VOL. 15, 1155-1165 (1976)
RIOPOLY MERS
Interaction of Water with Poly-a-Amino Acids. I. Relationships Between Conformation and Relaxation Processes H. SHIRAISHI,* A. HILTNER, and E. BAER, Department of Macromolecular Science, Case Western Reserve University, Cleveland,
Ohio 44106 Synopsis The relaxation behavior of the sodium salt of poly(L-glutamic acid) in the solid state has been examined by means of dynamic mechanical spectroscopy. Bound water was found to exert a profound influence on the relaxation behavior and on a hulk property, the rigidity. Certain features of the loss spectrum have been identified with the hydration-dependent fl-to-n conformational transition. Thus two side-chain relaxations are observed below ambient temperature, one associated with the fl form (018) and a second a t a lower temperature associated with the (Y form (01"). The greater rigidity of the (Y form below the relaxation temperature and the larger rigidity drop accompanying the 81" can be explained in terms of the structural differences of the two conformations.
INTRODUCTION The interaction of water and biological macromolecules is one of the most important phenomena of all life processes. Although the human body is 50-60% water, only 10%of this is found in the vascular system and is considered to have fluid properties. The rest is bound by poorly understood mechanisms in the cytoplasm of cells and in gels of extracellular connective tissue. T h e physical and mechanical properties of connective tissue are altered appreciably by changes in the water content. The most important protein in connective tissue is collagen, but despite the obvious significance many aspects of water-collagen interactions remain a mystery. In large part this is due to the complex chemical composition, which in collagen includes more than 19 different amino acids, and the equally complex structural organization of the protein. On a molecular level, water is probably associated through hydrogen bonds with the peptide linkages and with polar side groups, e.g., the hydroxyl groups of serine, tyrosine, and hydroxyproline; it can also form hydration spheres around ionized residues such as lysine and glutamic acid. In general, each type of association will differ as regards the energy of the interaction and the effect on the physical properties. Molecular models are essential if the individual molecular interactions are t o be separated and characterized. The most convenient * On leave from: Fuji Photo Film Company, Ltd., Minamiashigara, Kanagawa, .Japan. 1155 Q 1976 by John Wiley & Sons, Inc.
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SHIRAISHI, HILTNER, AND BAER
and versatile models are the synthetic poly(a-amino acids). They have the peptide backbone, can assume a variety of conformations, and can be synthesized as homopolymers, as random copolymers, or with more than one amino acid in a known sequence. In investigating the nature of water-macromolecule interactions, we have employed dynamic mechanical spectroscopy, complemented by other more conventional methods of analysis. The dynamic mechanical technique has application to problems involving the relationships between structural and mechanical change, and has also proven to be very sensitive to interaction of water with macromolecular substances. The influence of water on the relaxation behavior of native collagenous tissue has been described;l12 and in another paper we report the behavior of two synthetic polypeptides, Proline and hydroxypoly(L-proline 11) and poly(~-hydroxyproline).~ proline are important components of the crystalline regions of the collagen molecule and their homopolymers assume helical conformations closely resembling the collagen helix. From the changes in temperature location and intensity of loss peaks, as many as four types of water have been distinguished in collagen and characterized with the aid of the model homopolymers. In this paper we pursue our interest in model poly(a-amino acids) with an investigation of the sodium salt of poly(L-glutamic acid) (Glu-Na+),. Glutamic acid is present in the polar, noncrystalline regions of collagen and is also common in the helical region of globular proteins. The hydration of (Glu-Na+), in the solid state has previously been followed using infrared spectroscopy4 and circular d i c h r ~ i s m .A~ reversible change of structure is observed in which the 0-sheet conformation is converted to the a-helix upon increasing relative humidity. An analogous phenomenon has been reported for poly(L-lysine hydrochloride).6 Few synthetic polymers undergo transformations between structural forms that are as different and well characterized as these. The work with (Glu-Na+), reported here represents the first attempt to include the conformational effect in a dynamic mechanical study of interaction of water with poly(a-amino acids).
