Comparison of Weight and Energy Changes in the Absorption of

14 C o m p a r i s o n o f Weight a n d E n e r g y Changes i n the A b s o r p t i o n o f Water by C o l l a g e n a n d K e r a t i n

Downloaded by UNIV OF PITTSBURGH on May 17, 2016 | http://pubs.acs.org Publication Date: August 19, 1980 | doi: 10.1021/bk-1980-0127.ch014

M. ESCOUBES Université Claude Bernard, 69621 Villeurbanne, France M. PINERI Centre d'Études Nucléaires de Grenoble, 38041 Grenoble Cédex, France The collagen and keratin constitute the two most important protective fibrous proteins in living systems. The role of water in connection with these polymers in biological processes has been the object of a number of studies for more than a century but remains controversial. We have shown, in preceeding article concerning collagen (1), that the analysis of data for weight absorption and kinetics of absorption of water as studied by torsion pendulum experiment of hydrated collagen permit an improvement on literature data and suggests a hydration scheme involving five stages. This article concerns collagen fibers from rat tail tendon. The object is to confirm, improve or modify the proposed model by studying different collagen samples and secondly fibrous proteins such as the α keratin of human hair which exhibits similar hydration properties. 1. Review of results on collagen fiber from rat tail tendon This type of collagen has the advantage of illustrating the most recent structural data. It is necessary to review briefly this data to describe the proposed hydration model, and to aid in characterizing other samples of fibrous protein discussed later. The molecule of collagen has the form of a rigid rod (2) with a length of 2900 A and a diameter of 12.5 A with a molecular weight of 300 000. This rod consists of three helical polypeptide chains with three parallel axes separated by 4.5 Â (triple helix or tropocollagen). Each sequence of three residues (taken as a weight reference) possesses one glycine residue (-NH- CIL? - CO - ) and on average one proline or hydroxyproline (-N-CH-C0-) and has a molecular weight of about 270 gm. The three chain complex is stabilized essentially by hydrogen bonding between the CO and NH units. Models have been proposed C3,4) involving one and two hydrogen bonds for each group of three residues. In each model the stabilization is completed by two bridged water molecules. The destrucc

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0-8412-0559-0/ 80/47-127-235505.00/ 0 © 1980 American Chemical Society

Rowland; Water in Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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t i o n of hydrogen bonds by heating represents the majority of the enthalpy involved i n the h e l i x - c o i l or collagen-gelatin t r a n s i tions .


- Collagen molecules spontaneously form aggregates i n the form of m i c r o f i b r i l s . Electron microscopy data (5) suggest an assembly of f i v e molecules i n which each two adjacent molecules are s h i f t e d by a quarter of f h e i r length (allignment "quarter stagger") on a distance of 670 A. The succession of the two molecules assures an i n t e r v a l of 350 Â. This gives a free volume of the order of 10 % and a p e r i o d i c i t y of 670 A i n the dehydrated state. - F i n a l l y the j u x t a p o s i t i o n of m i c r o f i b r i l s i n a tetragonal arrangement l i m i t e d to the sublattices and largely associated with the amorphous region r e s u l t s i n a f i b e r diameter between 1000 and 5000 A. Molecular cohesion i n the m i c r o f i b r i l as w e l l as i n the amorphous regions i s due to two types of bonding. F i r s t l y weak covalent bonds (of the type ester, imide, peptide) between l a t e r a l groups of c e r t a i n residues as p a r t i e a c i d , glutamic a c i d , lysine) and secondly from very strong linkages located only at the non h e l i c a l end areas of the molecule (telopeptidique areas) which cause the eventual condensation of the two aldehyde derivatives of the l y s i n e and hydroxylysine forms. These two types of linkages can also be present between chains of the same molecule. The l a t t e r i s also largely responsible f o r the i n s o l u b i l i t y i n acid media of old collagen. Fibers of r a t - t a i l tendon contain few linkages i n the terminal telopeptidique areas. We have shown (J_) that these f i b e r s r e t a i n 1 % of water under a vacuum of 10~ t o r r at 20°C and that the hydration i n the vapor phase at 20°C shows 3 r e l a t i v e l y sharp steps i n terms of the energetic and mechanical data, before the appearance of free water near 90 % r e l a t i v e humidity. 4

