INFRARED STUDY OF POLY-L-PROLINE IN AQUEOUS SOLUTION
4073
Infrared Study of Poly-L-proline in Aqueous Solution1
by Charles A. Swenson and Robert Formanek Department of Biochemistry, University of Iowa, Iowa Cdy,Iowa
(Received May 4, 1987)
Infrared spectra were measured for aqueous solutions (H20 and DzO) of form I1 polyL-proline in the fundamental region as a function of temperature. The temperature range includes the region where a reversible phase transition is known to occur. The spectral changes in the carbonyl absorption as this temperature range is approached are interpreted as a breakdown of solute-solvent interactions which result in destabilization of the solute in the solution phase. Simultaneously, there occurs a change in the C-H bending absorption which is interpreted as a small conformational change. Both of these absorptions show apparent isosbestic behavior as a function of temperature. A van't Hoff AHo of 60 10 kcal/mole (residue) was estimated for the over-all process from the temperature dependence of the carbonyl absorption.
*
Introduction Infrared spectroscopy in the fundamental region has been shown to be useful in elucidating the conformations of various proteins and synthetic polypeptides in oriented and unoriented dry film^.^^^ These studies have led to the assignment of some of the bands, notably amide I and amide 11, to various conformation^.^^^ Few studies of this type have been carried out in aqueous solutions (H20 or DzO) primarily because of experimental difficulties arising from the large extinction coefficient of water throughout the region of interest. A considerable amount of specific information can be gained from such a study, some of which cannot be obtained in any other way. I n the present communication we report the results of an infrared study of solute-solvent interactions for the peptide poly-Lproline in aqueous solution. Poly-L-proline is of interest because of its similarities with the structural protein collagen which has proline as its second most abundant amino acid. It is well suited as a model for an infrared study because of its limited capabilities for hydrogen bonding. Inter- and intrapeptide-chain hydrogen bonding cannot occur as it can act only as a base in this interaction. Thus a study of hydrogen bonding with the solvent can be carried out and interpreted without the usual complications. I n spite of the lack of intramolecular hydrogen bonds, the conformation of polyproline in solution might be expected to be semirigid due to the existence of a
resonance stabilized imide peptide linkage and a pyrrolidine ring in the structure. These restrictions leave the polymer with rotational freedom only a t the C,--C=O linkage, and this is thought to be hindered.6 Thus very few conformations are available to the polymer in aqueous solution. Poly-L-proline is known to exist in two forms, designated I and 11, which differ in their configuration at the imide linkage? Crystallographic analysis has shown that form I1 is a left-handed helix with all-trans imide linkages and a repeat distance of 3.1 A. Form I is a right-handed all-cis helix with a repeat distance of 1.9 A.8 Forms I and I1 can be interconverted in solution by simply changing the solvent. When an aqueous solution of poly-L-proline is heated to 55", precipitation occurs. This process is completely reversible and is analogous to the heat precipita(1) This research was supported by a grant from the National Science Foundation, GB 4385. (2) A. Elliott and E. J. Ambrose, Nature, 165, 921 (1950). (3) M. Beer, G. B. B. M. Sutherland, K. N. Tanner, and D. L. Wood, Proc. Roy. Soc. (London), A249, 147 (1959). (4) T. Miyazawa and E. R. Blout, J. Am. Chem. Soc., 83, 712 (1961). (5) S. Krimm, J . Mol. BWZ., 4, 528 (1962). (6) W. F. Harrington and P. H. von Hippel, Advan. Protein Chem., 16, 1 (1961). (7) F. Gornick, L. Mandelkern, A. F. Diorio, and D . E. Roberts, J . Ant. Chem. Soc., 86, 2549 (1964). (8) W. Traub and U. Shmueli, Nature, 198, 1165 (1963). (9) I. 2. Steinberg, A. Berger, and E. Katchalski, Biochem. Bwphya. Acta, 28, 647 (1958).
