Applied Chemistry at Protein Interfaces

4 A Physicochemical Approach to the

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Characterization of Stratum Corneum RICHARD H. WILDNAUER, DAVID L. MILLER, and WILLIAM T. HUMPHRIES Department of Skin Biology, Johnson & Johnson Research, New Brunswick, N. J. 08903

Stratum corneum is the principal diffusion barrier of the skin to molecules and is also a protective surface against mechanical insults; their function depends on morphological and macromolecular organization of the membrane. Its physical behavior is a two-phase system of oriented, amorphous, and crystalline regions, principally fibrous proteins associated with lipids. Thermally induced viscoelastic, dimensional, enthalpic, spectral, and diffusional changes occurring at 30°-50°C and 190°-220°C suggest transformations in both amorphous and crystalline regions, respectively. The magnitude and temperature of these transitions depend on moisture content, solvent exposure, chemical crosslinking, and degree of orientation. The fundamental and empirical parameters derived from the physical characterization methods discussed here aid in understanding the influence of physical and chemical factors on stratum corneum properties. Skin, ^

the m o s t expansive h u m a n o r g a n , envelops the entire surface of

t h e b o d y s u c h that its e p i t h e l i u m is c o n t i n u o u s w i t h the e p i t h e l i a

of the e x t e r n a l orifices of the d i g e s t i v e , sweat, sebaceous, r e s p i r a t o r y , a n d u r i n o g e n i c systems. A s a r e s u l t of its a n a t o m i c a l l o c a t i o n , the s k i n f u n c tions as the p h y s i c a l interface b e t w e e n the b o d y tissues a n d the e n v i r o n m e n t . T h e p h y s i o l o g i c a l f u n c t i o n s of the s k i n are p r o t e c t i o n , c o n t a i n m e n t , and thermoregulation. T h e s k i n is t w o discrete tissue layers, b o t h p o l y m e r i c b u t d i f f e r i n g i n p r o t e i n c o m p o s i t i o n , m o r p h o l o g y , a n d thickness (1, 2)

(Figure

1).

E p i d e r m i s , the o u t e r l a y e r , is c e l l u l a r a n d is c o m p o s e d p r i m a r i l y of the intracellular

fibrous

p r o t e i n k e r a t i n associated w i t h l i p i d s .

I n contrast,

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


4.

wiLDNAUER E T A L .

Characterization

of Stratum

75

Corneum

"Comparative Anatomy of the Vertebrates"

Figure

1.

Diagramatic

transverse

section of full thickness

human

skin (2)

the d e r m i s , o r i n n e r l a y e r is a n o r d e r o f m a g n i t u d e t h i c k e r , p r i n c i p a l l y a c e l l u l a r , a n d c o m p o s e d o f e x t r a c e l l u l a r proteins s u c h as c o l l a g e n a n d elastin c o m p l e x e d w i t h m u c o p o l y s a c c h a r i d e s .

T h e d e r m i s also contains

the v a s c u l a r system a n d nerve endings as w e l l as p r o t e i n - s y n t h e s i z i n g cells. It is the p r i n c i p a l l o a d - b e a r i n g tissue o f the s k i n .

Physicochemical

c h a r a c t e r i z a t i o n o f the p o l y m e r i c n a t u r e o f d e r m i s has b e e n s t u d i e d extensively a n d w i l l not b e c o n s i d e r e d i n this w o r k (3, 4, 5). F e w syst e m a t i c studies have b e e n m a d e to c h a r a c t e r i z e t h o r o u g h l y the e p i d e r m i s i n terms o f its f u n c t i o n a l l y r e l a t e d p h y s i c a l a n d c h e m i c a l (6-15).

properties

T h i s f o r m o f c h a r a c t e r i z a t i o n is the t o p i c o f this discussion.

S t r a t u m c o r n e u m , the outermost l a y e r o f m a m m a l i a n e p i d e r m i s , f u n c tions p h y s i o l o g i c a l l y as the p r i n c i p a l d i f f u s i o n b a r r i e r t o molecules p e n e t r a t i n g the s k i n a n d as a p r o t e c t i v e p h y s i c a l b a r r i e r to m e c h a n i c a l insults at t h e s k i n surface.

D a t a suggest that these f u n c t i o n s

are critically

d e p e n d e n t o n the specific m o r p h o l o g i c a l a n d m a c r o m o l e c u l a r o r g a n i z a t i o n o f the m e m b r a n e m o s a i c (16, 17, 18, 19, 20). T h u s , alterations o f b i o p h y s i c a l properties arise f r o m e n v i r o n m e n t a l factors a c t i n g d i r e c t l y o n the m e m b r a n e

o r u p o n t h e k e r a t i n i z a t i o n process, a n d t h e y

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

affect

76

A P P L I E D

significantly b i o l o g i c a l p e r f o r m a n c e flexibility

C H E M I S T R Y

(21).

A

T

P R O T E I N

I N T E R F A C E S

S i m i l a r l y , the smoothness a n d

of s t r a t u m c o r n e u m are i m p o r t a n t to c o s m e t i c aspects.

It is

thus r e a s o n a b l e t h a t c e r t a i n r e q u i r e m e n t s of s t r a t u m c o r n e u m s t r e n g t h a n d e l a s t i c i t y are essential properties to m a i n t a i n a contiguous

membrane

a n d to p e r m i t a d e q u a t e p h y s i o l o g i c a l f u n c t i o n . P r a c t i c a l a n d f u n d a m e n t a l i n f o r m a t i o n c o m e f r o m studies of

the

p h y s i c a l a n d c h e m i c a l p r o p e r t i e s of i s o l a t e d samples of s t r a t u m c o r n e u m . F i r s t , the gross manifestations of m a n y s k i n disorders are a l t e r e d p h y s i c a l Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

p r o p e r t i e s of t h e c o r n e u m s u c h as c r a c k i n g , s c a l i n g , roughness, bility, and increased permeability.

inflexi-

Studies c a n separate factors w h i c h

influence k e r a t i n i z a t i o n f r o m those w h i c h affect the m e m b r a n e d i r e c t l y . S e c o n d , the q u a l i t y of s t r a t u m c o r n e u m is a n i n d i c a t o r of e p i d e r m a l f u n c t i o n , since the s t r a t u m c o r n e u m is t h e epidermopoesis.

The

composition

final

differentiated product

a n d s t r u c t u r a l o r g a n i z a t i o n of

of the

k e r a t i n i z e d s t r a t u m c o r n e u m cells c o n t a i n the i n s c r i b e d h i s t o r y of t h e i r b i o l o g i c a l f o r m a t i o n a n d are a p o t e n t i a l source of p r a c t i c a l c l i n i c a l i n f o r m a t i o n . R e c o g n i t i o n of the c l i n i c a l i m p l i c a t i o n s of p h y s i c a l a n d c h e m i c a l studies of s t r a t u m c o r n e u m to d e r m a t o l o g y w a s p i o n e e r e d b y

Kligman

(17). T h e p h y s i c a l p r o p e r t i e s affecting p h y s i o l o g i c a l f u n c t i o n a r e p r i n c i p a l l y d e t e r m i n e d at the m a c r o m o l e c u l a r l e v e l b y the t h r e e - d i m e n s i o n a l n e t w o r k s t r u c t u r e of c o m p o n e n t l o n g - c h a i n p o l y m e r i c m o l e c u l e s p l e x e d w i t h s m a l l m o l e c u l e s s u c h as l i p i d s a n d p o l y s a c c h a r i d e s . n e t w o r k structures are best c h a r a c t e r i z e d b y p h y s i c o c h e m i c a l ments of i s o l a t e d s t r a t u m c o r n e u m to establish a profile of

comThese

measure-

parameters

r e l a t i n g s t r u c t u r e a n d p r o p e r t i e s to functions. C h a r a c t e r i z a t i o n of t h e k e r a t i n i z e d cells b y c l a s s i c a l h i s t o l o g i c a l a n d biochemical

approaches

has b e e n difficult b e c a u s e of the i n t r a c t a b l e

n a t u r e of the tissue. Y e t i t is p r e c i s e l y these p r o p e r t i e s of strength, i n s o l u b i l i t y , m a c r o m o l e c u l a r character, a n d l a c k of

mechanical metabolic

a c t i v i t y a l o n g w i t h its ease of i s o l a t i o n w h i c h makes s t r a t u m c o r n e u m a m e n a b l e to analysis b y p h y s i c a l m e t h o d s .

T h e extreme c o m p l e x i t y

of

c o m p o s i t i o n , m o l e c u l a r s t r u c t u r e , a n d o r g a n i z a t i o n of s t r a t u m c o r n e u m m a k e i n t e r p r e t a t i o n of these m a c r o s c o p i c p r o p e r t i e s i n terms of m o l e c u l a r s t r u c t u r e a n d events d e p e n d e n t

h e a v i l y o n analogous

studies

of

m o d e l s y n t h e t i c p o l y m e r systems a n d the m o r e t h o r o u g h l y c h a r a c t e r i z e d , keratin-containing Sample

wool. 'Preparation

S t r a t u m c o r n e u m u s e d i n these studies w a s i s o l a t e d f r o m h u m a n , n e w b o r n rat, c a l l u s , a n d g u i n e a p i g foot p a d . T h e m e t h o d s of i s o l a t i n g the v a r i o u s tissues h a v e b e e n d i s c u s s e d elsewhere (10,11). E v e n t h o u g h the v a r i o u s c o r n e u m tissues differ s o m e w h a t i n m o r p h o l o g y a n d c h e m -



4.

W I L D N A U E R

E

T

A L .