- The f i r s t step between 0 and 10 % with a energy of 17 k c a l / mole (corresponding to the formation of double hydrogen bond without breaking other préexistant bonds) i s a t t r i b u t e d to i n t r a molecular water and corresponds q u a n t i t a t i v e l y to two water molecules per reference u n i t . - The second step between 10 and 25 % with an energy of 13 k c a l / mole may correspond to bridging of water between the collagen molecules occurring with a diminution of the cohesive energy of the microfibril. - The t h i r d step between 25 and 50 % with an energy of 10 k c a l / mole does not correspond to the presence of free water according to most of the data i n the l i t e r a t u r e . The decrease i n part arranged

l a t t e r occurs near 50 %. This step i s associated with a i n the c o e f f i c i e n t of d i f f u s i o n . We have attributed t h i s to the formation of water clusters w i t h i n the p e r i o d i c a l l y free volume zones i n the m i c o f i b r i l .


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Collagen and Keratin

237

Thus the hydration of r a t - t a i l tendon collagen f i b e r s appears to be connected with d i f f e r e n t s t r u c t u r a l l e v e l s of the protein. It i s i n t e r e s t i n g to confirm t h i s pattern by studying other f i brous proteins. 2. Sample characterization


2.1 - Collagen samples - Turkey leg tendon at 22 months. This tissue i l l u s t r a t e s the c a l c i f i c a t i o n of collagen produced with time. The mineral-protein i n t e r a c t i o n has been l a r g e l y studied by small angle X-ray studies (10), electron microscopy (11) and neutron d i f f r a c t i o n (12). I t seems to be established that calcium phosphate (mostly apatite crystals) i s d i s t r i b u t e d along the length of f i b e r with same p e r i o d i c i t y of 670 À which characterizes the s h i f t of two adjacent collagen molecules. Despite the absence of information concerning the l a t e r a l arrangement, i t seems reasonable to speculate that the mineral i s developed not only at the l e v e l of the f i b e r but also i n the holes of the m i c r o f i b r i l as much as the s i z e of the c r y s t a l s calculated from d i f f r a c t i o n spots agree with the s i z e of the holes. The highly c a l c i f i e d apatite i n the tissues i s found between the f i b r i l s without any p e r i o d i c i t y and i s not observed by d i f f r a c t i o n . - Films of reconstituted collagen at 20°C and 50°C The remarkable properties of collagen have led to many d i v e r se a p p l i c a t i o n : s o l u t i o n s , gels, f i l m s , tubes, sponges. In p a r t i cular the f i l m i s used i n a p p l i c a t i o n as diverses as the f a b r i c a t i o n of sausage skins and the treatment of burns or blood d i a l y s i s and a r t i f i c i a l kidneys. I t i s thus e s p e c i a l l y i n t e r e s t i n g to understand the behaviour with reference to the water present i n reconst i t u t e d collagen. The samples analysed here have been prepared by the method developed by the Centre Technique de Cuir'at Lyon (France) from tannery products (Brevet Français n° 1596790 of Nov. 27.1968) : the protective s k i n of veal i s ground, washed with a phosphate pad and exchanged with water to obtain at f i r s t f i b e r i n a 0.1M s o l u t i o n of a c e t i c a c i d , r e p r e c i p i t a t e d by addition of NaCl, dialysed with exchanged water and l y o p h i l i c agents. The lyophylic acid soluble product i s dissolved i n a 0.1 II a c e t i c acid s o l u t i o n at a concentration of 3 %, contrifuged, deaerated and f i n a l l y f i l m s are formed by evaporation at d i f f e r e n t temperatures i n special frames. The biochemical and biophysical studies and mechanical properties of the f i l m have been compared with those of the l y o p h y l i c acid soluble products (11, 12) : . a p a r t i a l i n s o l u b i l i t y , or an increase i n i n t r a and intermolecul a i r e bonds i s present i n the end regions of the f i b e r ; . a more i r r e g u l a r placement of molecules i s present i n the i n t e r i o r of the network of f i b e r which can be interpreted by a d i sappearance of the "quarter stagger" allignment ;


