e or a c i d soluble.

T h e remainder,

i n s o l u b l e c o l l a g e n , c a n b e almost c o m p l e t e l y s o l u b i l i z e d b y treatment w i t h p r o t e o l y t i c enzymes w i t h o u t d e s t r o y i n g its n a t i v e m o l e c u l a r s t r u c ture (25).

E a c h c o l l a g e n m o l e c u l e contains three s u b u n i t s or chains.


158

APPLIED

Table I .

F r o m References

N e w l y formed

INTERFACES

Content, wt %

Acid

5.9-11.6 4.6-9.3 4.4 - 7.0 0 - 0.1 7.0-12.4 20.2-33.5 0.4-1.0 0.4-2.7 9.3-15.7 1.1-1.9 2.3-4.0 2.5-5.6 0.4-1.0 1.2-3.6 12.4-18.0 2.9-4.2 1.5-2.4 0.0 0.1-1.1 2.0-3.3

Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Hydroxylysine Hydroxyproline Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine a

AT PROTEIN

Amino A c i d Composition of Collagens and Gelatins °

Amino


CHEMISTRY

12-23.

c o l l a g e n e x t r a c t e d w i t h c o l d , aqueous

consists of three e q u a l - s i z e d chains c o m p o s i t i o n types (a-1 a n d a - 2 ) .

(α-components)

NaCl

solutions

of t w o

different

T h e t w o chains of s i m i l a r c o m p o s i t i o n

are the a-1 chains. T h e a-2 c h a i n differs f r o m the a-1 i n a n u m b e r of a m i n o acids, p a r t i c u l a r l y h y d r o x y p r o l i n e , p r o l i n e , l y s i n e , a n d h i s t i d i n e (26).

A s the c o l l a g e n m o l e c u l e m a t u r e s , the α-chains c r o s s l i n k i n t r a m o -

l e c u l a r l y i n p a i r s ; this o l d e r p r o t e i n c a n b e r e a d i l y extracted w i t h a c i d i c solutions s u c h as d i l u t e acetate a n d c i t r a t e buffer, b u t n o t w i t h salt s o l u tions. T h e c r o s s l i n k e d c h a i n s are c a l l e d β c o m p o n e n t s ; the crosslinks are p r o b a b l y covalent bonds

(26)

that arise b y c o n d e n s a t i o n of t h e side

chains of strategic l y s y l residues after e n z y m a t i c o x i d a t i v e d e a m i n a t i o n . O l d e r collagen* also forms i n t e r m o l e c u l a r b o n d s , b u t the n a t u r e of this crosslink has not yet b e e n d e t e r m i n e d

(27).

O n e of the most i m p o r t a n t a n d u s e f u l p r o d u c t s d e r i v e d f r o m c o l l a g e n is g e l a t i n . G e l a t i n is a c o l l e c t i v e n a m e c o v e r i n g a w i d e r a n g e of m a t e r i a l s that are d i s t i n g u i s h e d b y g o o d c l a r i t y a n d the a b i l i t y to f o r m t o u g h gels at r e l a t i v e l y l o w concentrations (18).

G e l a t i n is d e r i v e d f r o m c o l l a g e n

b y irreversible hydrolytic procedures.

T h e properties of a g e l a t i n s a m p l e

d e p e n d o n p H , electrolytes present, the c o l l a g e n source, the m e t h o d of m a n u f a c t u r e , a n d its t h e r m a l h i s t o r y , a g i n g , a n d c o n c e n t r a t i o n . P u r e , d r y


7.

BAiER A N D ziSMAN

Collagen

and Gelatin

159

Surfaces

c o m m e r c i a l g e l a t i n is tasteless, odorless, t r a n s p a r e n t , b r i t t l e , glasslike, a n d v e r y f a i n t y e l l o w to a m b e r i n color. Y e t g e l a t i n is e x t r e m e l y h e t e r o geneous—composed

of

many-sized polypeptides

(18).

Its

molecular

w e i g h t ranges f r o m 15,000 to several h u n d r e d t h o u s a n d , reflecting t h e degree of h y d r o l y s i s of t h e h i g h e r m o l e c u l a r w e i g h t c o l l a g e n material.

source

S o u r c e c o l l a g e n a n d p r o d u c t g e l a t i n differ l i t t l e since g e l a t i n

has the same a m i n o a c i d c o m p o s i t i o n as t h e c o l l a g e n f r o m w h i c h i t w a s derived.

Differences

among

gelatins of d i v e r s e origins a p p e a r to

be


r e l a t e d to t h e d e g r e e of d e g r a d a t i o n of the p a r e n t c o l l a g e n . C r y s t a l l i t e s of c o l l a g e n l i k e s t r u c t u r e m a y f o r m after the g e l a t i o n of gelatin.

W h e n g e l a t i n is d r i e d d o w n f r o m a g e l ( g e l - d r i e d ) , its w i d e

a n g l e x - r a y d i f f r a c t i o n p a t t e r n is s i m i l a r to t h a t of c o l l a g e n w h e r e a s , w h e n g e l a t i n is d r i e d f r o m a w a r m s o l u t i o n ( s o l - d r i e d ) , there is no e v i d e n c e of c r y s t a l l i n i t y (24).

A s a result, t h e tensile strength of g e l - d r i e d layers

is t w i c e that of s o l - d r i e d layers. F u r t h e r m o r e , the I R a b s o r p t i o n of g e l d r i e d layers is s i m i l a r to that of c o l l a g e n

(24).

I n the process of p r o d u c i n g g e l a t i n f r o m c o l l a g e n , the c o l l a g e n coils are s p l i t a p a r t b o t h l a t e r a l l y a n d l o n g i t u d i n a l l y i n t o separate strands a n d g r o u p s of strands (21).

T a n g l e d p r o t e i n strands are r a v e l l e d o r s n i p p e d

short, or are present as a diverse c o m b i n a t i o n of r a v e l l e d structures; g e l a t i n is therefore d e s c r i b e d as b e i n g r a n d o m l y c o i l e d .

T h e r e are t w o

p r i n c i p a l m e t h o d s for m a n u f a c t u r i n g g e l a t i n . I n one, g e l a t i n is d e r i v e d f r o m a n a c i d - p r o c e s s e d c o l l a g e n stock, p r i m a r i l y p i g s k i n .

I n t h e other,

c o l l a g e n stock ( s u c h as calf, c a t t l e , a n d w a t e r buffalo hides a n d d e m i n e r a l i z e d b o n e ) is treated w i t h l i m e . T h e l i m e treatment m e t h o d , u s e d e s p e c i a l l y to m a n u f a c t u r e p h o t o g r a p h i c g e l a t i n , i n v o l v e s the f o l l o w i n g steps (24):

( a ) c o l l a g e n stock is

treated w i t h a l i m e s l u r r y at 1 0 ° - 2 0 ° C for weeks or m o n t h s ; ( b ) the stock is w a s h e d a n d n e u t r a l i z e d w i t h a c i d ; a n d t h e n ( c ) the g e l a t i n is extracted w i t h w a r m w a t e r at n e u t r a l or s l i g h t l y a c i d p H u s i n g successively h i g h e r temperatures i n a series of cooks. T h e l i m e keeps the p H at a b o u t 12 a n d h y d r o l y z e s some of t h e p e p t i d e b o n d s . amide

groups

of

glutamine and

a s p a r t i c a c i d residues

It also h y d r o l y z e s the side c h a i n

asparagine

yielding glutamine

and

(24).

T h e a c i d process of g e l a t i n p r e p a r a t i o n is as f o l l o w s (21):

(a)

the

h i d e s , u s u a l l y p i g , are w a s h e d w i t h w a t e r a n d soaked i n d i l u t e s u l f u r i c acid ( p H ~ 2 ; (b) (c)

t h e y are w a s h e d free of a c i d a n d s o l u b l e p r o t e i n s ;

they are p l a c e d i n e x t r a c t i o n kettles a n d h y d r o l y z e d w i t h successive

portions of hot w a t e r at a n a c i d p H ; ( d ) and

evaporated; (e)

the d i l u t e s o l u t i o n is

filtered

c o n c e n t r a t e d solutions are c h i l l e d to a g e l ; a n d

(f)

the g e l is d r i e d w i t h filtered a n d c o n d i t i o n e d a i r i n d r y i n g tunnels or i n continuous driers.