Characterization

of Stratum

Corneum

77

"Epidermal Wound Healing"

Figure

2. Dilute ΉaOH-swollen transverse section of mouse ear epidermis illustrating the morphological organization of the corneum (22)

istry, p h y s i c a l c h a r a c t e r i z a t i o n d a t a o b t a i n e d o n o n e tissue c a n often be e x t r a p o l a t e d t o t h e others. Significant differences i n p h y s i c a l or c h e m i c a l b e h a v i o r a m o n g the v a r i o u s c o r n e u m tissues s t u d i e d w i l l b e n o t e d w h e r e t h e y occur. S o l v e n t - e x t r a c t e d samples w e r e p r e p a r e d b y i m m e r s i n g d r y c o r n e u m samples i n the specified solvent for 90 m i n unless o t h e r w i s e stated. S a m p l e s w e r e not tested u n t i l at least 48 hrs f o l l o w i n g e x t r a c t i o n to a l l o w f o r e v a p o r a t i o n o f r e s i d u a l solvent. S a m p l e s w e r e p r e s t r e t c h e d b y m o u n t i n g d r y strips o f c o r n e u m i n a s t r e t c h i n g d e v i c e a n d s u b m e r g i n g the e n t i r e a p p a r a t u s i n w a t e r for 1 h r . T h e strips w e r e t h e n s t r e t c h e d b y m a n u a l a d j u s t m e n t o f the d e v i c e t o t h e r e q u i r e d extension a n d a l l o w e d to d r y p r i o r to testing. H y d r a t i o n i n c r e a s e d s a m p l e l e n g t h b y 4 - 8 % , a n d this is t a k e n i n t o c o n s i d e r a t i o n w h e n c a l c u l a t i n g s t r a i n (18). T h e d r y w e i g h t u s e d t o c a l c u l a t e the extent o f s o r b e d w a t e r ( w t % ) is b a s e d o n the s a m p l e w e i g h t at 1 1 0 ° C i n a n a t m o s p h e r e o f d r y a i r (less t h a n 10 p p m H 0 ) . D e s i c c a t e d samples refer t o c o r n e u m stored over calcium sulphate at room temperature and containing approximately 5 wt % HoO. 2

Morphology

and Biology

Stratum corneum is a multicellular membrane of acutely-flattened, m e t a b o l i c a l l y - i n a c t i v e cells s t a c k e d i n v e r t i c a l c o l u m n s (22). T h i s s t r a t i fied m o r p h o l o g i c a l o r g a n i z a t i o n is d e m o n s t r a t e d i n a f r e s h l y f r o z e n t r a n s verse

section o f e p i d e r m i s

visualization.

(Figure 2) where

t h e s w e l l i n g aids t h e

T h e m o r p h o l o g i c a l a r c h i t e c t u r e is s u c h t h a t the cells i n

one c o l u m n i n t e r d i g i t a t e w i t h those i n adjacent c o l u m n s t o f o r m a c o n -


78

A P P L I E D

t i g u o u s a n d coherent m e m b r a n e .

C H E M I S T R Y

A

T

P R O T E I N

T h e compactness

I N T E R F A C E S

of this c e l l u l a r a r

r a n g e m e n t is s h o w n i n a d i f f e r e n t i a l i n t e r f e r e n c e m i c r o g r a p h ( F i g u r e 3 ). O n g e n e r a l b o d y areas, the m e m b r a n e is c o m p o s e d o f 1 0 - 1 5 s t a c k e d cells a n d is a b o u t 10/x t h i c k w h e n d r y . C a l l u s areas of t h e h a n d s a n d feet are a t y p i c a l , b e i n g c o n s i d e r a b l y t h i c k e r (~200μ) stacking.

w i t h m u c h less r e g u l a r

I n m a n y of t h e s c a l i n g diseases, a t h i c k e n e d c o r n e u m w i t h a

p o o r c e l l - s t a c k i n g p a t t e r n is o b s e r v e d c l i n i c a l l y

(16).

T h e s t r a t u m c o r n e u m is d y n a m i c . T h e cells at the surface are c o n Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

t i n u a l l y lost t h r o u g h d e s q u a m a t i o n ( i n t e r c e l l u l a r f r a c t u r e of s m a l l c e l l aggregates) a n d r e p l a c e d b y d i f f e r e n t i a t e d e p i d e r m a l cells, m a i n t a i n i n g a r e a s o n a b l y constant n u m b e r of c e l l layers. S t r a t u m c o r n e u m cells are the

final

d i f f e r e n t i a t e d p r o d u c t of the k e r a t i n i z a t i o n process w h i c h is

i n i t i a t e d b y m i t o t i c d i v i s i o n s of cells l o c a t e d i n t h e g e r m i n a t i v e b a s a l layer. F o l l o w i n g d i v i s i o n , one of the d a u g h t e r cells begins to differentiate a n d joins the s t r e a m of v i a b l e cells p r o c e e d i n g to the surface of t h e s k i n . D u r i n g this t r a n s i t to the s k i n surface, a n u m b e r of i m p o r t a n t b i o c h e m i c a l a n d b i o p h y s i c a l events o c c u r w h i c h result i n the dense, a c u t e l y c o r n e u m cells (24, 25).

flattened

I n the e a r l y stages of this transit f r o m b a s a l l a y e r

to t h e s t r a t u m c o r n e u m ( l a s t i n g 14 d a y s ) , the m a j o r m e t a b o l i c a c t i v i t y is the synthesis of fibrous p r o t e i n . I n the u p p e r layers of t h e e p i d e r m i s , t h e cells g r a d u a l l y d e h y d r a t e , lose m e t a b o l i c a c t i v i t y , a n d flatten a l o n g the p l a n e p a r a l l e l to the s k i n surface r e s u l t i n g i n a b i a x i a l o r i e n t a t i o n of

Figure

3.

Interference

micrograph

of human stratum

corneum surface


(23)

4.

W I L D N A U E R

E

T

Characterization

A L .

t h e fibrous proteins (26).

of Stratum

79

Corneum

T h e polygonal (principally hexagonal)

shaped

cells v a r y i n size w i t h b o d y l o c a t i o n r a n g i n g f r o m a d i a m e t e r of 34μ o n t h e f o r e h e a d a n d h a n d s to 46> o n the t h i g h a x i l l a (27).

T h e cells are

g e n e r a l l y a b o u t 0.8/x t h i c k w h e n d r y . I n spite of the fact t h a t s t r a t u m c o r n e u m cells are m e t a b o l i c a l l y inert, changes i n k e r a t i n structure a n d o r g a n i z a t i o n o c c u r as e a c h c e l l transits t h r o u g h the s t r a t u m c o r n e u m p r i o r to d e s q u a m a t i o n (28).

This

suggests some a s y m m e t r y i n p h y s i c a l a n d c h e m i c a l p r o p e r t i e s t h r o u g h Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

the thickness of the c o r n e u m .

O n e d e m o n s t r a t i o n of this is the s w e l l i n g

of f r e s h f r o z e n transverse sections of c o r n e u m i n d i l u t e a c i d or base.

The

most m a t u r e surface cells s w e l l c o n s i d e r a b l y m o r e s l o w l y a n d to a lesser extent t h a n the l o w e r layers of the c o r n e u m (18).

S u c h a s y m m e t r y is of

p a r t i c u l a r i m p o r t a n c e i n s t u d y i n g the d i f f u s i o n a n d m e c h a n i c a l p r o p e r t i e s of this m e m b r a n e . Chemistry

and Supramolecular

Structure

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

by

the m e m b r a n e ' s c o m p o s i t i o n , f o r m a t i o n , a n d structure. S o m e gross c h e m i c a l c h a r a c t e r i z a t i o n s h a v e d e t e r m i n e d the p r i m a r y c h e m i c a l c o m p o n e n t s of the tissue w h i c h are s h o w n i n T a b l e I ( 2 9 ) . cellular w i t h approximately 1 0 % l i p i d a n d mucopolysaccharides. intracellular

fibrous

T h e tissue is p r i m a r i l y

extracellular components

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

g e n e r a l b o d y c o r n e u m d e n s i t y of 1.35-1.40 g m / c m gas d i s p l a c e m e n t t e c h n i q u e Table I .

3

as d e t e r m i n e d b y a

(30).

Composition of Stratum Corneum

(27) %

Tissue

w h i c h are

T h e b u l k of the tissue is d e n s e l y p a c k e d

Component

Cell membrane Intercellular

Chemical

Composition

of Stratum Corneum

lipid, protein lipid, protein, and mucopolysaccharides lipid, 2 0 % fibrous p r o t e i n , 7 0 % non-fibrous p r o t e i n , 1 0 %

Intracellular

Electron microscopic

5 10

85

studies f u r t h e r substantiate the presence

h i g h l y - o r d e r e d m a c r o m o l e c u l a r structures w i t h the c o r n e u m cells 31).

The

filaments,

fibrous

of (24,

k e r a t i n s t r u c t u r e has b e e n d e s c r i b e d as l o w - d e n s i t y

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

interfilamentous m a t r i x . T h e k e r a t i n

fibrils

organize into lipid-covered

b u n d l e s p r e f e r e n t i a l l y o r i e n t e d i n the l o n g i t u d i n a l p l a n e of the a c u t e l y flattened

c e l l (16).