EXPERIMENTAL Poly(L-glutamic acid) ( M , 50,000-80,000) was obtained as the sodium salt (Glu-Na+), from Pierce Chemical. Film specimens were cast from aqueous solution. After drying under vacuum for several days, the specimens were equilibrated over saturated salt solutions at the desired humidities. The chain conformation was determined by infrared spectroscopy using a Digilab Fourier Transform Spectrometer. A standard 25-mm cylindrical cell was modified to include a small reservoir, which contained the saturated salt solution. The polymer film was cast on one of the removable AgCl windows and equilibrated for several days. After the spectrum was recorded the sample window was replaced by an AgCl blank and the spectrum
WATER-AMINO ACID INTERACTION. I
1157
of the moist air also recorded. The air spectrum was subtracted from that of the specimen using the sharp, fine-structured water peak centered a t 1600 cm-l. The heat of fusion of the sorbed water was measured with a DuPont 990 DTA with DSC cell. Heating scans were made from 160' to 300°K under dry nitrogen flow a t a rate of 10°/min. The 3-5-mg samples were weighed into standard crimped pans with a small hole in the lid through which hydration was controlled. The heat of fusion was obtained by weighing the scissored peak. Dynamic mechanical measurements were made with an inverted free oscillating torsional pendulum at about 1 Hz over the temperature range 80°-3000K.7 Because (Glu-Na+), forms intractable films, the torsional braid technique was used in which the polymer is cast from solution onto a viscoelastically inert glass braid support. In order to avoid loss of water it was necessary to quench the hydrated specimens in liquid nitrogen before loading into the instrument. With this procedure less than 5% of the total water was lost over an entire run. No attempt was made to obtain G' from the dynamic mechanical data. Instead the parameter used for comparison was the relative rigidity defined as Grel' = ( f / f g o 0 ) 2 where f is the observed frequency and f90° is the frequency of the same specimen dry a t 90°K. Typically f90" was about 0.5 Hz.
RESULTS Infrared Spectroscopy Conformational assignments were made from the infrared band positions using the conformationally sensitive amide I, 11, and V regions. Spectra of unoriented films equilibrated at 46,66, and 84% relative humidity are shown in Figure 1. In accord with Fasman et al.,5 the film equilibrated a t 46% relative humidity is found to be predominately in the conformation while the film a t 84% relative humidity is in the a conformation. A film equilibrated a t 66% relative humidity shows a mixture of the a and @ conformations, the characteristic amide I and I1 bands of both conformations are observed, and the amide V is shifted to an intermediate position. Identical spectra were obtained whether the film was dried or rehydrated to a given relative humidity, confirming the previous conclusion that the conformational transition is a reversible one. A film equilibrated at 12% relative humidity showed a disordered conformation as reported previou~ly.~
Differential Scanning Calorimetry (DSC) All the (Glu-Na+), samples with more than 0.9 g/g water exhibited a broad DSC endotherm over the temperature region -25O-O"C. The heat associated with the melting endotherm is plotted as a function of the total water content in Figure 2. The slope of this plot is the heat of fusion of the
1158
SHIRAISHI, HILTNER, AND BAER
v)
A
0 9
Fig. 1. Infrared absorption spectra of (Glu-Na+), film at several relative humiditiesshowing the p form (46%RH), a form (84%RH), and a mixture of 0 and a forms (66%RH).
melting water and the intercept gives the amount of water that does not melt. A least squares fit of the data yields AH/ = 75.3 cal/g, in close agreement with the heat of fusion of bulk water (79.7 cal/g), and a nonmelting water content of 0.9 g/g. We conclude that a characteristic amount of water (0.9 g/g) does not crystallize upon cooling to -115OC and is assumed to be closely associated with the polymer molecule. Additional water over this amount crystallizes and melts with the normal heat of fusion. The DSC thermograms of some specimens also show a heat capacity change below the temperature of the melting endotherm (Fig. 3). A similar heat capacity change has been observed in esters of (Glu), and is associated with a large side-chain relaxation.* The dynamic mechanical measurements will show that the heat capacity change in hydrated (Glu-Na+), is also associated with a large side-chain relaxation, @I" a t -65°C.
Dynamic Mechanical Spectroscopy (Glu-Na+), is essentially viscoelastically inert when dry, but with water present shows two relaxations regions 01 and 0 2 (Fig. 4). The @2 is the
1159
WATERoAMINO ACID INTERACTION. I
p2 50 c f
7
8
I
1
H,O moloculos /poptido
,.;I ,
I
0
1.00
1.50
2.00
WATER CONTENT
2.50
3.00
(g/g)
Fig. 2. T h e integral heat of fusion of the water melting transition as a function of water content.
EX0
T
I
180
I
I
200
1
1
I
220
TEMPERATURE [ K O ]
Fig. 3. DSC thermograms of (Glu-Na+), showing a heat capacity change. The specimen contains 1.36 g/g water.
1160
SHIRAISHI, HILTNER, AND BAER
3 .6
4
c.4
5a
1
4
.2
TEMPERATURE
(K’)
Fig. 4. Dynamic mechanical loss spectra of (Glu-Na+), at various water contents showing the and p2 relaxation regions.