Volume 71. Number I.?? November 1967
4074
tion observed with tropocollagen. lo Structurally there is also a similarity; X-ray crystallographic studies have shown that the conformational arrangement of poly-L-proline, form 11,is the basis for the structure of collagen.1° Our purpose for this study was to investigate the interactions of polyproline with the solvent as the precipitation temperature is approached and relate them to the stability of the solution phase and/or changes in the conformation.
Experimental Section Poly-L-proline (mol wt 34,000) was obtained from Mann Research Laboratories (Lot R 1416). It existed entirely in form I1 as judged from the absence of the band a t 965 cm-’ in the infrared spectrum of the solid, Two other lower molecular weight samples were also investigated. One was obtained from New England Nuclear (mol wt l500), and the other was prepared in this laboratory (mol wt 2000). These two samples were predominantly form I and were converted to form I1 by dissolution in anhydrous formic acid prior to study. All the samples, after conversion to form 11, had identical solid-state infrared spectra and were completely soluble in cold water. Although all three samples were investigated, the data reported here are almost exclusively on the Mann sample. Solutions in water and deuterium oxide were prepared by weighing the dry polyproline. Complete dissolution was effected in a few minutes by cooling to about 0”. A concentration of 0.5% was used for most of these studies; however, a limited number of experiments were performed with concentrations as low as 0.1% and as high as 5%. Spectra were recorded with a Perkin-Elmer Model 521 spectrometer a t a spectral slit of approximately 4 cm-1. The monochromator and the sample chamber were purged with dry air. This reduced atmospheric absorption and prevented the cells from sweating at low temperatures. The sample cells were thermoststed by circulating water through a coil which was in thermal contact with the cell windows. The cell and water jacket were insulated from the surroundings by a layer of styrofoam. A thermistor in thermal contact with the cell window was used to monitor the temperature in the range 0-70”. Two matched cells with CaFz windows and optical paths of 0.05 mm were used. The reference cell contained pure solvent. For the region 1700-1500 cm-1 the solvent was DzO, and for the region 1550-1300 cm-’, H 2 0was used. DzOwould have been the solvent of choice for all of our studies except for the fact that HDO, which is difficult to eliminate entirely, absorbs The Journal of Physical Chemistry
CHARLESA. SWENSON AHD ROBERT FORMANEK
a t 1450 cm-I and complicates the interpretation of changes in the band shapes in this region.
Results The infrared spectrum of a 0.5% (w/v) solution of poly-L-proline form 11 is shown in Figure 1. Both water and deuterium oxide were employed as solvents to cover the spectral region of interest. The concentration was varied from 0.1 to 5% with no apparent changes in the details of the spectrum. It is notable that the spectra obtained in solution are slightly sharper than those obtained in the solid phase. No absorptions due to bands characteristic of form I at 965 and 1365 em-’ were observed.11 It is possible that these bands, which are often used to distinguish between form I and form 11, are characteristic only of the solid-state spectrum. Several intense bands are present in the spectrum of polyproline in aqueous solution. Only two of these can be assigned with any degree of certainty; the band at 1624 em-’, which is predominantly a carbonyl-stretching motion, and the band at 1456 cm-I, which is predominantly a C-H bending motion of the pyrrolidine ring.12 A variation in the solution temperature causes no observable effects on the spectrum until the precipitation temperature is approached. All the bands are then observed to change; the effects on most bands are changes in intensity with little or no shifting of the absorption frequencies. Large changes in intensity and frequency are observed to occur in the carbonyl and the C-H bending regions. Two new bands appear in these regions: one on the high frequency side of the original carbonyl absorption, and one on the low frequency side of the C-H bending absorption. I n both cases the new band increases in intensity as the temperature is raised with simultaneous decreases in the original band. The isosbestic behavior of these two bands is shown in Figures 2 and 3. Preliminary observations concerning the precipitation of the polymer showed the rate of heating to be critical. The nature of the precipitation has recently been studied carefully by Ciferri and Orofino so that further details need not be presented here.13 The important point to note is that equilibrium is established only very slowly in aqueous poly-L-proline solutions a t or near the precipitation temperature. Thus the curves ~~
-
(10) A. Veis, “The Macromolecular Chemistry of Gelatin,” Academic Press Inc., New York, N. Y., 1964. (11) (a) E. R. Blout and G. D. Fasman, Recent Aduan. Gelatin Glue Rea., Proc. Conf. Univ. Cambridge, 122 (1958); (b) J. Kurta, A. Berger, and E. Katchalsky, ibid., 131 (1958). (12) F. A. Miller, “Organic Chemistry,” H. Gilman, Ed., Vol. 3, John Wiley and Sons, Inc., New York, N. Y., 1953,p 143. (13) A. Ciferri and T. A. Orofino, J. Phys. Chem., 70,3277 (1966).