160


INTERFACES

T h e a c i d d e g r a d a t i o n m e t h o d is m u c h faster t h a n l i m e t r e a t m e n t since the a c i d - s o a k i n g r e q u i r e s o n l y a f e w hours. T h e r e are, h o w e v e r , a n u m b e r of i m p o r t a n t p h y s i c a l differences derived gelatin.

between

acid- and alkaline-

T h e r e a c t i v e g r o u p sites v a r y i n n u m b e r ,

different p r o p e r t i e s (21).

producing

I m p o r t a n t differences i n s t r u c t u r e are i n d i c a t e d

b y v a r i a t i o n s i n the isoelectric p o i n t ( w h e r e the net c h a r g e of the g e l a t i n is z e r o ) . I n the a c i d process, a m i d e groups of g l u t a m i n e a n d asparagine are n o t h y d r o l y z e d so the g e l a t i n has f e w e r free c a r b o x y l groups t h a n Downloaded by UNIV OF MARYLAND COLL PARK on October 15, 2014 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch007

l i m e - p r o c e s s e d g e l a t i n a n d , therefore, a h i g h e r isoelectric p o i n t (24). isoelectric p o i n t of a c i d - p r o d u c e d

g e l a t i n is b e t w e e n

p H 7.0 a n d

whereas that of l i m e - t r e a t e d g e l a t i n is b e t w e e n 4.7 a n d 5.1

The 9.0

(21).

N o t w o g e l a t i n p r e p a r a t i o n s w i l l h a v e the same p r o p e r t i e s unless the stock a n d m e t h o d of p r e p a r a t i o n are i d e n t i c a l . T h e g e l p r e p a r e d f r o m e a c h successive extract is less r i g i d t h a n the p r e v i o u s b a t c h ; the v i s c o s i t y m a y increase o r decrease f r o m extract to extract d e p e n d i n g o n the stock used. E i g h t c o m p a n i e s i n t h e U n i t e d States a n n u a l l y p r o d u c e a t o t a l of a p p r o x i m a t e l y 60 m i l l i o n p o u n d s of g e l a t i n . T h e p h o t o g r a p h i c i n d u s t r y exploits almost every p r o p e r t y of g e l a t i n i n c l u d i n g its g e l a t i o n , p r o t e c t i v e c o l l o i d , viscous

flow,

and

film-forming

f u n c t i o n s as w e l l as the p o o r l y

u n d e r s t o o d c h e m i c a l properties l e a d i n g to s e n s i t i z a t i o n (28).

T h e gelatin

l a y e r coats i n d i v i d u a l m i c r o s c o p i c particles of silver b r o m i d e a n d prevents their agglutination and

flocculation;

it also helps to r e g u l a t e the size a n d

g r o w t h of the s i l v e r h a l i d e particles (21).

I n the p a p e r i n d u s t r y , the

tensile strength of t h i n g e l a t i n films increases the b u r s t i n g strength of p a p e r . P a p e r c a n accept i n k w i t h o u t s m u d g i n g because of gelatin's h y d r o p h i l i c n a t u r e a n d i n k c a n be r e m o v e d f r o m l e d g e r p a p e r w i t h a p e n k n i f e because of gelatin's brittleness (28). film-forming

I n the textile i n d u s t r y , t h e adhesive

properties of g e l a t i n strengthen w a r p fibers a n d p r e v e n t the

s h e d d i n g of m i n u t e filaments d u r i n g w e a v i n g . G e l a t i n g l u e forms a strong t a c k y g e l o n c o o l i n g since its p o l a r groups w e t m a n y surfaces a n d its l o n g c h a i n m o l e c u l a r structure p r o v i d e s a t o u g h joint. Materials

and Methods

T h e p r i m a r y e x p e r i m e n t a l m a t e r i a l was a s a m p l e of w a t e r - s o l u b l e c o l l a g e n a c i d - e x t r a c t e d f r o m rat s k i n (29) that w a s d o n a t e d b y K a r l A . P i e z , N a t i o n a l Institute of D e n t i s t r y . T h e o r i g i n a l s a m p l e was a d r y , p u r e w h i t e , fibrous, c o t t o n l i k e mass t h a t h a d b e e n d e h y d r a t e d b y f r e e z e - d r y i n g . T h e c o l l a g e n s a m p l e was u l t r a c e n t r i f u g a l l y h o m o g e n e o u s ( F i g u r e 1 A ) ; i t s e d i m e n t e d w i t h a single, h y p e r s h a r p p e a k a n d h a d a s e d i m e n t a t i o n co efficient of 2.82 χ 10" . S o l u t i o n of the s a m p l e i n w a t e r w a r m e r t h a n 40 ° C l e d to the f o r m a t i o n of a m i l d l y d e n a t u r e d c o l l a g e n f o r m , d e s c r i b e d here as g e l a t i n , w h i c h also s e d i m e n t e d w i t h a single p e a k ( F i g u r e I B ) . 13



7.

BAIER A N D ZISMAN

Collagen

and Gelatin

Surfaces

161

Figure 1. Ultracentrifuge Schlieren photographs of sedimentation behavior of highly purified rat skin collagen and its mildly gelatinized product at corre sponding times Top, hypersharp sedimentation peak for 0.15% solution of rat skin collagen in water (sedimentation coefficient = 2.82 X 10~ ); and bottom, broader sedimentation peak for 0.16% solution of rat skin collagen in water after heating to 40°C (sedimentation coefficient = 2.35 Χ 10 ) n

13

T h e g e l a t i n p e a k w a s c o n s i d e r a b l y b r o a d e r , a n d the s e d i m e n t a t i o n coeffi cient was lower—2.35 Χ 10" . 13

A second c o l l a g e n s a m p l e w a s b o v i n e A c h i l l e s t e n d o n c o l l a g e n ( S c h w a r t z M a n n , I n c . ) . T h i s m a t e r i a l resisted s o l v a t i o n i n a l l s i m p l e aqueous solutions a n d even i n strong acids, b u t it c o u l d b e s o l u b i l i z e d w i t h t i m e b y d i c h l o r o a c e t i c a c i d ( D C A ) . T h u s , films f o r m e d f r o m this s a m p l e h a d to be cast f r o m D C A . E x c e p t for a c o m m e r c i a l s a m p l e of K n o x g e l a t i n , a l l g e l a t i n samples w e r e o b t a i n e d f r o m F i s h e r Scientific C o r p . : 1099, g e l a t i n ( p u r i f i e d calf s k i n ) ; 1099-P, g e l a t i n ( p r a c t i c a l ) ; 5247, g e l a t i n ( p u r i f i e d p i g s k i n ) ; G - 5 , white gelatin (silver l a b e l ) ; and bacteriological gelatin. A l l films p r e p a r e d for contact angle studies w e r e cast f r o m a n excess of d r y p o l y m e r s w o l l e n a n d fluidized b y a s m a l l q u a n t i t y of a d d e d l i q u i d ( u s u a l l y t r i p l e - d i s t i l l e d w a t e r , the last t w o distillations b e i n g i n a n a l l q u a r t z a p p a r a t u s ) . T h e p H of these w e a k gels at the i n i t i a t i o n of film f o r m a t i o n was a l w a y s 5 - 6 . A l l o u r surface films w e r e g e l - d r i e d r a t h e r t h a n s o l - d r i e d . A l t h o u g h s w e l l i n g m a y not h a v e r e a c h e d e q u i l i b r i u m p r i o r to film d r y i n g , the films w e r e o n l y a b o u t 1 m m t h i c k a n d t h e i r surface p r o p e r t i e s , as d e t e r m i n e d b y contact angle measurements, w e r e always reproducible w i t h i n 5°. T h i n films of t h e c o l l a g e n a n d g e l a t i n samples w e r e f o r m e d o n f r e s h l y flamed p l a t i n u m sheets b y the d r o p s p r e a d i n g m e t h o d ( 1 ). A f e w grains


162

APPLIED CHEMISTRY

Table II.

