13
Density of Elastin-Water System MARIASTELLA SCANDOLA and GIOVANNI PEZZIN
Downloaded by MONASH UNIV on March 1, 2016 | http://pubs.acs.org Publication Date: August 19, 1980 | doi: 10.1021/bk-1980-0127.ch013
Centro di Studio per la Fisica delle Macromolecole del C.N.R., Via Selmi 2, 40126 Bologna, Italy
A variety of different experimental techniques has been used to investigate the hydration of fibrous proteins: among them calorimetric (1,2,3), dielectric (4) and dynamic-mechanical measurements (5,6), equilibrium sorption data (7,8,9), infrared spectroscopy (10) and Nuclear Magnetic Resonance (11,12). Little information is however available in the literature on the specific volume of hydrated fibrous proteins. To the best of the authors' knowledge, the only set of data published to date refers to hydrated keratin, was published by King (13), and later discussed by Rosenbaum (14). Volumetric data can be essential in the thermodynamic treatment of the "polymer-solvent" interaction process. The lack of them for many important fibrous proteins is due to the difficulty of measuring the density, at controlled temperature and hydration degree, for these systems. As far as elastin is concerned, it has been reported that when completely hydrated this protein has a negative and very large coefficient of thermal expansion (15), a result which has been interpreted as evidence of a hydrophobic character of the protein (16). Elastin, which is substantially amorphous but fibrous at all levels of investigation (starting from the largest filaments which are about 6 μια in diameter and down to about 10 nm (17,18)), is a fragile, glassy substance when dry and has a glass-to-rubber transition temperature at about 200°C (19); upon hydration or solvation with appropriate solvents, it becomes a rubbery system with the glass transition below room temperature (20). Calorimetric data have shown that only half of the total water sorbed by elastin (about 0.6 g water / g dry protein) is really "bound", the remaining water being freezable (_1). The volumetric data reported in the literature (15,16) refer therefore to an essentially heterophase system, so that the negative and very large coefficient of thermal expansion of the fully hydrated protein does not appear to be suitable for the interpretation of the thermoelastic data and calculation of the 0-8412-0559-0/ 80 / 47-127-225S05.00/ 0 © 1980 American Chemical Society In Water in Polymers; Rowland, Stanley P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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protein-water i n t e r a c t i o n parameter. I t i s the purpose of the present work to c o l l e c t r e l i a b l e v o l u m e t r i c data on the system e l a s t i n - w a t e r and to compare them w i t h the scarce l i t e r a t u r e data on s i m i l a r n a t u r a l polymer systems or on s y n t h e t i c polymeric systems. The measurements have been purposely l i m i t e d to the r e g i o n of low water contents where the system can be considered homogeneous.
Downloaded by MONASH UNIV on March 1, 2016 | http://pubs.acs.org Publication Date: August 19, 1980 | doi: 10.1021/bk-1980-0127.ch013
Experimental Samples of n a t i v e ox ligamentum nuchae e l a s t i n and of c o l l a g e n - f r e e e l a s t i n were obtained as reported p r e v i o u s l y (19). In order to c a r r y out the d e n s i t y measurements i n a wide range of temperatures, a s u i t a b l e g l a s s pycnometer was used: the pycnometer c a p i l l a r y i s placed l a t e r a l l y w i t h respect to the chamber c o n t a i n i n g the sample and the f i l l i n g l i q u i d , and i s provided w i t h an upper (expansion) and a lower ( c o n t r a c t i o n ) s p h e r i c a l c a v i t y . A low molecular weight s i l i c o n o i l (Dow Corning 200/20) was used as the f i l l i n g l i q u i d . Dry