WATER IN POLYMERS

238

. an i n c r e a s e i n t h e d e n a t u r a t i o n temperature ( f o r t h e 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 ) w h i c h i m p l i e s an i n c r e a s e i n bonds s t a b i l i z i n g the t r i p l e h e l i x ; . the e l a s t i c p r o p e r t i e s a t t r i b u t e d t o the t r i p l e h e l i x a r e main t a i n e d f o r f i l m p r e p a r a t i o n temperatures l e s s t h a n 46°C c o n f i r ming t h e r e t e n t i o n o f t r i p l e h e l i x s t r u c t u r e below t h i s tempera ture.


I t s h o u l d be added t h a t t h e two f i l m s a n a l y s e d a r e e s p e c i a l l y c r o s s - l i n k e d and do not c o n t a i n the " q u a r t e r s t a g g e r " s h i f t and t h a t the f i l m formed a t 50*£ no l o n g e r r e t a i n the t r i p l e h e l i x structure. 2.2 - K e r a t i n

samples

- α-keratin from h a i r Human h a i r o c c u r s i n t h e f o r m o f a f i b e r w i t h a c e n t r a l c o r e c o n t a i n i n g t h e k e r a t i n w i t h i n an e x t e r n a l c u t i c u l e h a v i n g t h e form of s c a l e s w h i c h c o n t a i n s 10 % o f i t s mass. C h e m i c a l l y , k e r a t i n i s formed f r o m p o l y p e p t i d e c h a i n s formed from 20 d i f f e r e n t amino a c i d s . The r e l a t i v e p r o p o r t i o n o f w h i c h v a r y f o r d i f f e r e n t k e r a t i n s . Some p o s s e s s an a c i d s i d e group (12 % of w h i c h a r e g l u t a m i c a c i d ) o t h e r s a amine f u n c t i o n (30 % o f w h i c h are l y s i n e ) , o t h e r s a h y d r o x y l (10 % o f w h i c h a r e s e r i n e ) . B u t t h e k e r a t i n i s above a l l c h a r a c t e r i z e d b y a n i m p o r t a n t q u a n t i t y o f s u l f u r due t o t h e p r e s e n c e o f c y s t i n e u n i t s w h i c h form a d i s u l f i d e b r i d g e between two c h a i n s g r e a t l y c o n t r i b u t i n g t o t h e s t a b i l i t y o f the p r o t e i n s . I n c o n c l u s i o n , t h e c h a i n s a r e connected by a number of d i v e r s e i n t e r a c t i o n s i n c l u d i n g hydrogen bonds, s a l t bonds and c o v a l e n t bonds. K e r a t i n has a c r y s t a l l i n i t y o f about 30 %. S i n c e 1950,X-ray d i f f r a c t i o n and e l e c t r o n m i c r o s c o p y have l e d t o a model i n w h i c h the s t r u c t u r a l elements a r e (13,14) : t h e α-helix w i t h a d i a m e t e r of 10 Â , t h e e l e m e n t a r y f i b r i l c o n t a i n i n g two t o t h r e e hélicoïdal c h a i n s o f 20 Â d i a m e t e r , the m i c r o f i b r i l o r a s s o c i a t i o n o f t e n e l e m e n t a r y f i b r i l s t h r o u g h t h e amorphous r e g i o n s ( o f 80 A diame t e r ) and f i n a l l y t h e f i b r i l s o r s t r u c t u r e s o f s e v e r a l m i c r o f i b r i l s w i t h i n an amorphous m a t r i x . The s t r u c t u r e o f t h e amorphous r e g i o n i n unknown b u t i t i s known t o be r i c h i n s u l f u r w i t h a number o f d i s u l f i d e b r i d g e s and absorb more w a t e r t h a n t h e m i c r o f i b r i l s . The a b s o r p t i o n o f w a t e r b y k e r a t i n has been s t u d i e d by N.M.R., I.R., d i e l e c t r i c and c a l o r i m e t r i c methods ( 1 5 - 2 0 ) . K e r a t i n e x h i b i t s a s i n g l e l i n e spectrum b y N.M.R. w i t h l i n e - w i d t h depending on o r i e n t a t i o n . T h i s i s i n c o n t r a s t t o c o l l a g e n w h i c h shows a t h r e e l i n e spectrum. Lynch and H a l y (15) have s t u d i e d the i n f l u e n c e o f o r i e n t a t i o n on t h e s p i n - s p i n r e l a x a t i o n time o f absorbed w a t e r i n k e r a t i n from r h i n o c e r o s h o r n . A p r i v i l e d g e d r o t a t i o n o f the w a t e r i s o b s e r v e d around an a x i s a p p r o x i m a t e l y p a r a l l e l t o the f i b e r d i r e c t i o n . From I.R. r e s u l t s B e n d i t proposes a weak a s s o c i a t i o n o f w a t e r w i t h c a r b o n y l groups i n t h e c r y s t a l l i n e phase. I.R. experiment on d e u t e -