INFRARED STUDY OF POLY-L-PROLINE IN AQUEOUS SOLUTION
4075
0.0
I
I
W 0
z
U
m K
0.2
0
cn
m
a
0.4
O'*
0.8 1.0
1
SOLVENT- Or0
SOLVENT- H p O
1 I
c
1
1500
I700
I300
FREOUENCY
I
I
I
0
z
a m a 0 cn m a
1
1
1
I
I
1650 1625 1600 FREQUENCY
I500
I450
I
1400 CM-I
FREQUENCY Figure 3. Spectra of the C-H bending region as a function of temperature: curve a, 45'; curve b, 52'; curve c, 57"; curve d, 67".
1
w
1675
1
I100 CM-I
Figure 1. Infrared spectrum of a 1.0% aqueous solution of poly-tproline in a 0.05-mm CaF2 cell 31". Solvents: D20,1800-1530 cm-1; HzO, 1550-1000 cm-1.
4
I
I
1575 CM-I
Figure 2. Spectra of the carbonyl region aa a function of temperature: curve a, 45'; curve h, 53'; curve m, 65'.
cells when maintained at these temperatures would not permit precise measurements. Reversal of the precipitation or the spectral changes associated with it could not be achieved by cooling to room temperature. However, complete reversal occurs in a few minutes upon cooling to 0-5", in agreement with earlier observation^.'^ Only a very slight turbidity could be seen in the cells at the highest temperature during the time span of our study. If the solution was permitted to remain for 24 hr or longer at a temperature in the range of precipitation, precipitate was noted at the bottom of the cell. Spectra of this dried precipitate confirmed the earlier observation that intraconversion of form 11-form I does not occur upon heating. l 3 Two other observations are notable. The precipitation temperature seemingly was dependent on concentration. At the higher concentrations it was apparently decreased by several degrees. No attempt was made to study this effect further. We also noted that the precipitation temperature in DzO was several degrees lower than in water for equal concentrations of polymer.
Discussion shown in Figures 2 and 3 may not represent equilibrium values of the absorbance. I n order to obtain them, a standard rate of heating of l"/hr was adopted. We attempted to approach the 1°/day used by Ciferri and Orofino ; however, the evaporation of solvent from the
The temperature effect shown by the carbonyl absorption is typical of hydrogen bonding. l4 N-Methyl (14) G. C. Pimentel and A. L. McClellan, "The Hydrogen Bond," W. H. Freeman and Co., San Francisco, Calif., 1960.