samples (about 3 g of e l a s t i n s t r i p s l e s s than 1 mm t h i c k ) were stored under vacuum over 1*2^5 ^ ^ days, c a r e f u l l y weighed on an automatic balance, placed i n the pycnometer, covered w i t h s i l i c o n o i l and degassed by means of a r o t a t i v e pump u n t i l no gas r e l e a s e by the sample was observed. The pycnometer was then c l o s e d , immersed i n a bath maintained a t the measurement temperature and, when the thermal e q u i l i b r i u m was reached, the l i q u i d l e v e l i n the l a t e r a l c a p i l l a r y was adjusted w i t h a small s y r i n g e . Weighing of the pycnometer, a f t e r c a r e f u l d r y i n g , was c a r r i e d out a t room temperature and the d e n s i t y of the p r o t e i n sample ρ was c a l c u l a t e d from the equation: o r
V
Ρ
s e v e r a
(1)
where: V i s the volume of the pycnometer m^is the weight of the sample m^ i s the t o t a l weight of the pycnometer c o n t a i n i n g sample and f i l l i n g l i q u i d m i s the weight of the empty pycnometer i s the d e n s i t y of the s i l i c o n o i l The d e n s i t y of samples of p u r i f i e d e l a s t i n c o n t a i n i n g appropriate amounts of water was determined w i t h the same procedure. The h y d r a t i o n l e v e l s chosen are those corresponding to water weight f r a c t i o n s of 0.05, 0.14, 0.18 and 0.29. Due to the d i f f i c u l t i e s i n reproducing the same water weight f r a c t i o n , only one measurement of d e n s i t y was c a r r i e d out a t each water content. However, the experimental s c a t t e r i s s m a l l , as seen from the l i n e a r i t y of the data p l o t t e d i n F i g u r e s 1 and 2.
In Water in Polymers; Rowland, Stanley P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
Downloaded by MONASH UNIV on March 1, 2016 | http://pubs.acs.org Publication Date: August 19, 1980 | doi: 10.1021/bk-1980-0127.ch013
SCANDOLA AND P E Z Z i N
Figure 1.
Elastin-
Water System
227
Temperature dependencies of the density and specific volume of dry elastin: (O) native; (φ) purified.
Figure 2. Density and specific volume of dry and hydrated purified elastin samples as a function of temperature at water weight fractions of: (—) 0; (A) 0.05; (O) 0.14; (φ) 0.18; (A) 0.29
In Water in Polymers; Rowland, Stanley P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
228
WATER IN
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Downloaded by MONASH UNIV on March 1, 2016 | http://pubs.acs.org Publication Date: August 19, 1980 | doi: 10.1021/bk-1980-0127.ch013
Results and D i s c u s s i o n The temperature dependence of the d e n s i t y of dry samples of n a t i v e and p u r i f i e d e l a s t i n i s shown i n F i g u r e 1. In the temperature range explored, the n a t i v e p r o t e i n shows a higher d e n s i t y than the p u r i f i e d one, t y p i c a l values being 1.245 g/ml and 1.232 g/ml, r e s p e c t i v e l y , at 25°C. In a f i r s t approximation, t h i s d i f f e r e n c e i n d e n s i t y can be accounted f o r by the d i f f e r e n t composition of the n a t i v e and p u r i f i e d p r o t e i n . I f n a t i v e e l a s t i n i s considered a two phase composite m a t e r i a l (approximately 80% e l a s t i n and 20% c o l l a g e n ) , d i s r e g a r d i n g other minor components whose d e n s i t y data are not a v a i l a b l e , the d e n s i t y of the composite can be c a l c u l a t e d from the equation: 1 w - = _ Q e Ε E
+
w _C
( 2 )
e C
Assuming w = 0.80, w = 0.20 and t a k i n g ρ = 1.232 g/ml and Qç= 1.32 g/ml (21), a c a l c u l a t e d d e n s i t y of tne composite ρ= 1.247 g/ml i s obtained, which compares s a t i s f a c t o r i l y with the experimental d e n s i t y of n a t i v e e l a s t i n . The volumetric expansion c o e f f i c i e n t s , c a l c u l a t e d from the equation: c
a =
-