T h e s e filaments t e r m i n a t e at the p e r i p h e r y of t h e c e l l


80

A P P L I E D

C H E M I S T R Y

A

T

P R O T E I N

I N T E R F A C E S

Biochimica et Biophysica Acta

Figure 4. Wide angle x-ray diffraction patterns of newborn rat stratum corneum with beam normal and parallel (edge) to the corneum plane (13)

NORMAL

EDGE


adjacent t o desmosomes, t h e s p e c i a l i z e d i n t e r c e l l u l a r a t t a c h m e n t p l a q u e s I n a d d i t i o n t o t h e desmosomes, m u c o p o l y s a c c h a r i d e s

(32).

form the

g r o u n d substance t h a t fills t h e i n t e r c e l l u l a r spaces a n d aids i n i n t e r c e l l u l a r c o h e s i o n (33). A h y p o t h e t i c a l m o d e l f o r the m o l e c u l a r o r g a n i z a t i o n o f t h e

fibril

u n i t o f k e r a t i n w i t h l i p i d s consists o f p r o t e i n c y l i n d e r s , s u r r o u n d e d b y a l i p i d l a y e r , w i t h the l i p i d c h a i n s a r r a n g e d r a d i a l l y o n the p r o t e i n c y l i n d e r . S u p p o r t f o r this m o d e l comes f r o m w i d e - a n d s m a l l - a n g l e x-ray d i f f r a c t i o n studies ( 1 3 , 19, 34) as w e l l as s m a l l - a n g l e l i g h t - s c a t t e r i n g studies (35). T h e r e l a t i v e c o n t r i b u t i o n o f this c o m p l e x t o s t r a t u m c o r n e u m properties a n d f u n c t i o n s is not w e l l u n d e r s t o o d .

T h e s e l i p i d s are present i n t h e

l o w e r e p i d e r m a l cells associated w i t h 1000g p r e c i p i t a t e o f h o m o g e n i z e d e p i d e r m i s (36). T h e s e

findings

suggest a n e a r l y association o f l i p i d s

w i t h the p r o t e i n filaments w h i c h m a y h a v e a r o l e i n d e t e r m i n i n g t h e i r final o r g a n i z a t i o n . S w a n b e c k has suggested o n t h e basis o f x - r a y diffract i o n studies that one o f the defects i n the d e r m a t o l o g i c a l c o n d i t i o n s o f psoriasis a n d i c h t h y o s i s is r e l a t e d t o the l a c k o f p r o p e r l i p i d - p r o t e i n filament

c o m p l e x f o r m a t i o n (37).

F i g u r e 4 shows the 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 f o r n e w b o r n rat s t r a t u m c o r n e u m w i t h the b e a m n o r m a l a n d p a r a l l e l to t h e p l a n e o f the flattened c o r n e u m

cell.

T h e p a t t e r n shows

t w o sharp

reflections

of l i p i d o r i g i n at 4.2 A a n d 3.7 A a n d t w o diffuse halos at 4.6 A a n d 9.8 A a t t r i b u t a b l e to p r o t e i n . T h e i n t e n s i t y o f the a z i m u t h a l reflections suggests t h a t the l i p i d s are associated w i t h the p r o t e i n s w h i c h are o r i e n t e d p a r a l l e l t o t h e l o n g axis o f the

flattened

cell.

T h e reflections are n o t

present i n samples w h i c h h a v e b e e n e x t r a c t e d w i t h c h l o r o f o r m - m e t h a n o l ( 3 / 1 b y v o l u m e ) o r after l o n g exposure to ether. X - r a y d i f f r a c t i o n p a t terns o f c o r n e u m specimens

a t v a r i o u s temperatures demonstrate t h e

t h e r m a l s t a b i l i t y o f these l i p i d c o m p o n e n t s (13). T w o m e l t t e m p e r a t u r e s are o b s e r v e d b y the d i s a p p e a r a n c e o f the 3.7-A s p a c i n g at 40 ° C a n d the 4.2-A one at a b o u t 70 ° C . U p o n c o o l i n g t h e h e a t e d n e w b o r n r a t c o r n e u m samples to r o o m t e m p e r a t u r e , b o t h reflections r e a p p e a r . T h e r e c r y s t a l l i z a t i o n process w a s not m o i s t u r e - d e p e n d e n t . T h e r e also appears t o b e some v a r i a t i o n i n t h e r e v e r s i b i l i t y o f the melts, n u m b e r o f reflections, a n d t h e i r i n t e n s i t y a m o n g the v a r i o u s types


4.

W I L D N A U E R

E

T

Characterization

A L .

of Stratum

Corneum

81

of c o r n e u m tissues. F o r e x a m p l e , h u m a n c o r n e u m e x h i b i t e d reflections at 3.7,4.2, a n d 4.6 A , of w h i c h t h a t at 3.7 A d i d not r e t u r n w h e n the s a m p l e was h e a t e d a n d c o o l e d b a c k to r o o m t e m p e r a t u r e . D i f f r a c t i o n patterns f r o m p s o r i a t i c scales a n d h u m a n c a l l u s e x h i b i t w e a k l i p i d reflections w h i l e h u m a n h a i r exhibits no l i p i d reflections

(38).

T h e 4.2-A spacings of other l i p i d - c o n t a i n i n g m e m b r a n e structures are i n t e r p r e t e d as the average i n t e r c h a i n s e p a r a t i o n i n d i r e c t i o n s p e r p e n d i c u l a r to the l o n g axis of the h y d r o c a r b o n p o r t i o n of t h e m o l e c u l e s Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

(39).

T h i s explains t h e i n d e p e n d e n c e

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

of the c h a i n a n d the n a t u r e of t h e p o l a r g r o u p . Lipid Characterization. T h e l i p i d extracts of r a t s t r a t u m c o r n e u m r e c o v e r e d f r o m solvents of v a r y i n g p o l a r i t i e s h a v e b e e n

characterized

f u r t h e r b y d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y ( D S C ) a n d s h o w n to d i s p l a y two major melting endotherms: 3 7 ° - 4 0 ° C and 5 8 ° - 6 2 ° C

(Figure

5).

T h e f a c t t h a t the melts of the i n t a c t c o r n e u m d e t e r m i n e d b y x - r a y d i f f r a c t i o n are b o t h s o m e w h a t h i g h e r i n t e m p e r a t u r e t h a n the D S C melts of the extract suggests a d d i t i o n a l specific i n t e r a c t i o n of the l i p i d m o l e cules w i t h m e m b r a n e p r o t e i n s . T h e extracts are q u i t e w a x y l o o k i n g a n d are c o m p o s e d of

a complex

m i x t u r e of

esters

i n s o l u b l e alcohols a n d h i g h e r f a t t y acids (40,

of

long-chain, water-

41).

Ο x w (A)

•(B)

LU Ο

20

40 60 80 T E M P E R A T U R E (°C )

100

120

Figure 5. DSC analysis of ether extract from A: newborn rat stratum corneum and B: human hair (N atm). Data from Ref. 18. g


82

A P P L I E D

C H E M I S T R Y

A

T

P R O T E I N

I N T E R F A C E S

T h e l i p i d extract f r o m w o o l o r h a i r e x h i b i t s o n l y one m e l t i n g e n d o t h e r m at 3 5 ° - 4 0 ° C ( F i g u r e 5 ) a n d is c o m p o s e d o f a b o u t 9 0 % esters o f l o n g - c h a i n acids a n d alcohols w i t h 1 0 %

free acids a n d alcohols. T h e

a c i d f r a c t i o n o f this h y d r o l y s a t e contains p r i n c i p a l l y b r a n c h e d c h a i n a n d h y d r o x y acids m e l t i n g a t 4 0 ° - 4 5 ° C . melts at 5 5 ° - 6 5 ° C (40).

T h e long-chain alcoholic fraction

I R a n d D S C d a t a o f the extracts f r o m h a i r

a n d corneum indicate that corneum

contains c o n s i d e r a b l y m o r e

free

alcohols t h a n w o o l or h a i r (42). Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

Keratin Structure and Orientation. A c u t e protein-filled

cells i n t h e final stages

flattening

o f the

fibrous

o f k e r a t i n i z a t i o n establishes a

biaxial orientation. A s w o u l d b e expected, n o birefrigence is observed n o r m a l to the p l a n e o f the c o r n e u m surface, b u t significant b i r e f r i g e n c e is o b s e r v e d p a r a l l e l t o t h e p l a n e o f the c o r n e u m surface ( I , 42). T h e x - r a y d i f f r a c t i o n p a t t e r n o f this i s o l a t e d e p i d e r m a l p r o t e i n , w h e n h i g h l y d r a w n , e x h i b i t s the c l a s s i c a l a l p h a p a t t e r n (7, 43). T h e w i d e angle x - r a y d i f f r a c t i o n p a t t e r n o f u n d e f o r m e d

corneum

exhibits diffuse halos at 4.6 A a n d 9.8 A c o m m o n to proteins ( F i g u r e 4 ). T h e l a c k o f the 5.1-A reflection c h a r a c t e r i s t i c o f a l p h a - k e r a t i n structures i n u n d e f o r m e d c o r n e u m suggests t h a t the p r o t e i n is c o n s i d e r a b l y less o r i e n t e d a n d p e r h a p s o f a l o w e r a l p h a content t h a n w o o l .

This is sup-

p o r t e d b y t h e fact t h a t t h e 5.1-A reflection begins to a p p e a r i n samples of c o r n e u m w h i c h w e r e h y d r a t e d a n d s t r e t c h e d to 1 0 0 % or m o r e ( F i g u r e 6 ) a n d a l l o w e d to d r y i n the e x t e n d e d state. T h e i n c r e a s e d o r i e n t a t i o n of t h e l i p i d reflections i n t h e s t r e t c h e d s a m p l e demonstrates f u r t h e r t h e i r association w i t h the o r i e n t i n g p r o t e i n

fibrils.