“water dispersion” characteristic of many hydrated polymers including poly(L-proline), poly(L-hydroxyproline), and nylon 6. The p2 peak shifts to lower temperatures, from 245’ to 160’K, as the water content increases to about 0.9 g/g. Above this water content the p2 temperature remains constant. The intensity is essentially constant throughout and the accompanying rigidity drop is very small. In another paper3 we discuss the origins of this dispersion and show that the p 2 temperature typically decreases as the noncrystallizable or bound water content increases but is not affected by additional water that crystallizes. The important relaxation region in (Glu-Na+), is the PI. The loss spectrum is dominated by the 01 peaks, which may be orders of magnitude more intense than the p2 and are accompanied by large drops in the rigidity. In the drier samples, 0.035 and 0.10 g/g, the is seen a t 270’K. For higher water content samples, the ,& region is comprised of two peaks. We will show that these are associated with the two conformations, a and 0,and are therefore designated and for the low- and high-temperature components, respectively. In parallel with the 02, both peaks shift to lower temperatures as the bound water content increases (Fig. 5). Where a single peak is observed a t low water content, 0.13 and 0.17 glg, the data fit on the curve in Figure 5. They are included as such although the infrared spectra indicate that a disordered conformation predominates here.
WATERoAMINO ACID INTERACTION. I H,O
1161
molo~ulor/p.ptido
WATER C O N T E N T
(a/g)
Fig. 5. Temperature of t h e various relaxation peaks of (Glu-Na+), as a function of water content.
Figure 4 also shows that as the water content increases the relative intensities of the two peaks are reversed. At low water content the 010 is the predominate peak, but progressive changes are noted as the water content is increased (Fig. 6): initially the intensity increases rapidly, reaches a maximum, and then drops off until the peak has essentially disappeared. While the @I@intensity decreases, the @ I a steadily increases and this becomes the predominate peak a t high water content. With the predominate a t the lower water content and the 01" a t higher water content, the changes in intensity parallel the conformational p a transition observed by infrared. We have indicated the @ a transition in Figure 6 at 0.65 glg (5.5 water molecules per peptide) although the dynamic mechanical technique reveals that the a and p forms coexist over a fairly wide range of water contents and in actuality the transitions from t o a are gradual. T h e maximum in intensity occurs a t 0.5 g/g or about four water molecules per peptide, which is taken to be the maximum amount of water that can be accommodated before the @-sheetstructure breaks up with formation of a-helices. The a-helical structure can accommodate still more bound water, up to 0.9 g/g or about 7.5 water molecules per residue.
- -
1162
SHIRAISHI, HILTNER, AND BAER
3
1
.8
1
1
5 I
I
-
I
7 V
.
1
1
1
D-a
-
,pa-
-
.6
I
g a
-
A
.4-
.2
-
I
0 ' .
0
-oI--o-
0-0-
A3
.lo
'
d'/.
' i D
''/do
I
3
\ I
-
\
:
P2
,
O~-o-o-&oo-
.30
1.00
WATER C O N T E N T
3.00
(g/g)
Fig. 7. Cross plot of the relative rigidity as a function of water content at various temperatures.
WATER-AMINO ACID INTERACTION. I
1163
In addition to finger printing the dynamic mechanical loss spectrum, the and @ forms also show differences in a bulk property, the rigidity (Fig. 6). A t low water content a modest increase in rigidity accompanies the conversion of the disordered form to the more organized 0structure. Up to 0.5 g/g, where the /3 form predominates, the drop in rigidity at the /31 relaxation is relatively small. With conversion to the CY form the rigidity increases abruptly at low temperatures and a t the 01 relaxation drops by more than an order of magnitude. In the transition region this is manifest as first an increase in rigidity as 0is transformed to a then as a decrease in rigidity as the 61 relaxation is passed. Above 0.9 g/g addition of free or crystallizable water has little effect on either the loss spectrum or the rigidity. This water is apparently incorporated as filler without significantly affecting the properties. N
DISCUSSION It is apparent from the foregoing that interaction of bound water with the (Glu-Na+), macromolecule has a profound effect on the structure and properties in the solid state. The present paper does not provide new insight into the factors that determine the thermodynamically stable conformation, although the molecular mobility represented by the & relaxation clearly influences the rate (and the experimental reversibility) of the solid-state /3 a transition. This paper does have important implications as regards the effect of hydration and conformational structure on the properties. Relevant to this discussion is the mechanistic origin of the relaxation. Although this paper is a first attempt to study in depth a hydrophilic poly(a-amino acid) by dynamic mechanical spectroscopy, the hydrophobic esters of (Glu), have been well characterized. In the pure state these polymers exhibit a large p relaxation at about room temperature, which most workers assign to side-chain motions. The temperature of the relaxation increases as the size of the ester group increases, and for the methyl ester is found at a higher temperature for the p form than for the The relaxation is accompanied by a heat capacity step in DSC measurernentq8 and analysis of the viscoelastic behavior of the methyl and benzyl esters has shown that the 0 relaxation is described by the WFL equationlo [Eq (l)]:
-
-CI(T - T g ) C:!+T-Tg where aT is the shift factor; Tgis the glass transition temperature; and C1 and C2 are constants. For these reasons, the is frequently referred to as a side-chain glasslike transition. The 01 is almost certainly the analogous process in (Glu-Na+),. However, participation of the ionic side group in the relaxation is determined by the state of hydration. In the dry state, electrostatic forces essentially immobilize the side chains. Hydration of the ionic species and increased charge separation have the effect of lowering log aT
=
1164
SHIRAISHI, HILTNER, AND BAER
the barriers to reorientation and the relaxation correspondingly shifts to lower temperatures while increasing in intensity. The P I a a t the high water content most closely resembles the (Glu), ester 0relaxation with the intense loss peak, large rigidity drop, and heat capacity change in DSC measurements. With'the 01 assigned a side-chain mechanism, we have assumed that the 01relaxation has the effect of facilitating intermolecular shear when the primary intermolecular forces involve the side chains. Intermolecular forces that determine the rigidity of the 0 structure are the side-chain electrostatic forces between sheets and the intermolecular hydrogen bonds that stabilize the sheet itself. It is known that water molecules entering the 0 structure concentrate a t the layer of ions between the pleated sheets? When a sufficient quantity of water is present the onset of side-chain motion occurs a t the 01temperature. Following our assumption, this would allow the pleated sheets to slide past one another but the individual molecules would still be held in the sheet structure by the intermolecular hydrogen bonds. As the water content increases and the 0 structure breaks up to form a-helices the forces change. Hydrogen bonding is intramolecular, and now the primary forces between chains are electrostatic attraction and repulsion involving the side chains and counterions. At low temperatures this is a more rigid structure than the 0sheet, as evidenced by the abrupt increase in rigidity a t the 0 a transition. However, with only side-chain interactions between molecules, at the p1 temperature the individual molecules are able to slide past one another, and as a result, the drop in rigidity is much larger a t the 01" than the 010. A brief consideration of the esters of (Glu), shows a somewhat different situation. In this instance the sidechain interactions are nonbonded in nature and weaker than the intermolecular hydrogen bonds. Here our analysis predicts that the a form would be less rigid than the 0 form a t temperatures below the side-chain p relaxation. Finally, we can suggest at least one immediate implication of these results in regards to the proteins. The relaxation spectrum of native collagenous tissue shows a 01 process at 200"K, also associated with bound water, which shifts to lower temperatures as the water content increases.1*2 It now appears that this process can be identified with motion of the ionic side chains located in the noncrystalline regions of the collagen structure.
-
CONCLUSION The general conclusions of this paper can be summarized as follows. It is now well established that dynamic mechanical spectroscopy has unique application to the study of interaction of macromolecular substances with small molecules in the solid state. Complementing this technique with other more conventional methods of analysis, we have been able to study the interrelationships among structure, hydration, and properties of these systems.
WATER-AMINO ACID INTERACTION. I
1165
In the case of (Glu-Na+),, a relaxation process was found that is specific for the secondary structure. By identifying the process with side-group motion, and noting the effect of hydration, we have been able to explain the observed changes in a bulk property, the rigidity. Finally, we have shown that characterization of model poly(a-amino acids) enables us to begin interpreting the relaxation spectra of the much more complex proteinaceous materials. The authors thank Dr. Toshio Hayashi for his helpful discussions of the infrared data. One of them (H. S.) gratefully acknowledges the support of the Fuji Photo Film Company during a leave of absence. This work was sponsored by the National Institutes of Health, Grant HL-15195-01A1.
References 1. Hiltner, A,, Nomura, S. & Baer, E. (1974) in Peptides, Polypeptides and Proteins,Blout, E. R., Bovey, F. A., Goodman, M. & Lotan, N., Eds., Wiley, New York, p. 485. 2. Hiltner, A,, Shiraishi, H., Nomura, S. & Baer, E. (1976) Int. J . Polyrn. Muter. (in press). 3. Shiraishi, H., Hiltner, A. & Baer, E., manuscript in preparation. 4. Lenormant, H., Baudras, A. & Blout, E. R. (1958) J . Amer. Chern. Soc. 80, 61916195. 5. Fasman, G. D., Hoving, H. & Timasheff, S. N. (1970) Biochemistry 9,3316-3324. 6. Shmueli, V. & Traub, W. (1965) J . Mol. Biol. 12,205-2140. 7. Armeniades, C. D., Kuriyama, I., Roe, J. M. & Baer;E. (1967) J . Mucromol. Sci.-Phys. B1,777-791. 8. Pezzin, G., Ceccorulli, G., Pizzoli, M. & Peggion, E. (1975) Macromolecules 8, 762764. 9. Kajiyama, T., Kuroishi, M. & Takayanagi, M. (1975) J . Mucrornol. Sci.-Phys. B11, 195-217. 10. Tsutsumi, A,, Hikichi, K., Takahashy, T., Yamaschita, Y., Matsushima, N., Kanke, M. & Kaneko, M. (1973) J. Macromol. Sci.-Phys. B8,413-430.