AT PROTEIN

INTERFACES

Wettability of Purified Rat Skin Collagen Average Contact Angle,"


Wetting

Liquid

Water Glycerol Formamide Thiodigylcol M e t h y l e n e iodide s^ra-Tetrabromoethane 1-Bromonaphthalene o-Dibromobenzene 1- M e t h y lnaphthalene Dicyclohexyl n-Hexadecane n-Decane

Surface Tension at 20°C, dynes/cm

Film Formed at 20°C»

WaterSwollen 20°C Film

72.8 63.4 58.2 54.0 50.8 47.5 44.6 42.0 38.7 33.0 27.7 23.9

92 79 81 54 48 43 35 34 30 16 0 0

90 89 85 97 68 69 58 50 33 19 11 0

0

degrees Film Formed at80°C d

90 70 65 48 48 46 38 35 29 12 0 0

° Averages of at least 10 readings on at least two independently prepared films of each type. Drop spread from distilled water on platinum sheet and air dried in grease free container at 20°C. Drop-spread film swollen with distilled water. Drop spread from distilled water at 80°C (i.e. gelatinized) and air dried at 80°C in greasefree container. b

c

d

of d r y p o l y m e r are p l a c e d i n the center of a freshly flamed b u t c o o l p l a t i n u m p l a t e a n d a n a p p r o p r i a t e solvent is t r a n s f e r r e d to this s a m p l e d r o p w i s e w i t h a freshly flamed, b u t c o o l p l a t i n u m w i r e . T h e solvent drops a n d t h e i r d i s s o l v e d p o l y m e r b u r d e n s p r e a d spontaneously o v e r the h i g h e n e r g y m e t a l p l a t e , a n d , after s l o w a i r d r y i n g i n a c o v e r e d , greasefree container, a s p e c u l a r l y s m o o t h film is left o v e r most of the flat p l a t e surface a n d r e m n a n t s of t h e s o l i d p o l y m e r i n t h e p l a t e center. T h i s t e c h n i q u e has t h e o b v i o u s benefits of s i m p l i c i t y a n d g e n e r a l a p p l i c a b i l i t y , a n d it requires o n l y s m a l l amounts of t h e sometimes scarce p o l y m e r i c m a t e r i a l . I n a d d i t i o n , the t h i n films e q u i l i b r a t e most q u i c k l y w i t h t h e r o o m e n v i r o n m e n t , v a r i o u s test h u m i d i t i e s , a n d s w e l l i n g l i q u i d s s u c h as w a t e r . M A I R s p e c t r o s c o p y at I R w a v e l e n g t h s w a s u s e d to i d e n t i f y t h e p o l y m e r s , m o n i t o r a n y i n d u c e d s t r u c t u r a l transformations after the v a r i o u s treatments, a n d v e r i f y the f r e e d o m of t h e t h i n film f r o m r e s i d u a l t r a p p e d solvent or a d v e n t i t i o u s c o n t a m i n a n t s (1,2). A m o d e l 9 i n t e r n a l reflection accessory ( W i l k s Scientific C o r p . ) w a s u s e d i n c o n j u n c t i o n w i t h B e c k m a n I R - 1 2 a n d I R - 7 a n d P e r k i n - E l m e r 2 1 , 257, a n d 457 I R spectrometers to r e c o r d reflection spectra of t h e i m m e d i a t e interface of the s a m p l e w h i c h w a s c l a m p e d against the m u l t i p l e i n t e r n a l reflection p r i s m s m a d e f r o m the t h a l l i u m b r o m i d e salt K R S - 5 . S l o w l y a d v a n c i n g contact angles w e r e d e t e r m i n e d w i t h p u r e reference l i q u i d s p l a c e d d r o p w i s e o n e a c h s p e c i m e n surface (1, 2, 3); these liquids included hydrogen-bonding and nonhydrogen-bonding compounds w i t h a w i d e r a n g e of surface tension a n d s t r u c t u r a l t y p e . E a c h c i t e d



7.

A N D ziSMAN

BAiER

Collagen

and Gelatin

163

Surfaces

v a l u e f o r contact angle is t h e average v a l u e r e c o r d e d r e p r o d u c i b l y w i t h i n the first 1 0 - 2 0 sec after t h e d r o p w a s s l o w l y a d v a n c e d over a fresh s u r face r e g i o n . W i t h w a t e r , f o r m a m i d e , a n d ethylene g l y c o l , t h e contact angles sometimes c h a n g e d r a p i d l y after this i n t e r v a l as t h e result o f g r a d u a l p e n e t r a t i o n of t h e l i q u i d into t h e p l a s t i c s o l i d . W i t h g l y c e r o l , the contact angles also c h a n g e d i n some experiments, b u t m u c h m o r e s l o w l y . W i t h t h i o d i g l y c o l , t h e contact angles w e r e often constant f o r m a n y m i n u t e s b e f o r e t h e y also b e g a n to d i m i n i s h . T h e contact angles measured w i t h the nonhydrogen-bonding organic liquids were generally constant f o r m a n y m i n u t e s , a n d u s u a l l y there w a s almost n o hysteresis w h e n r e c e d i n g angles w e r e m e a s u r e d . A l l d a t a w e r e r e c o r d e d w i t h s a m ples a n d l i q u i d s e q u i l i b r a t e d i n a c l e a n r o o m m a i n t a i n e d a t 20 ° C a n d 5 0 % r e l a t i v e h u m i d i t y unless specifically stated otherwise. Results A v e r a g e contact angle values f o r v a r i o u s h i g h l y p u r i f i e d d i a g n o s t i c l i q u i d s o n films p r e p a r e d f r o m p u r i f i e d r a t s k i n c o l l a g e n a r e p r e s e n t e d i n T a b l e I I . C o n t a c t angles w e r e m e a s u r e d w i t h the films ( a ) d r o p s p r e a d f r o m d i s t i l l e d w a t e r o n a p l a t i n u m sheet a n d a i r d r i e d t o a s p e c u l a r l y s m o o t h c o n t i n u o u s film i n a greasefree c o n t a i n e r a t a t e m p e r a t u r e t h a t never e x c e e d e d 2 0 ° C ; ( b ) p r e p a r e d as i n M e t h o d a b u t t h e n r e s w o l l e n w i t h d i s t i l l e d w a t e r f o r at least 1 h r a n d m e a s u r e d w h i l e s t i l l c o m p l e t e l y water swollen i n e q u i l i b r i u m w i t h relative humidities of 5 0 % SURFACE TENSION (y + 1.0 0.9 0.8 0.7 CD

UJ 0.6 ζ < /> g+0.5 Ο Ο

0.4 0.3 0.2 +0.1

I

10

1

15 20

25 ^30

1

ι1

-

ι ι LI' , III' ι in' 11

H

35

L!

) IN DYNES/CM

40 45

-

1 1 1 1 1 1 1 1

1

1

Il '

1

1

! ! 1!

%

ι\1 !i 1 IT li

I IΙ I Μι' ι

ι

1 \#

1 1 1 πV 1 ,l ι l ! H\ f ι Ί 1

v

60 65

III' I1

ι

1

I I I I I

ι I

ι 1 \ ι \! 1 1 \ 1 1 I \

ι

70 75

1

1

_

1ι

ι

ι ι

ι , ,

!

ι

ι 1

1

!

ι ί ι

+1.0 0.9

0.8

_

0.7 CD

0.6 UJ ζ

ι — (/> , +0.5 ο ο Ί ι 0.4 ! ι 0.3

!! Ii RAT SKIN COLLAGIENι |FM ILMS ι I AT 50% R.H., APPARENTLY DRY I 1 \ I ' 0.2 1 1 1 •™h • i • WATER-SWOLLEN t\ : 1 I I ι ! ϋ ! !j I ι \ 1 +0.1 ι njl 1 I1 1 ! \! .'.!! i !! 1 1 . ι Ιι,ι ι I li II 1 1 1 1 5011 55 !60 I ι65 ι 70 l\ ^75 L ι 0 10 15 20 25 30 35 1401 145 SURFACE TENSION (/ ) IN DYNES/CM ;

;

1

—

!'