A d d i t i o n a l e v i d e n c e for the presence o f a l p h a - h e l i c a l fibrous p r o t e i n i n s t r a t u m c o r n e u m is p r o v i d e d b y I R d i c h r o i s m studies

(42). T h e

transmission I R o f n e w b o r n r a t c o r n e u m ( F i g u r e 7 ) is diffuse b e c a u s e of a large v a r i e t y o f s i d e - c h a i n b a n d s , b u t i t is c h a r a c t e r i s t i c a l l y p r o t e i n .

Critical Reviews in Bioengineering

Figure 6. Wide angle x-ray diffraction pattern of newborn rat stratum corneum stretched to various elongations while hydrated and allowed to dry in extended state (normal to plane of membrane) (6)



4.

wiLDNAUER E T A L .

'—

1

4000

3500

Characterization

I

I

of Stratum

>

I

)

Figure 10. Water vapor adsorption isotherms as a function of % RH for A, collagen; B, RSC; C, silk; D, nylon. Data from Refs. 18, 5 1 , and 62.


4.

W I L D N A U E R

E

Table I I .

T

Characterization

A L .

of Stratum

Comparison of Water Content of Polyamides and Proteins with Polar Side Chain Content

Substrate

Polar Group X 10 moles

Ref.

S t r a t u m corneum

4.4

50

0.15

26

Collagen Silk

4.0

51

0.179

48

3.6

51

0.074

48

Nylon

0

0.031

48

g H 0/g Dry Tissue at 50% RH

s


87

Corneum

2

Ref.

regains f o r q u i t e s i m i l a r p o l a r a m i n o a c i d contents i n d i c a t e s that t h e other factors

mentioned

a b o v e m a y be

involved i n determining the

e q u i l i b r i u m w a t e r u p t a k e . T h e s e factors relate to the p h y s i c a l aspects of t h e system s u c h as b a c k b o n e c h a i n

flexibility

a n d crosslink d e n s i t y

(55).

I n spite of the c o m p o s i t e n a t u r e of the s t r a t u m c o r n e u m , its w a t e r s o r p t i o n i s o t h e r m is q u a l i t a t i v e l y i d e n t i c a l to those of t h e m o r e s i m p l e p r o t e i n systems s h o w n , suggesting that w a t e r interacts p r e d o m i n a t e l y w i t h the p r o t e i n c o m p o n e n t s

of the c o r n e u m .

T h i s c o n c l u s i o n is s u p

p o r t e d f u r t h e r b y the results of c h l o r o f o r m - m e t h a n o l ( 3 / 1 b y e x t r a c t i o n w h i c h r e m o v e d as m u c h as 2 5 %

volume)

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

( lipids a n d low molecular weight water-soluble components ) but d i d not q u a n t i t a t i v e l y alter the i s o t h e r m i n the l o w r e l a t i v e h u m i d i t i e s

(18).

T h e a p p l i c a t i o n o f the Z i m m - L u n d b e r g cluster t h e o r y (56, 5 7 )

to the

i s o t h e r m y i e l d s a d d i t i o n a l i n f o r m a t i o n as to the state of

sorbed

water i n the corneum.

the

T h e t e n d e n c y of w a t e r to cluster is expressed i n

this t h e o r y b y the cluster f u n c t i o n C i G n : CiG

u

=

(1—Φι) (d In 0 i / d In m) -

1

w h e r e φι is the v o l u m e f r a c t i o n of w a t e r i n the p o l y m e r a n d a\ is the a c t i v i t y of the w a t e r .

V a l u e s of C i G n

greater t h a n —1 i n d i c a t e the

t e n d e n c y of the w a t e r to p r e f e r self association. dependency

F i g u r e 11 shows

of t h e cluster f u n c t i o n o n r e l a t i v e h u m i d i t y for

the

newborn

rat s t r a t u m c o r n e u m at 2 5 ° C . A d r a m a t i c increase i n c l u s t e r i n g t e n d e n c y of w a t e r occurs over the r a n g e 4 0 - 6 0 %

R H . A s a c o n s e q u e n c e of this

t r a n s i t i o n , w a t e r s o r b e d at l o w r e l a t i v e h u m i d i t i e s is p r o b a b l y associated at i s o l a t e d sites i n the c o r n e u m w h e r e a s at h i g h r e l a t i v e h u m i d i t i e s a cooperative

effect i n t h e s o r p t i o n is o b s e r v e d .

T h e presence of

more

h i g h l y c l u s t e r e d w a t e r correlates h i g h l y w i t h a r a p i d decrease i n t h e t e n sile m o d u l u s of the c o r n e u m w i t h i n c r e a s i n g r e l a t i v e h u m i d i t y ( see F i g u r e 18).

F r o m this c o r r e l a t i o n , w a t e r clusters are s h o w n to b e m o r e efficient

p l a ^ t i c i z i n g agents t h a n the i n d i v i d u a l m o l e c u l a r species.

O n t h e other



88

A P P L I E D

C H E M I S T R Y

A

T

P R O T E I N

I N T E R F A C E S

-1

0

20

40

RELATIVE

60

80

100

HUMIDITY %>

Figure 11. Dependence of the water cluster function, C G , on relative humidity (newborn rat stratum corneum, 25°C). Data from Ref. 5 7 . 1

11

h a n d , b o t h the c l u s t e r i n g a n d the m o d u l u s effects m a y result f r o m w a t e r i n d u c e d a l t e r a t i o n i n the c o n f o r m a t i o n o f t h e p r o t e i n itself. Diffusion of Water Vapor i n Newborn

R a t Stratum Corneum.

M e a s u r e m e n t a n d i n t e r p r e t a t i o n o f d i f f u s i o n i n heterogenous b i o l o g i c a l systems s u c h as the s t r a t u m c o r n e u m are difficult c o m p a r e d w i t h s i m i l a r m e a s u r e m e n t s f o r w e l l - d e f i n e d s y n t h e t i c p o l y m e r systems, b u t studies of w a t e r d i f f u s i o n i n s t r a t u m c o r n e u m are essential to a better u n d e r s t a n d i n g o f those factors w h i c h c o n t r i b u t e t o t h e b a r r i e r f u n c t i o n of t h e corneum.

W a t e r d i f f u s i o n measurements u n d e r b o t h e q u i l i b r i u m a n d

n o n - e q u i l i b r i u m c o n d i t i o n s are u s e f u l to p r o b e the i n f l u e n c e o f t e m p e r a t u r e a n d o t h e r factors o n s t r a t u m c o r n e u m m a c r o m o l e c u l a r s t r u c t u r e . T h e r m a l d e s o r p t i o n is a d y n a m i c ( n o n - e q u i l i b r i u m ) t e c h n i q u e i n w h i c h a s a m p l e o f h y d r a t e d c o r n e u m is h e a t e d a t a constant rate i n a d r y atmosphere.

T h e w a t e r d e s o r p t i o n rate is p l o t t e d as a f u n c t i o n o f t e m -

p e r a t u r e . T h e g e n e r a l shape a n d t e m p e r a t u r e m a x i m a o f the d e s o r p t i o n rate vs. t e m p e r a t u r e curves ( F i g u r e 1 2 ) are c h a r a c t e r i s t i c o f t h e material's d i f f u s i o n a n d e q u i l i b r i u m s o r p t i o n b e h a v i o r as w e l l as e x p e r i m e n t a l c o n d i t i o n s s u c h as h e a t i n g rate. I n a s i m p l e d e s o r p t i o n process w h e r e


4.

W I L D N A U E R

E

T A

L

.

Characterization

of Stratum

there a r e n o significant t h e r m a l l y i n d u c e d changes

Corneum

89

i n the membrane

m a t r i x , there is a g r a d u a l increase i n t h e d e s o r p t i o n rate w i t h t e m p e r a ture.

T h e d e s o r p t i o n rate reaches a m a x i m u m a n d b e g i n s to decrease

w h e n t h e g r a d i e n t o f s o r b e d w a t e r i n t h e m e m b r a n e is r e d u c e d to t h e extent t h a t i t o v e r s h a d o w s

t h e influence o f t h e increase i n diffusion

coefficient w i t h t e m p e r a t u r e . I n m o r e c o m p l e x systems s u c h as s t r a t u m c o r n e u m , h e a t i n g p r o d u c e s s t r u c t u r a l changes i n t h e m a t r i x w h i c h a r e reflected i n the shape o f the r e s u l t a n t t h e r m a l d e s o r p t i o n curves. Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

E x a m p l e s o f t h e r e s u l t i n g plots o f t h e d e s o r p t i o n rate vs. t e m p e r a ture a r e s h o w n i n F i g u r e 12. U n t r e a t e d c o r n e u m samples e x h i b i t o n e m a x i m u m at a b o u t 8 0 ° C w h e r e a s ether e x t r a c t i o n ( 9 0 m i n ) p r o d u c e s a second l o w e r t e m p e r a t u r e m a x i m u m i n a d d i t i o n to the h i g h e r t e m p e r a t u r e peak.

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

of

loosely b o u n d w a t e r w h i c h c a n diffuse o u t o f t h e c o r n e u m m o r e e a s i l y than t h e p r i m a r y sorbed water.