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239


rated k e r a t i n also shows evidence f o r hydrogen-deuterium exchange processes (17). Leveque (18) has interpreted a maximum observed i n depolarisation experiments as being associated with a r e o r i e n t a t i o n of bound water molecules. Water molecules absorbed by k e r a t i n from wood have been c l a s s i f i e d as free or bound (21,22). A further subd i v i s i o n of the bound water molecules has been proposed (23). Feughelman (24) has proposed a model implying d i f f e r e n t types of water molecules with one to four degrees of association. The r e l a t i v e percentage of these d i f f e r e n t types changes with the l e v e l of water absorption.


No clear experimental proof has yet been given f o r the e x i s tence of these several types of water molecules. - Keratin of reduced h a i r and dyed h a i r Natural h a i r can be greatly changed i n terms of the cohesive bonds present. By treatment with t h i o g l y c o l i c acid i n ammonia, 33 % of the cystine (HOOC - CH - CHo - S - S - CHo - CH - COOH) i s t r a n s f o r NH NH2 (HOOC - CH - CH - SH) and 22 % to the form l a n 2

med to the form

2

NH2

thionine (HOOC - CH - CH - S - CH - CH - COOH). There i s a deNH NH2 crease i n the number of d i s u l f i d e linkages making the h a i r f i b e r temporarily p l a s t i c or i n e l a s t i c a l l y deformable. This reduction i s used i n hairdressing to create a permanent set i n which the reduced h a i r i s wound on a h a i r c u r l e r and f i x e d while the f i b e r r e constitute i t s i n i t i a l chemical texture. 2

2

2

- The dying of h a i r i s accomplished by the action of H 0 , the persulfate of sodium or ammonia which oxidize the cyst i n e . An analysis of t h i s treatment reveals the presence of sever a l intermediate oxidation products and the f i n a l transformation of 32 % of the cystine to cysteic acid (HOOC - CH - CH - SO3H). 2

2

NH2

The decrease i n c r o s s l i n k i n g i s comparable to that of reduced

hair.