• \ —;—rr \ '

« m

-

50 55

'\| | " i 1 l! 1\ I υ 1 * ι\ Ί 1 1 1 \ |! ^ 1 1

!>

and>99%

1

1

•

-

1 1

1

1

I 5

0

Lv

Figure 2.

Plot of contact angle data for rat skin collagen films at 50% rehtive humidity and when completely water swollen


164

APPLIED CHEMISTRY

A T PROTEIN

INTERFACES

1.0 0.9 0.8

ι I 0.7


j 0.6 ! 0.5 ,0.4

ι ; Q3 0.2 .1

MATERIAL:

GELATIN (DENATURED COLLAGEN) FORM: THIN FILM CAST ON PLATINUM SHEET FROM HOT DISTILLED WATER

.NOTE: VERTICAL LINES REPRESENT SPREAD OF DATA FROM ABOUT TWENTY MEASUREMENTS PER POINT 50/;RH 20°C

I

ι

30

0.0

Figure 3.

I

ι J

ι

I

40 50 60 SURFACE TENSION IN DYNES/CM

Plot of contact angle data for mildly rat skin collagen

ι

70

gelatinized

( n o significant differences w e r e o b s e r v e d at these t w o h u m i d i t i e s ) ; a n d (c) d r o p s p r e a d f r o m d i s t i l l e d w a t e r at 80 ° C a n d air d r i e d at 80° C i n a greasefree container.

T h e d a t a f o r t h e 20 ° C d r y a n d w e t c o l l a g e n films a r e

p l o t t e d i n F i g u r e 2. T h e d a t a for films g e l a t i n i z e d b y p r e p a r a t i o n m e t h o d c, w h i c h i n v o l v e d h e a t i n g the g e l a b o v e 40 ° C , are p l o t t e d i n F i g u r e 3; the d a t a s p r e a d w a s m o r e t h a n f o r r e p l i c a t e films of the n a t i v e c o l l a g e n p r e p a r a t i o n . T h e r e w e r e significant differences a m o n g these three types of films f o r m e d f r o m i d e n t i c a l s t a r t i n g m a t e r i a l (see T a b l e I I a n d F i g u r e s 2 a n d 3 ) . T h e a p p a r e n t l y d r y c o l l a g e n cast at l o w t e m p e r a t u r e h a d a c r i t i c a l surface t e n s i o n o f a b o u t 39 d y n e s / c m w h i c h decreased

signifi

c a n t l y to a b o u t 3 2 d y n e s / c m w h e n t h e film w a s t o t a l l y w a t e r s w o l l e n . I n contrast, w h e n the c o l l a g e n w a s m i l d l y r a n d o m i z e d i n the h o t w a t e r c a s t i n g t e c h n i q u e , a l l l i q u i d s c a p a b l e of r e a c t i n g across the interface b y h y d r o g e n b o n d f o r m a t i o n , i n a d d i t i o n to t h e m o r e u n i v e r s a l d i s p e r s i o n force, d i d so react.

D a t a f o r t h e Η - b o n d i n g l i q u i d s f a l l o n a separate

c r i t i c a l surface t e n s i o n l i n e i n d i c a t i n g that the surface free energy sensed b y s u c h l i q u i d s w a s i n c r e a s e d b y t h e r a n d o m i z a t i o n of t h e c o l l a g e n structure.

I n a l l p r e p a r a t i o n s , anomalous n o n s p r e a d i n g of l o w surface

tension, d i s p e r s i o n force o n l y l i q u i d s was n o t e d ; w e i n t e r p r e t this a n o m a l y — a s c o m p a r e d w i t h a l l s i m p l e r p o l y m e r s w e h a v e e x a m i n e d so f a r — a s r e f l e c t i n g the presence of a d s o r b e d a n d o r g a n i z e d w a t e r o n proteinaceous surfaces (see b e l o w ) .


7.

B A i E R A N D ziSMAN

Collagen

and Gelatin

165

Surfaces

W e suggested ( 2 ) that t h i o d i g l y c o l a n d m e t h y l e n e i o d i d e b e u s e d as i n d i c a t o r s o f the a v a i l a b i l i t y o f h y d r o g e n b o n d f o r m i n g groups at the s o l i d surface.

I n the absence o f h y d r o g e n b o n d f o r m a t i o n across t h e

interface, the h i g h e r surface tension l i q u i d s h o u l d p r o d u c e

the l a r g e r

contact a n g l e ; c o n s e q u e n t l y , e q u a l i t y o f the contact a n g l e values w i t h thiodiglycol a n d methylene iodide, or a reversal of their normal order, p r o v i d e s a q u i c k i n d i c a t i o n o f h y d r o g e n b o n d a c c e s s i b i l i t y across a s o l i d l i q u i d interface. B o t h these l i q u i d s h a v e large e n o u g h m o l e c u l a r sizes a n d Downloaded by UNIV OF MARYLAND COLL PARK on October 15, 2014 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch007

p o o r e n o u g h s o l v e n c y p o w e r s to m i n i m i z e p o t e n t i a l c o m p l i c a t i o n s s u c h as p e n e t r a t i o n i n t o the p o l y m e r surface a n d s o l u t i o n o f the p o l y m e r i n the l i q u i d d r o p l e t .

T h e contact angle values m e a s u r e d w i t h h y d r o g e n -

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

been

o m i t t e d f r o m the p l o t i n F i g u r e 2 ; a l l s u c h d a t a w e r e n o n e q u i l i b r i u m measurements o f angles a r o u n d 90° that r e s u l t e d f r o m a m a r k e d i n t e r a c t i o n w i t h the s w o l l e n p o l y m e r i m m e d i a t e l y after p l a c e m e n t o f c e r t a i n d r o p l e t s o n the test s p e c i m e n surfaces. T y p i c a l I R reflectance spectra of films f o r m e d b y c o l d w a t e r c a s t i n g a n d b y h o t w a t e r c a s t i n g are presented i n F i g u r e 4. T h e o n l y d i s c e r n a b l e difference b e t w e e n the n a t i v e a n d the d e n a t u r e d o r g e l a t i n i z e d samples was t h a t the latter m a t e r i a l w a s c h a r a c t e r i z e d b y s l i g h t l y b r o a d e r a b s o r p t i o n b a n d s i n d i c a t i v e o f the presence o f a w i d e r v a r i e t y of m o l e c u l a r c h a i n configurations. C o n t a c t angles w e r e m e a s u r e d w i t h t h e same series o f w e t t i n g l i q u i d s o n the surfaces o f D C A - s p r e a d films of the b o v i n e A c h i l l e s t e n d o n c o l l a g e n sample (Table I I I ) . 5.0

T h e films w e r e o f three t y p e s : ( a ) those MICRONS 6.0

7.0

8.0

9.0 1

10

12

1 1 -

A.

/

\

. \J 2000

1800 1600 1400 WAVENUMBER (CM" )

-

\ γ

1200

1000

800

1

Figure 4. Internal reflection IR spectra of the surface zones of cold water cast rat skin col lagen films (top) and hot water cast (i.e., gela tinized) films from the same original sample (bottom)


formed

166


Table III.

Wettability of Bovine Achilles Tendon Collagen Average Contact


Wetting

INTERFACES

Liquid

Water Glycerol Formamide Thiodiglycol M e t h y l e n e iodide syra-Tetrabromoethane 1-Bromonaphthalene o-Dibromobenzene 1- M e t h y lnaphthalene Dicyclohexyl n-Hexadecane n-Tetradecane n-Tridecane n-Dodecane n-Decane

degrees

Angle

Surface Tension at 20°C, dynes/cm

Film Formed at 20°C from DCA"

20°C Film after Water Wash"

20°C Film Heat Denatured at 80°C

72.8 63.4 58.2 54.0 50.8 47.5 44.6 42.0 38.7 33.0 27.7 26.7 25.9 25.4 23.9

87 80 72 65 60 50 41 39 33 25 14 13 10 0 0

79 76 70 64 51 45 37 35 33 21 11

56 70 60 53 51 47 40 34 30 15 0

d

— —8

— — — —

0

Averages of at least 10 readings on at least two independently prepared films of each type Drop spread from dichloracetic acid on platinum sheet and air dried at 20°C in covered, greasefree container. After extensive water wash and drying. Heat denatured at 80°C in water and redried. a

t

o

b

c

d

at 20 ° C f r o m D C A a n d a i r d r i e d for e x t e n d e d p e r i o d s of t i m e for solvent r e m o v a l ; ( b ) those f o r m e d l i k e T y p e a, t h e n w a s h e d w i t h d i s t i l l e d w a t e r ( w h i c h d i d not s o l u b i l i z e t h e m e v e n t h o u g h t h e y h a d a p p a r e n t l y b e e n a c i d i f i e d ) a n d a g a i n a i r d r i e d ; a n d ( c ) those w h i c h w e r e heat d e n a t u r e d or g e l a t i n i z e d w h i l e w a t e r s w o l l e n b y c o n d u c t i n g the w a t e r - s w e l l i n g a n d d r y i n g processes at 80 ° C .