T h e thermogram for the c h l o r o f o r m -

m e t h a n o l - e x t r a c t e d c o r n e u m reveals a single, b r o a d p e a k i n d i c a t i v e of a m o r e g e n e r a l solvent d a m a g e to t h e c o r n e u m m a t r i x . T h e t h e r m a l d e -

A;

\

1 f I

\ \

ι t

/ /

LU Ύ ζ / Ο ί α. ο: Ο CO

''n. / / \/ \

Ί

\

1

1

/ \ 1

\

\

/'

\

D\

y

LU Ω

ι

40

ι

1

60

I

\ \ \\ \ \\\

1

80

TEMPERATURE °C Figure 12. Thermal desorption of water from newborn rat stratum corneum. A: control new born rat, B: extracted 90 min with ether, C: formaldehyde crosslinked, D: extracted 90 min with chloroform-methanol (3:1); heating rate 5° C J min.


90

A P P L I E D

C H E M I S T R Y

A

T

P R O T E I N

I N T E R F A C E S

s o r p t i o n c u r v e for f o r m a l d e h y d e c r o s s l i n k e d c o r n e u m also peaks at a l o w e r t e m p e r a t u r e t h a n the c o n t r o l . T h i s effect is a s c r i b e d to the sample's reduced water-holding capacity coupled w i t h a higher initial desorption rate. A t this e a r l y stage of t e c h n i q u e d e v e l o p m e n t , c h a r a c t e r i z a t i o n of s t r a t u m c o r n e u m b y t h e r m a l d e s o r p t i o n is u s e f u l o n l y to s u r v e y q u a l i t a t i v e l y the effect agents h a v e o n the t h e r m a l b e h a v i o r of c o r n e u m . T h e m o r e c o n v e n t i o n a l m e t h o d for s t u d y i n g t h e energetics of d i f f u s i o n i n m e m b r a n e s is to p e r f o r m p e r m e a t i o n e x p e r i m e n t s as a f u n c t i o n Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

of e q u i l i b r i u m t e m p e r a t u r e . F i g u r e 13 illustrates the effect of t e m p e r a t u r e o n the a p p a r e n t d i f f u s i o n coefficient c a l c u l a t e d f r o m the w a t e r v a p o r permeation

time

lag established b y

steady-state

permeation

with

a

75 to 0 % R H g r a d i e n t across the m e m b r a n e . T h e p r i n c i p l e s of the t i m e l a g p e r m e a t i o n m e t h o d are a d e q u a t e l y d i s c u s s e d e l s e w h e r e ( 5 8 ) .

The

l o w e r c u r v e corresponds to a s a m p l e w h i c h w a s n o t m e c h a n i c a l l y s u p p o r t e d a n d w a s o b s e r v e d to d e f o r m i n t o a h e m i s p h e r i c a l shape. d e f o r m a t i o n is t h e c o m b i n e d result of a s m a l l pressure difference

This across

the m e m b r a n e a n d a decrease i n m o d u l u s of s t r a t u m c o r n e u m as t h e t e m p e r a t u r e is i n c r e a s e d . T h e u p p e r c u r v e corresponds to a s u p p o r t e d s a m p l e . P r e v i o u s to t h e e x p e r i m e n t , b o t h samples h a d i d e n t i c a l t h e r m a l histories.

Stresses a c c o m p a n y i n g d e f o r m a t i o n of the u n s u p p o r t e d

1 0 0 0 / Τ , °K"

1

Figure 13. Temperature dependence of apparent diffusion coefficient of water vapor in newborn rat stratum corneum, Oto 75% RH vapor gradient. Open circles, supported membrane; closed circles, unsup ported membrane. Thickness assumed constant for the purposes of calculation.


cor-

4.

W I L D N A U E R

E

T

A L .

Characterization

of Stratum

91

Corneum

Table III. Diffusion Coefficients for Water in Newborn Rat Stratum Corneum

Method

Conditions

Nominal Water Content, g H 0/g Dry Tissue

T h e r m a l desorption

bulk water vapor bulk water vapor

4.80 0.35 1.5 0.35

1.9 9.5 1.4 1

1.5 0.35

1.2 X 2.1 X

2


Permeation lag time S t e a d y state flux and sorption values a

3

bulk water vapor

a

a

D , cm 1 sec 2

Χ ΙΟ" Χ ΙΟ" Χ ΙΟ" Χ ΙΟ"

11

12

1010-

10 11

1 0 1 1

H - H 0 radio tracer method. 2

n e u m g r e a t l y alter the d i f f u s i o n b e h a v i o r .

T h e d e f o r m e d s a m p l e has a

d i f f u s i o n coefficient a n o r d e r of m a g n i t u d e l o w e r t h a n t h a t of the u n d e f o r m e d s a m p l e at 5 0 ° C . U p o n c o o l i n g the samples i n t h e d i f f u s i o n c e l l to r o o m t e m p e r a t u r e , the d i f f u s i o n coefficients of n e i t h e r the s u p p o r t e d n o r the u n s u p p o r t e d samples r e t u r n to the i n i t i a l l y o b s e r v e d m a g n i t u d e . T h e r m a l s o r p t i o n hysteresis f r o m a c c u m u l a t e d stresses c r e a t e d as the s a m p l e cools i n the m e c h a n i c a l constraints of the d i f f u s i o n c e l l c o u l d a c c o u n t for the a l t e r e d diffusion coefficients.

T h e i r r e v e r s i b l e c h a n g e i n the a p p a r e n t

d i f f u s i o n coefficient for the u n s u p p o r t e d m e m b r a n e is c a u s e d b y a l t e r a tions i n the c o r n e u m m a t r i x r e s u l t i n g f r o m the d e f o r m a t i o n .

T h e mean

energy

membrane

of a c t i v a t i o n f o r w a t e r d i f f u s i o n i n the s u p p o r t e d

o v e r t h e range s t u d i e d is 18 k c a l / m o l e .

T h i s agrees w e l l w i t h

that

r e p o r t e d for b u l k diffusion of w a t e r i n h i g h l y s w o l l e n h u m a n c o r n e u m (59).

T a b l e I I I lists d i f f u s i o n coefficients

methods:

c a l c u l a t e d via t h r e e different

i n i t i a l d e s o r p t i o n rate, p e r m e a t i o n t i m e l a g , a n d

steady-state

flux c o m b i n e d w i t h e q u i l i b r i u m d i s t r i b u t i o n values c a l c u l a t e d f r o m w e l l k n o w n formulas (58).

F o r e a c h m e t h o d , a c o m p a r i s o n is m a d e

between

the d i f f u s i o n coefficient for w a t e r f r o m v a p o r a n d t h a t f r o m b u l k l i q u i d . As observed

b y others

(15),

the d i f f u s i o n coefficient

of w e t

corneum

is s o m e w h a t h i g h e r t h a n t h a t of d r y corneum—i.e., t h e presence of l a r g e a m o u n t s of i m b i b e d w a t e r appears to p l a s t i c i z e the m a t r i x . E x c e p t for the d e s o r p t i o n v a l u e , t h e results represent g o o d agreement a m o n g t h e v a r i o u s methods e x p l o r e d a n d g o o d agreement w i t h a p r e v i o u s l y r e p o r t e d v a l u e of 5 χ

10"

11

c m / s e c for n e w b o r n rat c o r n e u m 2

(60).

The low

value

o b t a i n e d f r o m the d e s o r p t i o n of the w e t c o r n e u m most l i k e l y results f r o m its h i g h l y s w o l l e n c o n d i t i o n .

A s has a l r e a d y b e e n s h o w n , w a t e r tends

to cluster i n this system at v a p o r pressures w e l l b e l o w s a t u r a t i o n . B e cause the c l u s t e r e d w a t e r is n o t d i s s o l v e d i n the m a t r i x , i t c a n n o t t r i b u t e to the d r i v i n g force of the diffusion.

con

C o r r e c t i n g for the cluster


92

A P P L I E D

C H E M I S T R Y

A

T

P R O T E I N

I N T E R F A C E S

effect w i l l effectively increase the m a g n i t u d e o f the d i f f u s i o n coefficient. T h e a p p a r e n t d e p r e s s i o n o f the d i f f u s i o n coefficient o f v a p o r s i n s y n t h e t i c p o l y m e r systems has b e e n a t t r i b u t e d to a s i m i l a r c l u s t e r i n g effect (61). Physical

and Chemical

Properties

T h e r m a l B e h a v i o r . T h e most c h a r a c t e r i s t i c p a r a m e t e r o f a n a m o r p h o u s p o l y m e r is the glass t r a n s i t i o n t e m p e r a t u r e (T ).

I n the glass

g


t r a n s i t i o n r e g i o n , a viscoelastic t r a n s i t i o n occurs as a result o f t h e onset of m o t i o n s o f c h a i n segments i n the a m o r p h o u s r e g i o n of the p o l y m e r w h i c h transforms the m a t e r i a l f r o m a r i g i d state t o a r u b b e r y one. T h e m e c h a n i s m o f d e f o r m a t i o n response b y t h e m a t e r i a l is d e p e n d e n t o n T , g

a n d i t d e t e r m i n e s the d u c t i l i t y a n d brittleness o f the p o l y m e r . T h e u s u a l m e t h o d for d e t e r m i n i n g T is t o m e a s u r e the t e m p e r a t u r e a t w h i c h the g

specific v o l u m e - t e m p e r a t u r e p l o t shows a n i n f l e c t i o n i n d i c a t i n g a n i n creased t h e r m a l e x p a n s i o n coefficient.

I n general, a l l p h y s i c a l properties

of a m o r p h o u s p o l y m e r s w h i c h are d e p e n d e n t rate s u c h as viscous

flow,

o n segmental relaxation

m e c h a n i c a l a n d d i e l e c t r i c r e l a x a t i o n , creep,

a n d d i f f u s i o n s h o w a m a j o r c h a n g e o n h e a t i n g t h r o u g h the glass t r a n s i t i o n region.