Received October 2,1975 Returned for revision December 10, 1975 Accepted January 5,1976
RIOPOLY MERS
Interaction of Water with Poly-a-Amino Acids. I. Relationships Between Conformation and Relaxation Processes H. SHIRAISHI,* A. HILTNER, and E. BAER, Department of Macromolecular Science, Case Western Reserve University, Cleveland,
Ohio 44106 Synopsis The relaxation behavior of the sodium salt of poly(L-glutamic acid) in the solid state has been examined by means of dynamic mechanical spectroscopy. Bound water was found to exert a profound influence on the relaxation behavior and on a hulk property, the rigidity. Certain features of the loss spectrum have been identified with the hydration-dependent fl-to-n conformational transition. Thus two side-chain relaxations are observed below ambient temperature, one associated with the fl form (018) and a second a t a lower temperature associated with the (Y form (01"). The greater rigidity of the (Y form below the relaxation temperature and the larger rigidity drop accompanying the 81" can be explained in terms of the structural differences of the two conformations.
INTRODUCTION The interaction of water and biological macromolecules is one of the most important phenomena of all life processes. Although the human body is 50-60% water, only 10%of this is found in the vascular system and is considered to have fluid properties. The rest is bound by poorly understood mechanisms in the cytoplasm of cells and in gels of extracellular connective tissue. T h e physical and mechanical properties of connective tissue are altered appreciably by changes in the water content. The most important protein in connective tissue is collagen, but despite the obvious significance many aspects of water-collagen interactions remain a mystery. In large part this is due to the complex chemical composition, which in collagen includes more than 19 different amino acids, and the equally complex structural organization of the protein. On a molecular level, water is probably associated through hydrogen bonds with the peptide linkages and with polar side groups, e.g., the hydroxyl groups of serine, tyrosine, and hydroxyproline; it can also form hydration spheres around ionized residues such as lysine and glutamic acid. In general, each type of association will differ as regards the energy of the interaction and the effect on the physical properties. Molecular models are essential if the individual molecular interactions are t o be separated and characterized. The most convenient * On leave from: Fuji Photo Film Company, Ltd., Minamiashigara, Kanagawa, .Japan. 1155 Q 1976 by John Wiley & Sons, Inc.
1156
SHIRAISHI, HILTNER, AND BAER
and versatile models are the synthetic poly(a-amino acids). They have the peptide backbone, can assume a variety of conformations, and can be synthesized as homopolymers, as random copolymers, or with more than one amino acid in a known sequence. In investigating the nature of water-macromolecule interactions, we have employed dynamic mechanical spectroscopy, complemented by other more conventional methods of analysis. The dynamic mechanical technique has application to problems involving the relationships between structural and mechanical change, and has also proven to be very sensitive to interaction of water with macromolecular substances. The influence of water on the relaxation behavior of native collagenous tissue has been described;l12 and in another paper we report the behavior of two synthetic polypeptides, Proline and hydroxypoly(L-proline 11) and poly(~-hydroxyproline).~ proline are important components of the crystalline regions of the collagen molecule and their homopolymers assume helical conformations closely resembling the collagen helix. From the changes in temperature location and intensity of loss peaks, as many as four types of water have been distinguished in collagen and characterized with the aid of the model homopolymers. In this paper we pursue our interest in model poly(a-amino acids) with an investigation of the sodium salt of poly(L-glutamic acid) (Glu-Na+),. Glutamic acid is present in the polar, noncrystalline regions of collagen and is also common in the helical region of globular proteins. The hydration of (Glu-Na+), in the solid state has previously been followed using infrared spectroscopy4 and circular d i c h r ~ i s m .A~ reversible change of structure is observed in which the 0-sheet conformation is converted to the a-helix upon increasing relative humidity. An analogous phenomenon has been reported for poly(L-lysine hydrochloride).6 Few synthetic polymers undergo transformations between structural forms that are as different and well characterized as these. The work with (Glu-Na+), reported here represents the first attempt to include the conformational effect in a dynamic mechanical study of interaction of water with poly(a-amino acids).