T h e s e d a t a c o n f i r m the

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

findings

with

T a b l e I a n d F i g u r e s 1, 2

the }

3,

a n d 4 ) . T h e s e d a t a p a r t i c u l a r l y demonstrate the i n c r e a s e d r a n d o m i z a t i o n , as j u d g e d b y a c c e s s i b i l i t y of h y d r o g e n b o n d interactions across t h e i n t e r face, i n the h e a t e d sample. I R spectra w e r e m a d e of the t h i n films of the rat s k i n a n d the b o v i n e A c h i l l e s t e n d o n c o l l a g e n samples a c t u a l l y u s e d for the contact

angle

measurements ( F i g u r e 5 ). T h e c o n s i d e r a b l y greater b r e a d t h of the b a n d s f o r the b o v i n e t e n d o n s a m p l e belies the suggestion, w h i c h m i g h t h a v e d e r i v e d f r o m F i g u r e 4, t h a t s i m p l e b r e a d t h of d i a g n o s t i c p r o t e i n a b s o r p t i o n b a n d s m i g h t b e a n i n d e p e n d e n t l y r e l i a b l e i n d i c a t o r of the r a n d o m i z a t i o n or h y d r o g e n

bond

i n t e r a c t i o n p o t e n t i a l of

these

materials.


protein

7.

BAiER A N D z i s M A N

2.5

CoUagen

3.0

4.0

and Gelatin

5.0

1

1

MICRONS 6Ό

1

-

1

Η

\

y

167

Surfaces

1

ι

7Ό

8.0

r

9.0

10

12 1

-

.

Λ /

—


-

-

-

4000

3500

3000

i

2500

2000

1800 1600 1400 WAVENUMBER ( C M }

1200

1000

800

Figure 5. Internal reflection IR spectra of the surface zones of films of a highly purified rat skin collagen sample (top) ana a more heterogeneous collagen prep aration from bovine Achilles tendon collagen (bottom) T h e contact angles m e a s u r e d w i t h t h e series o f p u r i f i e d w e t t i n g l i q u i d s o n c o l d w a t e r cast, t h i n films o f gelatins o f v a r i o u s d e r i v a t i o n are l i s t e d i n T a b l e I V . T h e t a b l e has b e e n a r r a n g e d t o p r o v i d e i n t h e se q u e n c e o f t h e c o l u m n s a sequence

o f m a t e r i a l s w i t h p r o g r e s s i v e l y less

a p p a r e n t h y d r o g e n b o n d c a p a b i l i t y across t h e i r interfaces, a n d , c o n c o m i t a n t l y , a p p a r e n t l y l o w e r degrees o f r a n d o m i z a t i o n f r o m the n a t i v e s t r u c t u r e w h i c h masks s u c h h y d r o g e n - b o n d i n g

potential.

I n Figure 6 are

p l o t t e d t h e contact angle values f o r p u r i f i e d c a l f s k i n g e l a t i n ; as w i t h Table IV.

Wettability of Gelatin Films Average Contact Angle,"

Wetting

Liquid

Water Glycerol Formamide Thiodiglycol M e t h y l e n e iodide s2/m-Tetrabromoethane 1-Bromonaphthalene o-Dibromobenzene 1-Methylnaphthalene Dicyclohexyl n-Hexadecane

Surface Tension at 20°C, Calf dynes/ skin cm 72.8 63.4 58.2 54.0 50.8 47.5 44.6 42.0 38.7 33.0 27.7

72 59 51 37 41 36 24 22 15 0 0

degrees

Silver Label

Knox

Pig skin

Bacteriological

67 62 49 42 44 35 30 28 23 11 0

69 61 47 43 45 41 34 30 26 15 0

70 64 60 42 42 39 33 29 23 11 0

62 55 44 47 45 38 34 25 16 5 0

Prac tical 63 73 54 62 49 45 38 32 27 16 0

Averages of at least 10 readings on at least two independently prepared films of each type. α


168

APPLIED CHEMISTRY SURFACE TENSION (y )

0.9 0.8 0.7

ISO


1

15 1

20

1

25 1

ι1

-

0.6

30

1 1

I

1

i

I

ι 1

1 1

+ 0.5 0.4 0.3 0.2 +0.1

0

! "

-

i ;

!!

ι! ι 1 ι

Iι ιί ιι i'!!! l" PURIFIED CALF SKIN GELATIN FIIM

-

,

; HI

ι

!! I

ι

ι ι\

!! ιJ!

1

i l l ( M | M « ' " 1

ι M l

1 1

I

10

ι 15

1

^

1

1

I

ι

N

1

1

1

1

1

1

1

ι

1 1

1

1

1

; !

' 1 1 '' 1 ι l ι I I ι1 1 1 I ; ι1

1 ι ι ί «'I ι - ι .ΜΙ ι I ι ι li. ι ι 1 îi , ! . ! !- J ι 20 25 30 35 40 45 50 55 60 SURFACE TENSION (y ) IN DYNES/CM

!1

il

ι ι

ι

1 11 I ι ι !ι ι i \ ^ \ ! I ιι 1 1 \ l 1 1 ι ιι ! ·ν I ι ' \ I ι1 1 ! \ I ι

M i 1 ! II ι 1 ι11 I I ι

1 , 1 1

1

70 75 1

ι

ι.

60 65

1 l1 ι

Κ l\l

1

:;!:!

55

! 1. !' II !' 1 ! _

1

1

50

45

40

1

' I!1 ,ΐ,ι , ill ι in

35

1

-

CD UJ Ζ

10

INTERFACES

IN DYNES/CM

LV

5 1

+ 1.0

AT PROTEIN

! ι ~

1

! 1

\i -

'

' \ "

ι 1

1

-

1

ί ι 65

l!i

70 75

LV

Figure 6. Plot of contact angle data illustrating the duality of wetting behavior of a purified calfskin gelatin film when tested with hydrogen-bonding and nonhydrogen-bonding liquids the r a n d o m i z e d p u r e c o l l a g e n s a m p l e , there w e r e separate c r i t i c a l surface t e n s i o n intercepts for h y d r o g e n - b o n d i n g a n d n o n h y d r o g e n - b o n d i n g

liq

u i d s . I n F i g u r e 7 are the i n t e r n a l reflection I R spectra f o r these same six samples i n t h e i d e n t i c a l f o r m that w a s p r e p a r e d a n d u s e d for the c o n t a c t a n g l e m e a s u r e m e n t s ; there w a s great d i v e r s i t y i n the I R a b s o r p t i o n p a t terns w h i c h reflect the m o l e c u l a r species a n d t h e i r conformations.

There

w a s n o o b v i o u s c o r r e l a t i o n b e t w e e n p a r t i c u l a r features o f i n d i v i d u a l I R spectra a n d the w e t t i n g d a t a . T h u s t h e contact a n g l e d a t a reflect the a c t u a l outermost a t o m i c c o n s t i t u t i o n o f t h e v a r i o u s samples m u c h m o r e sensitively t h a n e v e n this r e m a r k a b l y surface sensitive s p e c t r a l t e c h n i q u e w h i c h characterizes n o m o r e t h a n a m i c r o n o r so o f the s a m p l e surface phase. Interpretation

and Discussion of Results

T h e r e are three m a j o r c o m p l i c a t i o n s i n c o n t a c t proteins a n d other w a t e r sensitive p o l y m e r s .

a n g l e studies o f

F i r s t , n o s i m p l e o r safe

m e t h o d o f g u a r a n t e e i n g t h e cleanliness o r u n i f o r m i t y o f s p e c i m e n sur faces i s a v a i l a b l e ; the i n i t i a l p u r i t y o f t h e s a m p l e m u s t b e r e l i e d u p o n as the p r i m a r y c r i t e r i o n o f surface u n i f o r m i t y . S e c o n d , there is a strong effect f r o m a d s o r b e d w a t e r m o l e c u l e s w h i c h r e m a i n o n the surface a n d


7.