Similarly, T can b e determined b y differential scanning calo g

r i m e t r y ( D S C ) f r o m t h e t e m p e r a t u r e at w h i c h t h e r e is a s u d d e n c h a n g e i n the specific heat o f the s a m p l e .

T depends g

also o n the m o l e c u l a r

w e i g h t o f the p o l y m e r , o n i n t e r n a l s t r a i n , a n d t o a lesser extent o n h e a t i n g rate. Q u e n c h e d , h i g h l y a m h o r p h o u s p o l y m e r s g e n e r a l l y d i s p l a y a m o r e p r o n o u n c e d i n f l e c t i o n w h e n h e a t e d t h r o u g h the T r e g i o n (63). g

Ο

u

L i

Ο

ι

ι

l l i

40 80 120 TEMPERATURE ( ° C )

ι

160

ι

200

—

ΐ -

240

Figure 14. DSC scan of guinea pig footpad: A , desiccated control; B, hydrated in water 45 min; and C , high sensitivity of dry control (hermetically sealed Ν atm, 20°C/min heating rate). Data from Ref. 42. 2



4.

W I L D N A U E R

E

T

A L .

1

Characterization

of Stratum

!

-40

0

Corneum

93

u_ 40

80

TEMPE RATURE(°C) Figure 15. DSC scan of guinea pig footpad: A, control; B, rerun of rapidly cooled; C , rerun of slowly cooled; and D, rerun of C after 48 hrs N atm (samples of comparable weight, 20°C/min heating rate). Data from Ref. 42. 2

A

D S C scan of

stratum corneum

( F i g u r e 14)

indicates several

t h e r m a l l y i n d u c e d transformations f r o m 0 ° to 2 5 0 ° C .

As standard con-

d i t i o n s , a l l samples w e r e c o o l e d to — 4 0 ° C a n d h e a t e d at 2 0 ° C / m i n i n a stream of d r y n i t r o g e n . T o a v o i d the e n d o t h e r m i c c o n t r i b u t i o n of w a t e r e v a p o r a t i o n , a l l samples w e r e h e r m e t i c a l l y sealed a n d w e i g h e d a n d after the scan. T h e t h e r m o g r a m for d e s i c c a t e d c o r n e u m ( ^ H 0) 2

before 5 wt %

exhibits a n a b r u p t c h a n g e i n specific heat at 48 ° C suggesting a

glass t r a n s i t i o n .

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

T y of v a r i o u s n y l o n s a n d other p o l y a m i d e s (63,

64).

T h i s 48 ° C t r a n s i t i o n i n d r y c o r n e u m is a n o m o l o u s i n t h a t i t does n o t fit a l l the c r i t e r i a u s u a l l y associated w i t h c l a s s i c a l glass transitions. F o r e x a m p l e , once t h e s a m p l e has b e e n h e a t e d t h r o u g h the T

0

is n o c o r r e s p o n d i n g specific heat c h a n g e o n c o o l i n g .

region, there

Immediate reheat-

i n g after a s l o w c o o l i n g c y c l e d i s p l a y s little or n o glass t r a n s i t i o n ( F i g u r e 1 5 ) . T h e t r a n s i t i o n appears at s l i g h t l y l o w e r t e m p e r a t u r e s o n the second c y c l e w h e n the s a m p l e is r a p i d l y q u e n c h e d b e l o w T

tJ

before reheating.

I n this case, the specific heat c h a n g e is c o n s i d e r a b l y r e d u c e d f r o m t h a t o b s e r v e d i n the first c y c l e , a n d a second glass appears at 9 2 ° C . T h e 4 8 ° C


94

A P P L I E D

C H E M I S T R Y

A

T

P R O T E I N

I N T E R F A C E S

t r a n s i t i o n does b e g i n to r e a p p e a r for t h e s l o w - c o o l e d samples after a f e w h o u r s b u t at s l i g h t l y l o w e r t e m p e r a t u r e s . F i g u r e 16 demonstrates the effect o n the glass t r a n s i t i o n of a n n e a l i n g stratum corneum

at 7 5 ° C for

18 hrs.

W h e n the s a m p l e is

immediately following slow cooling from o n l y t h e 9 2 ° C glass t r a n s i t i o n appears.

scanned

the a n n e a l i n g t e m p e r a t u r e ,

A n n e a l e d samples a l l o w e d to

rest i n a d e s i c c a t o r for 72 hrs e x h i b i t e d glass transitions at b o t h a n d 92 ° C .

The lowered T

g


arise f r o m i n d u c e d i n t e r n a l stress o n c o o l i n g t h r o u g h O n e possible

42°

g e n e r a l l y o b s e r v e d o n the second c y c l e m a y

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

this a n o m o l o u s

T. g

glass t r a n s i t i o n i n

terms of m o l e c u l a r s t r u c t u r e a n d r e a c t i v i t y is b a s e d o n the suggestions f r o m s i m p l e r p o l y a m i d e systems s u c h as n y l o n . It is g e n e r a l l y h e l d t h a t this t r a n s i t i o n originates w i t h the r u p t u r e of i n t e r c h a i n h y d r o g e n b y t h e m o t i o n of l o n g - c h a i n segments i n the a m o r p h o u s regions.

bonds The

u n u s u a l aspects of the glass t r a n s i t i o n is b e l i e v e d i n p a r t to b e r e l a t e d to the u n i q u e s t r u c t u r e of p o l y a m i d e s w i t h a l t e r n a t i n g n o n p o l a r c h a i n seg ments a n d s t r o n g l y h y d r o g e n - b o n d i n g sites a l o n g the p o l y m e r c h a i n ( 6 5 ) . S i n c e the h y d r o g e n - b o n d i n g sites o c c u r o n l y at i n t e r v a l s a l o n g t h e c h a i n , steric factors h i n d e r the f o r m a t i o n of the n e t w o r k a n d m a y e x p l a i n the time dependence

of its r e t u r n . T h i s e x p l a n a t i o n is f u r t h e r s u b s t a n t i a t e d

b y t h e f a c t that this t r a n s i t i o n has b e e n o b s e r v e d i n the 4 0 ° to 50 ° C r e g i o n for p o l y a m i d e s i n w h i c h the n u m b e r of m e t h y l e n e groups b e t w e e n

Figure 16. DSC scan of guinea pig footpad: A , control; B, annealed at 75°C 18 hrs; and C, rerun of Β after 72 hrs N atm (samples of com parable weight). Data from Ref. 42. 2



4.

W I L D N A U E R

E

T

A L .

0

Characterization

of Stratum

95

Corneum

40 80 120 160 ELONGATION (%>) Journal of Investigative Dermatology

Figure

17. Force-extension curves at various for human stratum corneum (9)

RH levels

p o t e n t i a l h y d r o g e n - b o n d i n g sites o n t h e p o l y m e r b a c k b o n e w a s v a r i e d f r o m 4 t o 11 (63). H e n c e , the t r a n s i t i o n o b s e r v e d at 4 0 ° - 5 0 ° C i n the p o l y a m i d e structures appears t o d e p e n d

not o n the

flexibility

of the

n e t w o r k b u t o n the existence a n d d i s r u p t i o n o f h y d r o g e n b o n d s at p o i n t s t h r o u g h o u t the a m o r p h o u s regions o f the p o l y m e r . S i n c e the p r i m a r y s t a b i l i z i n g forces i n the glass a p p e a r t o b e h y d r o g e n b o n d s , this t r a n s i t i o n is h i g h l y sensitive to t h e presence o f w a t e r a n d other h y d r o g e n - b o n d i n g

molecules

i n t h e amorphous

regions.

Water

t h e n acts as a p l a s t i c i z e r o f the a m o r p h o u s p r o t e i n regions o f c o r n e u m as n o t e d b y the r e l a t i o n s h i p b e t w e e n the extent o f w a t e r f o u n d a n d the degree to w h i c h T is r e d u c e d . A l s o , there a p p e a r to b e t w o a m o r p h o u s g

regions w h i c h differ i n t h e i r a c c e s s i b i l i t y b y w a t e r at a m b i e n t t e m p e r a ture. T h e T g for the m o s t accessible r e g i o n shifts t o —18° C at 4 0 %

H 0

w h i l e t h e less accessible is o n l y l o w e r e d t o a p p r o x i m a t e l y 35 ° C

(82).

2

T h e T g of c o r n e u m at w a t e r contents less t h a n 5 w t % is difficult to determ i n e since t h e r e m a i n i n g w a t e r is t i g h t l y h e l d b y p r i m a r y s o r p t i o n sites a n d t e c h n i c a l l y difficult to r e m o v e w i t h o u t i n t r o d u c i n g s t r u c t u r a l a l t e r a tions. H e a t i n g the c o r n e u m a b o v e 1 0 0 ° C to r e m o v e this f r a c t i o n o f w a t e r results i n a T at 9 2 ° C . g

It is n o t c l e a r w h e t h e r this shift i n T

g

a t t r i b u t a b l e to the l o w e r w a t e r content

t o h i g h e r t e m p e r a t u r e is

o r to the a n n e a l i n g process.

T h e r e is a n a b r u p t decrease i n the m a g n i t u d e o f the associated specific heat c h a n g e at a s a m p l e w a t e r content o f a b o u t 15 w t % . I n this w a t e r content r a n g e ( 1 5 - 2 0 w t % ) force-extension curves f o r s t r a t u m c o r n e u m first b e g i n to d i s p l a y a y i e l d phase ( F i g u r e 17) as w e l l as t h e i n i t i a t i o n


96

A P P L I E D

C H E M I S T R Y

of a d r a m a t i c d r o p i n tensile m o d u l u s as cluster c a l c u l a t i o n s ( 5 7 ) water

A T

( F i g u r e 18).