EXPERIMENTAL Poly(L-glutamic acid) ( M , 50,000-80,000) was obtained as the sodium salt (Glu-Na+), from Pierce Chemical. Film specimens were cast from aqueous solution. After drying under vacuum for several days, the specimens were equilibrated over saturated salt solutions at the desired humidities. The chain conformation was determined by infrared spectroscopy using a Digilab Fourier Transform Spectrometer. A standard 25-mm cylindrical cell was modified to include a small reservoir, which contained the saturated salt solution. The polymer film was cast on one of the removable AgCl windows and equilibrated for several days. After the spectrum was recorded the sample window was replaced by an AgCl blank and the spectrum
WATER-AMINO ACID INTERACTION. I
1157
of the moist air also recorded. The air spectrum was subtracted from that of the specimen using the sharp, fine-structured water peak centered a t 1600 cm-l. The heat of fusion of the sorbed water was measured with a DuPont 990 DTA with DSC cell. Heating scans were made from 160' to 300°K under dry nitrogen flow a t a rate of 10°/min. The 3-5-mg samples were weighed into standard crimped pans with a small hole in the lid through which hydration was controlled. The heat of fusion was obtained by weighing the scissored peak. Dynamic mechanical measurements were made with an inverted free oscillating torsional pendulum at about 1 Hz over the temperature range 80°-3000K.7 Because (Glu-Na+), forms intractable films, the torsional braid technique was used in which the polymer is cast from solution onto a viscoelastically inert glass braid support. In order to avoid loss of water it was necessary to quench the hydrated specimens in liquid nitrogen before loading into the instrument. With this procedure less than 5% of the total water was lost over an entire run. No attempt was made to obtain G' from the dynamic mechanical data. Instead the parameter used for comparison was the relative rigidity defined as Grel' = ( f / f g o 0 ) 2 where f is the observed frequency and f90° is the frequency of the same specimen dry a t 90°K. Typically f90" was about 0.5 Hz.
RESULTS Infrared Spectroscopy Conformational assignments were made from the infrared band positions using the conformationally sensitive amide I, 11, and V regions. Spectra of unoriented films equilibrated at 46,66, and 84% relative humidity are shown in Figure 1. In accord with Fasman et al.,5 the film equilibrated a t 46% relative humidity is found to be predominately in the conformation while the film a t 84% relative humidity is in the a conformation. A film equilibrated a t 66% relative humidity shows a mixture of the a and @ conformations, the characteristic amide I and I1 bands of both conformations are observed, and the amide V is shifted to an intermediate position. Identical spectra were obtained whether the film was dried or rehydrated to a given relative humidity, confirming the previous conclusion that the conformational transition is a reversible one. A film equilibrated at 12% relative humidity showed a disordered conformation as reported previou~ly.~
Differential Scanning Calorimetry (DSC) All the (Glu-Na+), samples with more than 0.9 g/g water exhibited a broad DSC endotherm over the temperature region -25O-O"C. The heat associated with the melting endotherm is plotted as a function of the total water content in Figure 2. The slope of this plot is the heat of fusion of the
1158
SHIRAISHI, HILTNER, AND BAER
v)
A
0 9
Fig. 1. Infrared absorption spectra of (Glu-Na+), film at several relative humiditiesshowing the p form (46%RH), a form (84%RH), and a mixture of 0 and a forms (66%RH).
melting water and the intercept gives the amount of water that does not melt. A least squares fit of the data yields AH/ = 75.3 cal/g, in close agreement with the heat of fusion of bulk water (79.7 cal/g), and a nonmelting water content of 0.9 g/g. We conclude that a characteristic amount of water (0.9 g/g) does not crystallize upon cooling to -115OC and is assumed to be closely associated with the polymer molecule. Additional water over this amount crystallizes and melts with the normal heat of fusion. The DSC thermograms of some specimens also show a heat capacity change below the temperature of the melting endotherm (Fig. 3). A similar heat capacity change has been observed in esters of (Glu), and is associated with a large side-chain relaxation.* The dynamic mechanical measurements will show that the heat capacity change in hydrated (Glu-Na+), is also associated with a large side-chain relaxation, @I" a t -65°C.
Dynamic Mechanical Spectroscopy (Glu-Na+), is essentially viscoelastically inert when dry, but with water present shows two relaxations regions 01 and 0 2 (Fig. 4). The @2 is the
1159
WATERoAMINO ACID INTERACTION. I
p2 50 c f
7
8
I
1
H,O moloculos /poptido
,.;I ,
I
0
1.00
1.50
2.00
WATER CONTENT
2.50
3.00
(g/g)
Fig. 2. T h e integral heat of fusion of the water melting transition as a function of water content.
EX0
T
I
180
I
I
200
1
1
I
220
TEMPERATURE [ K O ]
Fig. 3. DSC thermograms of (Glu-Na+), showing a heat capacity change. The specimen contains 1.36 g/g water.
1160
SHIRAISHI, HILTNER, AND BAER
3 .6
4
c.4
5a
1
4
.2
TEMPERATURE
(K’)
Fig. 4. Dynamic mechanical loss spectra of (Glu-Na+), at various water contents showing the and p2 relaxation regions.