BAiER A N D ziSMAN

Collagen

and Gelatin

169

Surfaces

u s u a l l y also w i t h i n the b u l k o f h y d r o p h i l i c materials s u c h as p o l y a m i d e s . T h i r d , w a t e r sensitive m a t e r i a l s m a y b e r a p i d l y s w o l l e n o r s o l u b i l i z e d b y m a n y l i q u i d s a p p l i e d to t h e i r surfaces whereas a m o r e s i m p l e w e t t i n g a n d s p r e a d i n g context is d e s i r e d f o r a n u m b e r o f b i o l o g i c a l a n d / o r i n d u s t r i a l purposes.

I n our previous work, such complications were overcome

i n other instances w h e n i t w o u l d h a v e b e e n d e s t r u c t i v e to a t t e m p t t o d e h y d r a t e t h i n film specimens c o m p l e t e l y .

Drastic water removal pro-

cedures almost c e r t a i n l y i n d u c e other u n d e s i r a b l e alterations i n t h e b a s i c Downloaded by UNIV OF MARYLAND COLL PARK on October 15, 2014 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch007

p o l y m e r c h e m i s t r y a n d structure. T h e d i a g n o s t i c c r i t e r i a p r e s e n t e d ( J , 2 ) as i n d i c a t o r s o f t h e accessib i l i t y o f h y d r o g e n - b o n d i n g sites at p o l y m e r interfaces are a p p l i c a b l e e v e n for h i g h l y w a t e r s o l u b l e p o l y a c r y l a m i d e . T h e p l o t o f contact a n g l e d a t a i n F i g u r e 8 i n c l u d e s values p u b l i s h e d earlier ( 2 0 ) a n d also t h e less r e p r o ducible data for water, glycerol, a n d formamide—three more important h y d r o g e n b o n d f o r m i n g l i q u i d s — r e c o r d e d o n these surfaces at t h e same t i m e (21). O u r d i a g n o s t i c c r i t e r i o n o f a split i n the d a t a plots f o r h y d r o gen-bonding a n d nonhydrogen-bonding

l i q u i d s i s m e t i n this instance,

reflecting t h e m a r k e d degree o f i n t e r a c t i o n b e t w e e n t h e 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 t h e surface accessible

a m i d e s w h i c h i n this p o l y m e r a r e

present i n t h e i m p o s s i b l e - t o - m a s k side chains. T h e contact angle v a l u e

2.5

4000

3.0

3500

3000

4.0

5.0

2500

2000

MICRONS 6.0

7.0

1800 1600 1400 WAVENUMBER ( C M )

8.0

1200

9.0

10

1000

- 1

Figure 7. Internal reflection IR spectra revealing the variety of structures dominating the surface zones of six different gelatin preparations cast from hot water into thin, smooth films


170

APPLIED CHEMISTRY

o b t a i n e d w i t h the h y d r o g e n - b o n d i n g

AT PROTEIN

INTERFACES

l i q u i d o f largest m o l e c u l a r

size,

t h i o d i g l y c o l , w a s b o t h stable a n d r e p r o d u c i b l e ; this suggests t h a t its i n ability to penetrate

t h e sample

surface

eliminates t h e c o m p l i c a t i o n s

( scatter i n d a t a o f a b o u t 5 - 1 0 degrees ) c a u s e d b y s i m p l e p e n e t r a t i o n o f the smaller, h y d r o g e n - b o n d i n g

molecules.

T h e r e are three p o s s i b l e c r i t i c a l surface t e n s i o n intercepts w h i c h m i g h t i n d e p e n d e n t l y c h a r a c t e r i z e w a t e r sensitive p o l y m e r s ,

depending

o n the i m a g e forces a n d s p e c i a l interactions that c a n b e m a n i f e s t e d across Downloaded by UNIV OF MARYLAND COLL PARK on October 15, 2014 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch007

the l i q u i d - s o l i d interface.

A n i n t e r c e p t a b o v e 40 d y n e s / c m is o b t a i n e d

b y e x t r a p o l a t i n g d a t a for h y d r o g e n - b o n d i n g l i q u i d s ; a v a l u e at a b o u t o r slightly b e l o w 4 0 d y n e s / c m is obtained b y extrapolating data for noninteracting organic liquids, and a m u c h lower intercept—from a plot w i t h a m a r k e d l y different s l o p e — a t a b o u t 30 d y n e s / c m is o b t a i n e d b y e x t r a p o l a t i n g d a t a f o r d i s p e r s i o n force o n l y l i q u i d s w i t h v e r y l o w

surface

tensions ( w h i c h g i v e a n o m a l o u s nonzero contact a n g l e v a l u e s ) .

A s was

d e m o n s t r a t e d e a r l i e r w i t h s i m p l e r p o l y a m i d e s a n d here w i t h the c o l l a g e n g e l a t i n t r a n s f o r m a t i o n , the separate c r i t i c a l surface tension i n t e r c e p t for the h y d r o g e n - b o n d i n g l i q u i d s is not o b s e r v e d w h e n the p o l y m e r structure is s u c h that a m i d e groups w h i c h c o u l d enter into s u c h s p e c i a l interactions are m a s k e d f r o m the surface.

W i t h e x c e p t i o n a l l y w a t e r sensitive p o l y

mers s u c h as the proteins a n d p o l y a c r y l a m i d e , h o w e v e r , the

apparent

SURFACE TENSION {γ^) IN DYNES/CM

+ 1.0

1

1

0.9 0.8 0.7 Φ UJ

ζ

0.6

Ο + 0.5 ο

CO

0.4 0.3 0.2 +0.1 0

10 15 20 25 30 35 40 45 50 55 60 65 70 75 l1 I ' l l ^ti Λ I H ι k ι · l 1

-

! jjj*

-

-

-

!

-

•

;

ί

'!

!

' 'NJ

I S ' ι, !!|;

ι ;

! !! | ϋ i

1

ϋ 1

!

1

ι

1

1 1

-

I

" •111 ι

-

POLY •~

-

•

1 ι ·! ' ι'" ACRYLAMIDE ι ''" 1 % R.h.

•-997, R.H.

• ' ! !1 !

1

ι

Ι

ι

ί

ι ii 1

ΐ

11

1

!

1

!'

'

1

1

SURFACE TENSION (y ) LV

\

!

• 11 >

ι

ι

•Ml 1 j 1 !, I li> ι ι ι I ι I ! «Μ , - , !.!!!! ι ι . i l ! . ! ί! ι: 25 30 35 40 45 50 1

I I I ! 10 15 20

!

1

ι

I

!

ι 1

1

1

ι 1 . , ι ι ' I !ι : ι ! . 55 60 65 :

~"

1 1

! \ ι \

1

! '

1 1

;

! \ ^

1 1

1

1

1

ι

!' ι

11

^

•

1

1 11

1

1

ι

1

:

;

\ t

1 I I 1

1

_

_

I

1 1

I ι1 70 75

IN DYNES/CM

Figure 8. Plot of contact angle data illustrating great disparity in the of a polyacrylamide surface by liquids capable of sensing different chemical groupings


wetting surface

7.

BAiER A N D z i s M A N

Collagen

and Gelatin

171

Surfaces

lowest c r i t i c a l surface tension i n t e r c e p t for the l o w surface t e n s i o n , o r g a n i c l i q u i d s remains constant e v e n w h e n a l a r g e v a r i e t y of configurations is present, as i l l u s t r a t e d w i t h o u r series of gelatins. W e a t t r i b u t e this a p p a r e n t a n o m a l y to the presence o n a l l s u c h surfaces of a strongly a d s o r b e d a n d o r g a n i z e d l a y e r of w a t e r . T h e d i f f i c u l t y of s w e l l i n g a n d s o l u b i l i z a t i o n of the p o l y m e r s b y cert a i n test l i q u i d s arises i n a n y system w h e r e the l i q u i d s a n d t h e p o l y m e r s are p o t e n t i a l l y or k n o w n to be m i s c i b l e .