P R O T E I N

I N T E R F A C E S

T h e s e d a t a as w e l l

s t r o n g l y suggest that w a t e r s o r b e d at these

contents p l a s t i c i z e t h e a m o r p h o u s

regions

associated

with

the

observed T . g

A n a d d i t i o n a l major t r a n s i t i o n w h i c h is o b s e r v e d i n D S C scans of stratum corneum

is a d o u b l e t

endotherm

w h i c h peaks

2 1 0 ° C i n d r y samples a n d at 1 2 0 ° - 1 3 0 ° C i n w e t samples

at 194 ° C

and

(Figure

14).

T h e s e transitions are also c h a r a c t e r i s t i c of t h e m o r e extensively i n v e s t i -


g a t e d k e r a t i n - c o n t a i n i n g w o o l (49, 6 5 ) .

P o l y a m i d e s s u c h as t h e v a r i o u s

n y l o n s also s h o w m e l t i n g endotherms a b o v e 200 ° C

(63).

B e l o w 200° C , t h e h e a t - i n d u c e d changes i n d r y w o o l s t r u c t u r e are c o n f i n e d to t h e a m o r p h o u s parts of t h e p r o t e i n . A b o v e 2 0 0 ° C t w o m e l t i n g e n d o t h e r m s are present, a s m a l l one at 215 ° C f r o m the m e l t i n g of a l o w c r o s s l i n k e d f r a c t i o n of the h e l i x a n d a major one at 2 3 5 ° C as a result of t h e m e l t i n g of t h e h i g h e r c r o s s l i n k e d f r a c t i o n of t h e h e l i x ( 4 9 ) .

In

w o o l , the m o s t d i r e c t e v i d e n c e for this i n t e r p r e t a t i o n of h e l i x m e l t i n g is p r o v i d e d

b y x-ray d i f f r a c t i o n patterns at the v a r i o u s

20 RELATIVE

60 HUMIDITY

temperatures.

100 (%»)

Figure 18. Tensile modulus as a function of RH for newborn rat stratum corneum. Data from Ref. 82.


4.

W I L D N A U E R

E

T

A L .

Characterization

of Stratum

Corneum

97

60

LU υ Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

:40

/Λ

/

Σ If)

< 20 ο:

ι

ι

1700 1600 1500 F R E Q U E N C Y (CM"' ) Figure 19. IR transmission spectra for newborn rat stratum corneum at various temperatures: A, 25°C; B, 125°C; C, 250°C. Data from Ref. 18. B o t h t h e 5.1-A s p a c i n g c h a r a c t e r i s t i c of the s p a c i n g of h e l i x turns a n d the 9.8 A of the l a t e r a l h e l i c a l s p a c i n g decrease i n i n t e n s i t y at 210° a n d 230°C (49).

T h e fiber also loses its b i r e f r i n g e n c e i n this t e m p e r a t u r e

range. T h e s e reflections are not present i n fibers w h i c h h a v e b e e n s u p e r contracted or otherwise rendered amorphous.

S i m i l a r l y , the x - r a y d i f

f r a c t i o n p a t t e r n of w o o l h e a t e d to 130 ° C w h i l e i m m e r s e d i n w a t e r shows the t o t a l d i s a p p e a r a n c e of the a l p h a - k e r a t i n reflections a n d the a p p e a r ance of a d i s o r d e r e d b e t a p a t t e r n (66). fiber

W h e n either the d r y or the w e t

is h e a t e d a b o v e these m e l t i n g t e m p e r a t u r e s , the t r a n s f o r m a t i o n

is i r r e v e r s i b l e . I n f l u e n c e of W a t e r . E v e n i n d e s i c c a t e d c o r n e u m there is a w e a k , b r o a d e n d o t h e r m c e n t e r i n g at a b o u t 1 2 0 ° - 1 3 0 ° C as w e l l as the h i g h e r t e m p e r a t u r e d o u b l e t melts at 1 9 4 ° C a n d 2 1 0 ° C .

B o t h melts a p p e a r to

b e s o m e w h a t l o w e r i n t e m p e r a t u r e for c o r n e u m t h a n for w o o l , w h i c h is consistent w i t h the a c c e p t e d h i g h e r degree of o r i e n t a t i o n a n d h e l i c a l content of w o o l .

T h e 130 ° C t r a n s i t i o n has b e e n r e p o r t e d i n other

p o l y p e p t i d e systems b y D S C , I R , a n d x - r a y d i f f r a c t i o n w h e r e i t w a s f o u n d to b e h i g h l y m o i s t u r e sensitive (67).

It has b e e n suggested t h a t

this e n d o t h e r m i c process w h i c h occurs b e t w e e n

110°

a n d 150 ° C i n a

n u m b e r of p o l y p e p t i d e systems is a p a r t i a l c o n v e r s i o n f r o m the a l p h a -


98

A P P L I E D

C H E M I S T R Y

A

T

P R O T E I N

I N T E R F A C E S

h e l i c a l f o r m to a b e t a c o n f i g u r a t i o n . T h i s is s u p p o r t e d b y t h e a p p e a r a n c e of a 4.7-A s p a c i n g a n d a shift i n t h e a m i d e I a n d I I b a n d s i n the I R spectra (68, 69). B o t h these changes are c h a r a c t e r i s t i c o f the c o n v e r s i o n f r o m the i n t r a c h a i n h y d r o g e n - b o n d e d a l p h a to t h e i n t e r c h a i n h y d r o g e n bonded beta configuration. Transmission I R of d r y corneum at various temperatures from 2 5 ° to 2 5 0 ° C are s h o w n i n F i g u r e 19. F r o m a b o u t 1 2 0 ° C to 2 5 0 ° C t h e r e is a shift i n a m i d e I a n d I I b a n d s f r o m 1660 to 1640 c m "

1

a n d f r o m 1550

to 1520 c m " , r e s p e c t i v e l y , w h i c h is consistent w i t h a n a l p h a - t o - b e t a t r a n s Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

1

formation (44).

T h i s t r a n s i t i o n coincides w i t h the b r o a d b u t w e a k e n d o -

t h e r m at 1 2 0 ° C i n t h e D S C scans o f d r y c o r n e u m ( F i g u r e 1 4 ) . T h e i n t e n s i t y o f t h e 1 2 0 ° - 1 3 0 ° C e n d o t h e r m increases as s t r a t u m c o r n e u m m o i s t u r e content increases; there is a c o r r e s p o n d i n g i n t h e h i g h t e m p e r a t u r e m e l t i n g s at 194° a n d 2 1 0 ° C ( F i g u r e 2 0 ) .

decrease Highly

h y d r a t e d c o r n e u m d i s p l a y s t h e 1 2 0 ° - 1 3 0 ° C e n d o t h e r m w h i l e b e l o w 15 to 2 0 % w a t e r content, the t r a n s i t i o n is q u i t e s m a l l . T h e s e D S C m e a s u r e ments w e r e p e r f o r m e d i n h e r m e t i c a l l y sealed pans to a v o i d t h e e n d o t h e r m i c loss o f w a t e r .

T h e t r a n s i t i o n i n t h e presence

of w a t e r is a

c o o p e r a t i v e o n e i n that w a t e r facilitates the m a g n i t u d e o f t h e o b s e r v e d transition.

Ο

T h e heat c h a n g e associated w i t h t h e 1 2 0 ° - 1 3 0 ° C t r a n s i t i o n

20 RELATIVE

40 HUMIDITY

60

80

100

(%>)

Figure 20. Rehtive energy associated with DSC 120°C endotherm in newborn rat stratum corneum at various RH levels (hermetically sealed system). Data from Ref. 42.


4.

W I L D N A U E R

E

T

A L .

Characterization

of Stratum

99

Corneum

is a m a x i m u m at 9 8 % R H a n d is n o t i n c r e a s e d f u r t h e r b y i m m e r s i o n i n water. T h e influence of v a r i o u s solvents

Influence of Nonpolar Solvent.

o n the p h y s i c a l a n d c h e m i c a l p r o p e r t i e s of c o r n e u m has b e e n s t u d i e d w i d e l y ( 70, 71 ) w i t h p a r t i c u l a r interest i n t h e i r influence o n w a t e r b i n d ing.

I n a d d i t i o n to t h e e x t r a c t i o n of l i p i d s b y the n o n p o l a r solvents, the

samples are s u b s e q u e n t l y m o r e s u s c e p t i b l e to f u r t h e r d a m a g e b y w a t e r . Extracted

samples

have

lowered


mechanical behavior (9, 70).

w a t e r - b i n d i n g affinity a n d

altered

L i p i d - s o l u b l e materials removed b y n o n -

p o l a r solvents p e r f o r m a p r o t e c t i v e r o l e i n p r e v e n t i n g the loss of w a t e r s o l u b l e c o m p o n e n t s r e s p o n s i b l e for the w a t e r b i n d i n g at h i g h r e l a t i v e humidities. DSC

scans of ether a n d c h l o r o f o r m - m e t h a n o l

(3/1

by

volume)

e x t r a c t e d samples e x h i b i t b r o a d e r a n d less d e t a i l e d m e l t i n g e n d o t h e r m s at 194° a n d 2 1 0 ° C t h a n u n t r e a t e d samples.

A n a d d i t i o n a l difference is

s h o w n b y c h l o r o f o r m - m e t h a n o l - e x t r a c t e d samples w h e n q u e n c h c o o l e d and rerun.