We have i n t h i s study t r i e d to analyse the e f f e c t of these chemical modifications on the hydration properties of h a i r . 3. Experimental r e s u l t s 3.1 - Comparison of d i f f e r e n t collagen samples The absorption isotherms f o r water at 20°C are shown i n f i g u re 1. We note : 1) that the c a l c i f i e d collagen absorbs less water than that of r a t - t a i l tendon. The change i s p a r t i c u l a r l y important i n the range of 20-80 % r e l a t i v e humidity ; 2) that the reconstituted collagen f i l m exhibits the same hydra-



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Figure 1. Water sorption isotherms of different collagen forms: (Q) rat tail tendon collagen; (O) reconstituted collagen (I); (A) reconstituted collagen (2); (φ) calci fied turkey leg tendon


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Collagen and

241

Keratin

t i o n as the r a t - t a i l tendon at low humidities but absorbs less water than the r a t - t a i l tendon above humidities of 50 % ; 3) that the reconstituted collagen f i l m at 50°C absorbs less water over the entire humidity range. The isotherm i s s i m i l a r to that of the mineralized collagen near P/P of 0.5. 0

The energy absorption data are shown i n figure 2. The d i f f e rent steps of absorption observed for r a t - t a i l tendon are not seen for the other samples. E s p e c i a l l y unusual i s that f o r the c a l c i f i e d collagen the energy of absorption i s c l e a r l y less than 10 kcal/mole as soon as the weight of absorbed water exceeds 10 %. For the two films of reconstituted collagen the energy of absorption goes through a weak maximum near a weight of 10 % (P/P 0.4 - 0.5) and then decreases r e g u l a r l y .


%

Q

3.2 - Comparison of d i f f e r e n t k e r a t i n samples The absorption isotherms of k e r a t i n from human hair shows a sigmoidal form as did those f o r collagen which are c l a s s i f i e d as type I I with a hysteresis over the e n t i r e humidity range as shown i n figure 3. A difference exists i n the k i n e t i c s of absorption when observed by successive increments. Longer times are required to a t t a i n equilibrium f o r the k e r a t i n i n the f i r s t increment than for those above P/P = 0.6 (figure 4). Q

A s h i f t of the absorption isotherm i s observed toward higher water levels f o r reduced or dyed hair (figure 5 ). #

The absorption energy p r o f i l e f o r natural h a i r or dyed h a i r shows i n a l l cases a s l i g h t l y increasing slope (below P/P = 0.6) with the molar heat below that f o r the l i q u e f a c t i o n of water, f o l lowed by a part c l e a r l y decreasing up to r e l a t i v e l y low values (figure 6). 0

4. Discussion The above consideration lead e s s e n t i a l l y to a comparison with the model proposed f o r collagen f i b e r from r a t - t a i l tendon. In e f f e c t , i f i t i s possible to r e l a t e the hydration properties to s t r u c t u r a l or t e x t u r a l chemical differences i n the samples, one can hope to explain the collagen-water i n t e r a c t i o n on a molecular basis. Collagen For c a l c i f i e d collagen the hypothesis can be made that the decrease i n hydration as compared to that f o r the r a t - t a i l tendon corresponds to the f i l l i n g of holes i n the m i c r o f i b r i l by apatite c r y s t a l s . This would be evident i n the t h i r d step defined above. But a s h i f t i s observed at the low l e v e l s . The s i z e of the s h i f t i s much more important than would be expected on the basis of the volume of the holes i n question since i t extends to r e l a t i v e humid i t i e s near 50 %. I t i s also accompanied by a r a d i c a l change i n the energy absorption p r o f i l e and an o v e r a l l decrease i n the d i f fusion s i t e .


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Figure 2.

Energy of hydration vs. the amount of water absorbed for different collagen forms



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243


Am(%) 25-

20-

15-

Ό

Figure 3.

0.^5

0.5

o.fe

\

P/PO

Human hair keratin: (-{-) water sorption isotherm and (A) water desorption isotherm



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Figure 4.

POLYMERS

Kinetics of hydration for different specimens.

a is the ratio of the amount of water absorbed after time t vs. that absorbed at equilibrium. Symbols are defined as follows. Rat tail tendon collagen: (9) 0 < P/P < 0.33; (X) 0.43 < P/P

Comparison of Weight and Energy Changes in the Absorption of

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