T h e effect of o r g a n i c


interactions w i t h p o l y s t y r e n e w a s n o t e d m a n y years ago

liquid I n the

(22).

c u r r e n t experiments, w h e n h y d r o g e n - b o n d i n g l i q u i d s w e r e a p p l i e d to the various c o l l a g e n a n d g e l a t i n films, s w e l l i n g a n d p a r t i a l s o l u b i l i z a t i o n d i d o c c u r n o t i c e a b l y i n some instances.

R e p r o d u c i b l e values c o u l d a l w a y s

b e o b t a i n e d , h o w e v e r , b y a d v a n c i n g the i n t e r a c t i n g d r o p l e t s o v e r fresh surface areas a n d r e c o r d i n g o n l y the i n i t i a l a d v a n c i n g contact

angles.

W h e n d r o p s w h i c h h a d b e e n o n the surface for a m i n u t e or m o r e w e r e c a r e f u l l y i n c r e m e n t e d , the d r o p l e t p e r i m e t e r r e m a i n e d constant r a t h e r t h a n a d v a n c i n g , a n d the a p p a r e n t contact a n g l e values i n c r e a s e d to w e l l over 100° a n d r e m a i n e d at s u c h a n o m a l o u s l y h i g h values for m a n y m i n utes. R e t r a c t i o n of these i n c r e m e n t e d drops of i n t e r a c t i n g l i q u i d s f r o m the p r o t e i n surface, for e x a m p l e b y s i m p l y t o u c h i n g t h e m w i t h a w i c k o f filter p a p e r , left b e h i n d a n o b v i o u s l y l i q u i d - s w o l l e n r e g i o n w h i c h w a s c a p p e d b y a s u r f a c e - s p r e a d p r o t e i n film w h i c h c o l l a p s e d i n t h e shape of a t r u n c a t e d l i q u i d d r o p l e t of t h e o r i g i n a l size. B e c a u s e of these m a n y c o m p l i c a t i o n s , it is most e n c o u r a g i n g t h a t the d a t a d e r i v e d f r o m this s t u d y w e r e r e m a r k a b l y o r d e r l y a n d that t h e y a g r e e d w i t h p r e d i c t i o n s b a s e d o n our studies of s i m p l e r m o d e l p o l y m e r s (1, 2, 3).

T h e f o l l o w i n g c a n be c o n c l u d e d a b o u t the w e t t i n g properties

of c o l l a g e n a n d g e l a t i n : ( a ) c o l l a g e n m a i n t a i n s a t e n a c i o u s l y b o u n d l a y e r of a d s o r b e d m o i s ture u n d e r the e x p e r i m e n t a l c o n d i t i o n s ( 5 0 % r e l a t i v e h u m i d i t y at 2 0 ° C ) , a n d i t is u n l i k e l y that this a d s o r b e d aqueous c o m p o n e n t c o u l d be c o m p l e t e l y e l i m i n a t e d w i t h o u t d e s t r o y i n g the p o l y m e r ; ( b ) c o l l a g e n has a n e s t i m a t e d c r i t i c a l surface tension of a b o u t 40 d y n e s / c m , a n d , i n its n a t i v e fibrous s t r u c t u r e , it does not expose accessible h y d r o g e n - b o n d i n g , b a c k b o n e a m i d e groups at the s o l i d - g a s i n t e r f a c e ; a n d ( c ) g e l a t i n , a r a n d o m i z e d a n d d e s t r u c t u r e d p r o d u c t of c o l l a g e n , c a n be i n d u c e d b y s i m p l e t e m p e r a t u r e increases, a n d i t c a n e x h i b i t m a r k e d changes i n surface interactions w i t h h y d r o g e n - b o n d i n g l i q u i d s e v e n t h o u g h no large changes i n the I R spectra or other parameters of the b u l k p o l y m e r are observed. T h e c o m b i n a t i o n of a h i g h c r i t i c a l surface t e n s i o n i n t e r c e p t a n d a split i n the contact a n g l e d a t a for the h y d r o g e n - b o n d i n g a n d 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 the best i n d i c a t o r of the presence of

accessible


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APPLIED CHEMISTRY

AT PROTEIN

INTERFACES

a m i d e groups at the surface of p o l y a m i d e m a t e r i a l s , e v e n w h e n materials are as c o m p l i c a t e d as n a t u r a l fibrous proteins. T h e of a n o m a l o u s

nonzero

such

occurrence

contact angles for s i m p l e o r g a n i c l i q u i d s w i t h

v e r y l o w surface tensions suggests the presence of a s t r o n g l y l a y e r of w a t e r o n s u c h surfaces.

adsorbed

D e p e n d i n g o n the p r a c t i c a l use to b e

m a d e of contact a n g l e d a t a , a n y or a l l of the three different c r i t i c a l surface tension values m i g h t b e the most i m p o r t a n t . It w o u l d b e u n w i s e to


a t t e m p t to d e d u c e i n d i c a t o r s of the surface free e n e r g y of s u c h b i o l o g i c a l macromolecules liquid.

b y r e l y i n g u p o n contact angles w i t h o n l y one t y p e of

A c o m m o n error of biologists a n d e v e n some p o l y m e r chemists

is to p l a c e c o m p l e t e f a i t h i n measurements of w a t e r contact angles alone. A l t h o u g h c o n s i d e r a b l e a d d i t i o n a l w o r k w i t h m o r e m o d e l systems, a greater v a r i e t y of w e t t i n g l i q u i d s , a n d proteins of t h e greatest possible p u r i t y a n d u n i f o r m i t y is necessary, the o r d e r l y p a t t e r n of the d a t a o b t a i n e d so f a r deserves

attention.

T h i s a r t i c l e illustrates o n l y t h e

first

successful a p p l i c a t i o n of these m e t h o d s to the p r o t e i n c o l l a g e n a n d its v a r i o u s l y m o d i f i e d s t r u c t u r a l forms i n c o m m o n g e l a t i n p r e p a r a t i o n s .

Col-

l a g e n - d e r i v e d proteins constitute the b u l k of the o r g a n i c m a t e r i a l i n c o n n e c t i v e tissue a n d i n teeth, so the w e t t i n g a n d s p r e a d i n g of a v a r i e t y of l i q u i d s o n e a c h of these materials is of great p r a c t i c a l i m p o r t a n c e . S u r g i c a l adhesives, for e x a m p l e , m u s t efficiently w e t a n d s p r e a d u p o n the p r o t e i n of c u t tissue a n d f o r m s t r o n g b o n d s w i t h it. S i m i l a r l y , d e n t a l adhesives, e s p e c i a l l y those for use w i t h i n the p r e p a r e d tooth c a v i t y , m u s t w e t the proteinaceous c o m p o n e n t r i v e d f r o m the c o l l a g e n - b a s e d

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

d e n t i n e i f the restorative m a t e r i a l is to

m a k e a firm, v o i d - f r e e b o n d . T h e p r e l i m i n a r y w e t t i n g studies r e p o r t e d here describe a n d delineate the types of s p r e a d i n g b e h a v i o r w h i c h are to b e expected w i t h l i q u i d compositions of v a r i o u s types. F u r t h e r , o u r w o r k demonstrates the p r a c t i c a l u t i l i t y w h i c h i n d u c t i o n of modest c o n f i g u r a t i o n a l changes of proteins m i g h t h a v e i n p r e d i s p o s i n g a p r o t e i n - d o m i n a t e d surface to either f a v o r or reject spontaneous w e t t i n g b y a g i v e n l i q u i d or class of l i q u i d s . T h e p o t e n t i a l range of a p p l i c a b i l i t y of these

findings,

after t h e y are e x t e n d e d

a n d c o n f i r m e d , is e x c e p t i o n a l l y l a r g e w h e n one recalls that the surface properties of proteins h a v e a l r e a d y b e e n i m p l i c a t e d i n the catalysis or c o n t r o l of most b i o c h e m i c a l events; i n the p e r m s e l e c t i v i t y of b i o l o g i c a l m e m b r a n e s ; i n the a c c e p t a b i l i t y of o r g a n grafts a n d grafts of e n g i n e e r i n g or s t r u c t u r a l m a t e r i a l s ; i n t h e i r r e v e r s i b l e d a m a g e

occurring i n extra-

c o r p o r e a l devices for b l o o d h a n d l i n g ; i n the acceptance

of m e d i c a t i o n s ,

cosmetic agents, a n d other treatments b y the s k i n ; a n d i n t h e resistance of s k i n surfaces to adverse p e n e t r a t i o n b y c o n t a m i n a t i n g agents.