I n reruns of c o n t r o l a n d ether-extracted samples, T

is still

g

rather s h a r p a l t h o u g h r e d u c e d i n a m o u n t a n d t e m p e r a t u r e w h i l e the c h l o r o f o r m - m e t h a n o l - e x t r a c t e d s a m p l e is q u i t e b r o a d w i t h a s e c o n d a p p a r ent glass at a b o u t

90°-100°C

(42).

E i t h e r the w a t e r - s o l u b l e

mate

rials act as p l a s t i c i z e r s or t h e i r loss t h r o u g h solvent e x t r a c t i o n causes s t r u c t u r a l changes i n the proteins w h i c h i n h i b i t r e f o r m i n g of the o r i g i n a l glass. It w i l l be s h o w n later that d y n a m i c s p e c t r o s c o p y demonstrates a h i g h e r tensile m o d u l u s for c h l o r o f o r m - m e t h a n o l - e x t r a c t e d samples t h a n the c o n t r o l suggesting a s t r u c t u r a l r e o r g a n i z a t i o n has o c c u r r e d

(14).

Influence of Orientation. I n d u c e d o r i e n t a t i o n i n s t r a t u m c o r n e u m was a c h i e v e d b y s t r e t c h i n g h y d r a t e d samples to v a r y i n g degrees a n d a l l o w i n g t h e m to d r y i n that e l o n g a t e d state. D S C scans of these o r i e n t e d samples d i s p l a y a n u m b e r of alterations i n the m e l t e n d o t h e r m s a n d glass t r a n s i t i o n w h i c h v a r y w i t h the extent of p r e s t r e t c h i n g a n d m a y p r o v i d e some i n s i g h t i n t o t h e m o l e c u l a r m e c h a n i s m s r e s p o n s i b l e for elasticity. T h e r e is a g r a d u a l r e d u c t i o n i n T w i t h some b r o a d e n i n g a n d loss of g

d e f i n i t i o n of t h e h i g h t e m p e r a t u r e e n d o t h e r m s n e a r 2 0 0 ° C as the a m o u n t of p r e s t r e t c h increases ( F i g u r e 2 1 ) .

T h e orientation produced b y

d e f o r m a t i o n s appears to p r o d u c e a n i n t e r n a l stress i n the

low

amorphous

regions w h i c h acts as a n external l o a d to l o w e r the softening t e m p e r a t u r e (72). Secondary Transitions. I n a d d i t i o n to the a n o m a l o u s glass t r a n s i t i o n i n the 4 0 ° - 5 0 ° C r e g i o n because of t h e m o t i o n of large segments of the polymer

chain, stratum corneum

like many

other

s m a l l e r s e c o n d a r y transitions at l o w e r temperatures.

polymers

displays

T h e s e arise g e n

e r a l l y f r o m the m o t i o n of side chains or the s m a l l segments of t h e b a c k bone.

T h e t a n δ b e h a v i o r f r o m d y n a m i c m e c h a n i c a l studies of


dry

100

A P P L I E D

C H E M I S T R Y

s t r a t u m c o r n e u m i n d i c a t e s m a l l peaks (14)

A

T

P R O T E I N

I N T E R F A C E S

at — 1 0 ° a n d — 6 0 ° C s u g

g e s t i n g s e c o n d a r y transitions. T h e t a n δ f o r the d i e l e c t r i c p r o p e r t i e s of n e w b o r n r a t s t r a t u m c o r n e u m also demonstrates a t r a n s i t i o n at —10° C w i t h a n a p p a r e n t a c t i v a t i o n e n e r g y of 7 k c a l / m o l e

(6).

S i m i l a r r e l a x a t i o n p h e n o m e n a are o b s e r v e d i n other p o l y a m i d e sys 50/50


tems s u c h as the p r o t e i n c o l l a g e n a n d the s y n t h e t i c p o l y p e p t i d e

E L O N G A T I O N

(%>)

Figure 21. Glass transition temperature of prestretched guinea pig footpads (N

2

(from DSC) vs. elongation aim). Data from Ref. 42.


4.

wiLDNAUER E T

AL.

Characterization

of Stratum

101

Corneum


Amorphous oriented

Journal of Polymer Science

Figure 22.

Diagramatic

representation of the longitudinal of oriented fibers (77)

L-glutamic

acid-L-leucine.

The

shrinkage

behavior

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

m a t e r i a l s d i s p l a y h i g h l y m o i s t u r e - s e n s i t i v e relaxations a t —13° tween - 6 0 °

and be-

a n d - 9 0 ° C (73, 74).

Spontaneous Dimensional Changes.

O n e technique used to approxi-

m a t e the degree o f o r i e n t a t i o n i n o p a q u e a m o r p h o u s m a t e r i a l s is t o m e a s u r e t h e a m o u n t o f s h r i n k a g e o r m a g n i t u d e o f r e t r a c t i v e force w h e n t h e y are h e a t e d .

W h e n a n oriented amorphous

developed

p l a s t i c sheet is

h e a t e d a b o v e T , i t s h r i n k s b a c k to t h e a p p r o x i m a t e shape i t h a d b e f o r e g

orientation.

T h e r m o d y n a m i c a l l y this represents a n a t t e m p t b y t h e o r i -

e n t e d p o l y m e r s to a t t a i n a state o f m a x i m u m d i s o r d e r ( e n t r o p y ) .

I f the

film is h e l d at constant l e n g t h , i t generates a r e t r a c t i v e f o r c e w h e n h e a t e d a b o v e T s u c h that a n i n c r e a s e d r e t r a c t i v e f o r c e means a greater degree o f g

o r i e n t a t i o n i n the s a m p l e . I n the latter case, the r e t r a c t i v e f o r c e g e n e r a t e d is also d e p e n d e n t o n the m o d u l u s o f the m a t e r i a l (4, 72, 75, 76). T h e s h r i n k a g e b e h a v i o r o f a h y p o t h e t i c a l , o r i e n t e d fiber c o m p o s e d o f varying proportions F i g u r e 22.

of amorphous

S h r i n k a g e first occurs

a n d c r y s t a l l i n e phases is s h o w n i n i n the r e g i o n o f T . A m o d e l g

fiber

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


102

A P P L I E D

C H E M I S T R Y

A

T

P R O T E I N

I N T E R F A C E S

w o u l d undergo partial shrinkage around T but w o u l d show n o further g

s h r i n k a g e u n t i l the c r y s t a l l i n e m e l t .

A completely amorphous

oriented

system w o u l d o n l y d i s p l a y s h r i n k a g e at T (77). g

I t is a p p a r e n t f r o m F i g u r e 23 that the t h e r m a l l y i n d u c e d spontane ous contractions o f s t r a t u m c o r n e u m are q u i t e s i m i l a r to the h y p o t h e t i c a l m o d e l fiber c o m p o s e d o f a t w o - p h a s e system o f o r i e n t e d a m o r p h o u s a n d c r y s t a l l i n e phases.

T h e i n i t i a l c o n t r a c t i o n begins a t 5 0 ° C as p r e d i c t e d

f r o m the D S C d e t e r m i n a t i o n o f T . g

A s w o u l d be e x p e c t e d for a c r y s t a l l i n e


m e l t , there is a r a p i d loss o f m o d u l u s ( F i g u r e 2 4 ) , a l a r g e increase i n transverse thickness

(Figure

25),

and

a n endothermic

heat

process

a c c o m p a n y i n g the 196 ° C c o n t r a c t i o n . S i m i l a r b e h a v i o r is o b s e r v e d i n the α-keratin-containing h a i r (78).

A n e n e r g y o f a c t i v a t i o n o f 110 k c a l / m o l e

was c a l c u l a t e d f r o m the f r e q u e n c y d e p e n d e n c e o f d y n a m i c m e c h a n i c a l spectra o f c o r n e u m i n the 207 ° C r e g i o n . T h i s h i g h a c t i v a t i o n e n e r g y is i n d i c a t i v e o f t h e m o t i o n o f r a t h e r l a r g e segments o f the p o l y m e r c h a i n a n d is consistent w i t h a n a l p h a - t o - b e t a t r a n s f o r m a t i o n (14). A d d i t i o n a l e v i d e n c e for this t w o - p h a s e m o d e l for s t r a t u m c o r n e u m c a n b e d e m o n s t r a t e d b y m e a s u r i n g s h r i n k a g e as a f u n c t i o n o f tension. T h e degree o f s h r i n k a g e corresponds to d i s o r i e n t a t i o n o f the a m o r p h o u s phase a n d t h e successive

decrease i n s h r i n k a g e w i t h i n c r e a s e d

corresponds t o a n extension f r o m that d i s o r i e n t e d state ( 7 7 ) .

loads

As would

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

UJ S LJ Ο < -I Û. CO Q

Ο CO

X Ιϋ I Ο

UJ O CD
1 0 0 % ) .

T h e s e d a t a suggest that

e l o n g a t i o n is a c c o m p l i s h e d b y d e c r e a s i n g the d e g r e e o f o r i e n t a t i o n i n Downloaded by IMPERIAL COLLEGE LONDON on September 15, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch004

t h e a m o r p h o u s regions a n d p r o d u c i n g i n c r e a s e d o r i e n t a t i o n i n t h e c r y s t a l l i n e regions (72). T h e p r o p o s e d m e c h a n i s m agrees w i t h the i n c r e a s e d d i c h r o i s m o f the I R a m i d e I a n d I I b a n d s o f p r e s t r e t c h e d ( ~ 100%

samples

extension ) d i s c u s s e d e a r l i e r .

t

Ζ

ο

Iz UJ UJ

ο

Applied Chemistry at Protein Interfaces

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