“water dispersion” characteristic of many hydrated polymers including poly(L-proline), poly(L-hydroxyproline), and nylon 6. The p2 peak shifts to lower temperatures, from 245’ to 160’K, as the water content increases to about 0.9 g/g. Above this water content the p2 temperature remains constant. The intensity is essentially constant throughout and the accompanying rigidity drop is very small. In another paper3 we discuss the origins of this dispersion and show that the p 2 temperature typically decreases as the noncrystallizable or bound water content increases but is not affected by additional water that crystallizes. The important relaxation region in (Glu-Na+), is the PI. The loss spectrum is dominated by the 01 peaks, which may be orders of magnitude more intense than the p2 and are accompanied by large drops in the rigidity. In the drier samples, 0.035 and 0.10 g/g, the is seen a t 270’K. For higher water content samples, the ,& region is comprised of two peaks. We will show that these are associated with the two conformations, a and 0,and are therefore designated and for the low- and high-temperature components, respectively. In parallel with the 02, both peaks shift to lower temperatures as the bound water content increases (Fig. 5). Where a single peak is observed a t low water content, 0.13 and 0.17 glg, the data fit on the curve in Figure 5. They are included as such although the infrared spectra indicate that a disordered conformation predominates here.
WATERoAMINO ACID INTERACTION. I H,O
1161
molo~ulor/p.ptido
WATER C O N T E N T
(a/g)
Fig. 5. Temperature of t h e various relaxation peaks of (Glu-Na+), as a function of water content.
Figure 4 also shows that as the water content increases the relative intensities of the two peaks are reversed. At low water content the 010 is the predominate peak, but progressive changes are noted as the water content is increased (Fig. 6): initially the intensity increases rapidly, reaches a maximum, and then drops off until the peak has essentially disappeared. While the @I@intensity decreases, the @ I a steadily increases and this becomes the predominate peak a t high water content. With the predominate a t the lower water content and the 01" a t higher water content, the changes in intensity parallel the conformational p a transition observed by infrared. We have indicated the @ a transition in Figure 6 at 0.65 glg (5.5 water molecules per peptide) although the dynamic mechanical technique reveals that the a and p forms coexist over a fairly wide range of water contents and in actuality the transitions from t o a are gradual. T h e maximum in intensity occurs a t 0.5 g/g or about four water molecules per peptide, which is taken to be the maximum amount of water that can be accommodated before the @-sheetstructure breaks up with formation of a-helices. The a-helical structure can accommodate still more bound water, up to 0.9 g/g or about 7.5 water molecules per residue.
- -
1162
SHIRAISHI, HILTNER, AND BAER
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Fig. 7. Cross plot of the relative rigidity as a function of water content at various temperatures.
WATER-AMINO ACID INTERACTION. I
1163
In addition to finger printing the dynamic mechanical loss spectrum, the and @ forms also show differences in a bulk property, the rigidity (Fig. 6). A t low water content a modest increase in rigidity accompanies the conversion of the disordered form to the more organized 0structure. Up to 0.5 g/g, where the /3 form predominates, the drop in rigidity at the /31 relaxation is relatively small. With conversion to the CY form the rigidity increases abruptly at low temperatures and a t the 01 relaxation drops by more than an order of magnitude. In the transition region this is manifest as first an increase in rigidity as 0is transformed to a then as a decrease in rigidity as the 61 relaxation is passed. Above 0.9 g/g addition of free or crystallizable water has little effect on either the loss spectrum or the rigidity. This water is apparently incorporated as filler without significantly affecting the properties. N
DISCUSSION It is apparent from the foregoing that interaction of bound water with the (Glu-Na+), macromolecule has a profound effect on the structure and properties in the solid state. The present paper does not provide new insight into the factors that determine the thermodynamically stable conformation, although the molecular mobility represented by the & relaxation clearly influences the rate (and the experimental reversibility) of the solid-state /3 a transition. This paper does have important implications as regards the effect of hydration and conformational structure on the properties. Relevant to this discussion is the mechanistic origin of the relaxation. Although this paper is a first attempt to study in depth a hydrophilic poly(a-amino acid) by dynamic mechanical spectroscopy, the hydrophobic esters of (Glu), have been well characterized. In the pure state these polymers exhibit a large p relaxation at about room temperature, which most workers assign to side-chain motions. The temperature of the relaxation increases as the size of the ester group increases, and for the methyl ester is found at a higher temperature for the p form than for the The relaxation is accompanied by a heat capacity step in DSC measurernentq8 and analysis of the viscoelastic behavior of the methyl and benzyl esters has shown that the 0 relaxation is described by the WFL equationlo [Eq (l)]:
-
-CI(T - T g ) C:!+T-Tg where aT is the shift factor; Tgis the glass transition temperature; and C1 and C2 are constants. For these reasons, the is frequently referred to as a side-chain glasslike transition. The 01 is almost certainly the analogous process in (Glu-Na+),. However, participation of the ionic side group in the relaxation is determined by the state of hydration. In the dry state, electrostatic forces essentially immobilize the side chains. Hydration of the ionic species and increased charge separation have the effect of lowering log aT
=
1164
SHIRAISHI, HILTNER, AND BAER
the barriers to reorientation and the relaxation correspondingly shifts to lower temperatures while increasing in intensity. The P I a a t the high water content most closely resembles the (Glu), ester 0relaxation with the intense loss peak, large rigidity drop, and heat capacity change in DSC measurements. With'the 01 assigned a side-chain mechanism, we have assumed that the 01relaxation has the effect of facilitating intermolecular shear when the primary intermolecular forces involve the side chains. Intermolecular forces that determine the rigidity of the 0 structure are the side-chain electrostatic forces between sheets and the intermolecular hydrogen bonds that stabilize the sheet itself. It is known that water molecules entering the 0 structure concentrate a t the layer of ions between the pleated sheets? When a sufficient quantity of water is present the onset of side-chain motion occurs a t the 01temperature. Following our assumption, this would allow the pleated sheets to slide past one another but the individual molecules would still be held in the sheet structure by the intermolecular hydrogen bonds. As the water content increases and the 0 structure breaks up to form a-helices the forces change. Hydrogen bonding is intramolecular, and now the primary forces between chains are electrostatic attraction and repulsion involving the side chains and counterions. At low temperatures this is a more rigid structure than the 0sheet, as evidenced by the abrupt increase in rigidity a t the 0 a transition. However, with only side-chain interactions between molecules, at the p1 temperature the individual molecules are able to slide past one another, and as a result, the drop in rigidity is much larger a t the 01" than the 010. A brief consideration of the esters of (Glu), shows a somewhat different situation. In this instance the sidechain interactions are nonbonded in nature and weaker than the intermolecular hydrogen bonds. Here our analysis predicts that the a form would be less rigid than the 0 form a t temperatures below the side-chain p relaxation. Finally, we can suggest at least one immediate implication of these results in regards to the proteins. The relaxation spectrum of native collagenous tissue shows a 01 process at 200"K, also associated with bound water, which shifts to lower temperatures as the water content increases.1*2 It now appears that this process can be identified with motion of the ionic side chains located in the noncrystalline regions of the collagen structure.
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CONCLUSION The general conclusions of this paper can be summarized as follows. It is now well established that dynamic mechanical spectroscopy has unique application to the study of interaction of macromolecular substances with small molecules in the solid state. Complementing this technique with other more conventional methods of analysis, we have been able to study the interrelationships among structure, hydration, and properties of these systems.
WATER-AMINO ACID INTERACTION. I
1165
In the case of (Glu-Na+),, a relaxation process was found that is specific for the secondary structure. By identifying the process with side-group motion, and noting the effect of hydration, we have been able to explain the observed changes in a bulk property, the rigidity. Finally, we have shown that characterization of model poly(a-amino acids) enables us to begin interpreting the relaxation spectra of the much more complex proteinaceous materials. The authors thank Dr. Toshio Hayashi for his helpful discussions of the infrared data. One of them (H. S.) gratefully acknowledges the support of the Fuji Photo Film Company during a leave of absence. This work was sponsored by the National Institutes of Health, Grant HL-15195-01A1.
References 1. Hiltner, A,, Nomura, S. & Baer, E. (1974) in Peptides, Polypeptides and Proteins,Blout, E. R., Bovey, F. A., Goodman, M. & Lotan, N., Eds., Wiley, New York, p. 485. 2. Hiltner, A,, Shiraishi, H., Nomura, S. & Baer, E. (1976) Int. J . Polyrn. Muter. (in press). 3. Shiraishi, H., Hiltner, A. & Baer, E., manuscript in preparation. 4. Lenormant, H., Baudras, A. & Blout, E. R. (1958) J . Amer. Chern. Soc. 80, 61916195. 5. Fasman, G. D., Hoving, H. & Timasheff, S. N. (1970) Biochemistry 9,3316-3324. 6. Shmueli, V. & Traub, W. (1965) J . Mol. Biol. 12,205-2140. 7. Armeniades, C. D., Kuriyama, I., Roe, J. M. & Baer;E. (1967) J . Mucromol. Sci.-Phys. B1,777-791. 8. Pezzin, G., Ceccorulli, G., Pizzoli, M. & Peggion, E. (1975) Macromolecules 8, 762764. 9. Kajiyama, T., Kuroishi, M. & Takayanagi, M. (1975) J . Mucrornol. Sci.-Phys. B11, 195-217. 10. Tsutsumi, A,, Hikichi, K., Takahashy, T., Yamaschita, Y., Matsushima, N., Kanke, M. & Kaneko, M. (1973) J. Macromol. Sci.-Phys. B8,413-430.
Received October 2,1975 Returned for revision December 10, 1975 Accepted January 5,1976