7.

BAIER A N D ZISMAN

Summary and

CoUagen

and Gelatin

173

Surfaces

Conclusions

P r e v i o u s l y , w e e x a m i n e d the w e t t i n g properties of seven m o d e l p o l y amides, i n c l u d i n g the polypeptides poly ( m e t h y l glutamate) a n d poly( b e n z y l g l u t a m a t e ) ; p o l y g l y c i n e ; nylons 66, 6, a n d 11; a n d p o l y a c r y l a m i d e . A l l w e r e successfully c h a r a c t e r i z e d b y contact angle c r i t e r i a as a m i d e c o n t a i n i n g materials. W h e n s t r u c t u r a l transitions w e r e i n d u c e d i n these experimental

materials w h i c h

a l t e r n a t e l y exposed

hydrogen-bonding


a m i d e groups t o the surface a n d m a s k e d t h e m f r o m i t , the d i a g n o s t i c w e t t i n g c r i t e r i a w e r e v a l i d a t e d . T h e s e m o d e l m a t e r i a l s h a d the i n h e r e n t a d v a n t a g e of b e i n g s y n t h e t i c p r o d u c t s o f defined c o n s t i t u t i o n that c o u l d be f r e e d f r o m c o n t a m i n a t i o n b y s i m p l e techniques. W h e n the s t u d y w a s e x t e n d e d t o the m o r e c o m p l i c a t e d n a t u r a l p r o t e i n system o f c o l l a g e n g e l a t i n , i t w a s n o t e d that a n o m a l o u s contact a n g l e values for these w a t e r sensitive specimens w e r e i n a characteristic range of t h e i r o w n that d i d not interfere w i t h the contact angle diagnosis of accessible a m i d e groups at the p o l y m e r interface. T h e anomalous n o n z e r o contact angles for l o w surface tension, o r g a n i c l i q u i d s are i n d i c a t i v e o f the r e t e n t i o n o f a s t r o n g l y b o u n d w a t e r layer o n the p r o t e i n surfaces.

D e s p i t e this a d s o r b e d m o i s

ture, c o l l a g e n demonstrates a surface c o m p o s i t i o n w i t h a c r i t i c a l surface tension of a b o u t 4 0 d y n e s / c m w h e n i n its n a t i v e f o r m , a n d a second, h i g h e r c r i t i c a l surface tension i n t e r c e p t b e t w e e n 4 0 a n d 50 d y n e s / c m w i t h h y d r o g e n - b o n d i n g l i q u i d s o n l y w h e n r a n d o m i z e d t o its g e l a t i n f o r m . Acknowledgments H e l p f u l suggestions b y m e m b e r s of the S u r f a c e C h e m i s t r y B r a n c h of the N a v a l R e s e a r c h L a b o r a t o r y , especially G e o r g e L o e b , E l a i n e G . S h a f r i n , a n d M a r i a n n e K . B e r n e t t , are g r a t e f u l l y a c k n o w l e d g e d .

D . S.

C a i n o f the P h y s i c a l C h e m i s t r y B r a n c h r e n d e r e d v a l u a b l e assistance i n r e c o r d i n g t h e I R spectra.

A l b e r t J . F r y a r p r o v i d e d the d a t a o n s e d i

m e n t a t i o n coefficients.

Literature Cited 1. Baier, R. E., Zisman, W. Α., Macromolecules (1970) 3, 70. 2. Ibid. (1970) 3, 462. 3. Ellison, A. H., Zisman, W. Α., J. Phys. Chem. (1954) 58, 503. 4. Baier, R. E., Loeb, G. I., in "Polymer Characterization: Interdisciplinary Approaches," C. D. Craver, Ed., p. 79, Plenum, New York, 1971. 5. Shafrin, E. G., Zisman, W. Α., J. Amer. Ceram. Soc. (1967) 50, 478. 6. Bernett, M. K., Zisman, W. Α., J. Colloid Interface Sci. (1968) 28, 243. 7. "Gelatin," Gelatin Manuf. Inst. Amer., New York, 1973. 8. Stenzel, K. H., Miyata, T., Kohno, I., Schlear, S., Rubin, A. L., ADVAN. CHEM SER. (1975) 145, 26.

9. Stenzel Κ. H., Miyata, T., Rubin, A. L., Ann. Biophys. Bioeng., in press.



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APPLIED CHEMISTRY AT PROTEIN INTERFACES

10. Rubin, A. L., Drake, M. P., Davison, P. F., Pfahl, D., Speakman, P. T., Schmitt, F. O., Biochemistry (1965) 4, 181. 11. Stenzel, Κ. H., Dunn, M. W., Rubin, A. L., Miyata, T., Science (1969) 164, 1282. 12. Grahan, C. E.,J.Biol.Chem. (1949) 177, 529. 13. Neuman, R. E., Arch. Biochem. (1949) 24, 289. 14. Block, R. J., Boiling, D., "The Amino Acid Composition of Proteins and Foods," Charles C. Thomas, Springfield, 1951. 15. Hess, W. C., Lee, C., Neidig, Β. Α.,Proc.Soc. Exper. Biol. Med. (1951) 76, 783. 16. Tristram, G. R., in "The Proteins," H. Neurath, Ed., vol. 1A, p. 181, Aca demic, New York, 1953. 17. Eastoe, J. E., Biochem. J. (1955) 61, 589. 18. Idson, B., Braswell, E., Advan. Food Res. (1957) 7, 235. 19. Hughston, H. H., Earle, L. S., Binkley, F., J. Dent. Res. (1959) 38, 323. 20. Hess, W. C., Dhariwal, Α., Chambliss, J. F., Alba, Z. C., J. Dent. Res. (1961) 40, 87. 21. "Gelatin," Gelatin Manuf. Inst. Amer., New York, 1962. 22. Rubin, A. L., Drake, M. P., Davison, P. F., Pfahl, D., Speakman, P. T., Schmitt, F. O., Biochemistry (1965) 4, 181. 23. Knox Gelatine, Inc., unpublished data. 24. Curme, H. G., in "The Theory of the Photographic Process," 3rd ed., C. Ε. K. Mees and T. H. James, Eds., chap. 3, Macmillan, New York, 1966. 25. Nishihara, T., Rubin, A. L., Stenzel, Κ. H., Trans. Amer. Soc. Artif. Intern. Organs (1967) 13, 243. 26. Piez, Κ. Α., Lewis, M. S., Martin, G. R., Gross, J., Biochim. Biophys. Acta (1961) 53, 596. 27. Page, R. C., Bendrtt, E. P., Science (1969) 163 ,578. 28. Blake, J. N., in "Recent Advances in Gelatin and Glue Research," G. Stainsby, Ed., p. 219, Pergamon, New York, 1958. 29. Piez, Κ. Α., in "Treatise on Collagen," Vol. I: "Chemistry of Collagen," G. N. Ramachandran, Ed., p. 207, Academic, New York, 1967. 30. Jarvis, N. L., Fox, R. B., Zisman, W. Α., ADVAN. CHEM. SER. (1964) 43, 317. 31. Jarvis, N. L., private communication. 32. Ellison, A. H., Zisman, W. A., J. Phys. Chem. (1954) 58, 260. RECEIVED June 19, 1974. Robert E. Baier was supported as a Post-Doctoral Research Associate of the National Research Council, National Academy of Sciences in 1966-1968; during this time, most of the experimental work dis cussed here was completed.


Applied Chemistry at Protein Interfaces

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