Volume 71, Number 1% November 1967
4076
CHARLES A. SWENSON AND ROBERT FORMANEK
lactams which are suitable models for poly-L-proline show similar frequencies for the free and bonded states.16 Since inter- and intrachain hydrogen bonding cannot occur for poly-L-proline, the observed changes are a measure of this interaction between carbonyl and the solvent DzO. The temperatures at which an appreciable fraction of these hydrogen bonds are broken in D20 coincides qualitatively with the range of temperatures where precipitation is known to occur in water.13 At these temperatures the equilibrium may involve polyproline molecules which exist as an amorphous solid or a microcrystalline solid in suspension, in addition to those in true solution. The solid phase could exist even though no precipitate was discernible in the cell during the experiments. In spite of these various possibilities the experimental results suggest that the interaction of the carbonyl with the solvent is directly related to the stability of the solution phase. The results shown in Figure 2 were very reproducible as long as the rate of heating was duplicated. We were thus tempted to calculate the AH” for this process in spite of the large hysteresis effects known for the reverse process, which indicates that the system is slow to achieve equilibrium.’3 I n Figure 4 is shown a plot of In K os. 1/T. The equilibrium constants were calculated assuming two states as indicated by the spectra. The concentration of hydrated carbonyl, a, at a given temperature was calculated from the peak height of the band at 1624 cm-’, assuming that the peak height a t 30” represented the fully hydrated specie. Unhydrated carbonyl concentrations were then calculated by the difference, 1 - a. The equilibrium constant, K , is then a / ( l - cy). The overlapping bands were visually separated for this analysis. A value of 60,000 f 10,000 cal/mole (residue) was obtained for AH” from the slope. Although this A H o is only suggestive of the true energy due to the above indicated limitations, it still deserves comment. It is not likely to be AH” for the phase transition, as that is first order. The magnitude 60 kcal/mole (residue) is too large for just a dehydration of the carbonyl. This AH” could be associated with nucleation and/or some cooperative intramolecular process. Occurring simultaneously with the changes in the carbonyl absorption are changes in the C-H bending absorption of the pyrrolidine ring. These changes parallel exactly the behavior of the carbonyl absorption. Since the C-H vibration is not directly affected by hydrogen bonding, the frequency shift results either from a nonspecific solvent effect, an inductive effect, or a conformational change. Solvent effects for C-H vibration are not generally this large.lB Furthermore, a solvent effect such as raising the temperature would be The Journal of Phy&
Chemistry
Y
c
-I
3.0
3.05 I
x
3.1
103
Figure 4. Van’t Hoff plot for the carbonyl region.
expected to give rise to a gradually shifting frequency, not the isosbestic behavior observed here. An inductive effect is possible; however, the C-H bond is rather far removed from the carbonyl. It is also possible to explain the experimental observations in terms of a conformational change which occurs as the solvent interaction with the carbonyl group is decreased. Since the structure of polyproline is very rigid, the conformational change may be a slight rotation at the C,--0 bond, the only bond in the backbone with free rotation. At present we are not able to distinguish between these two possibilities. This infrared study of poly-L-proline in aqueous solution has permitted the observation of some specific solvent-solute interactions for this macromolecule. Poly-L-proline, form 11, in aqueous solution is likely to be a semirigid helix rather than a folded structure, and thus the solvent is able to interact with a large fraction of the peptide groups. Some of these interactions involve the solvent and the peptide carbonyl. They are easily broken by heating, presumably &s part of some intramolecular cooperative process. These hy(16) 0. E. Edwards and T. Singh, Can. J . Chem., 32, 683 (1964). (16) W. West, Ed., “Techniques of Organic Chemistry,” Vol. IX. “Chemical Applications of Spectroscopy,” Interscience Publishers. Ino., New York, N. Y., 1966.
THERMAL DECOMPOSITION OF NITRONIUM PERCHLORATE
drogen bonds between the solvent and the peptide carbonyl seem to be important for maintaining the balance of forces in favor of a stable solution form.
4077
Acknowledgment. The authors wish to thank Mrs. Mary Clemmer for her assistance with the experimental work and the preparation of the manuscript.
The Thermal Decomposition of Nitronium Perchlorate1
by J. N. Maycock and V. R. Pai Verneker Research Institute for Advanced Studies (RZAS), Martin Marietta Corporation, Baltimore, Maryland dld.97 (Received May 8, 1967)
The thermal decomposition of nitronium perchlorate has been studied in the temperature range 1O0-16O0 by isothermal constant-volume techniques and by mass spectrometry. Kinetic analyses have been performed for all the major decomposition species, e.g., Oz NO, and Clz. The activation energy is found to be 15 f 1 kcal mole-'. This is found to be in good agreement with the activation energy derived from E = hueo/€ where hv is the absorption edge and eo and E are the high- and low-frequency dielectric constants of the solid.
Introduction
ClO4'
The solid-state chemistry of the perchlorates is very important owing to their effectiveness as solid oxidizers. Considerable information is available relating to the metallic perchlorates,28but the only nonmetallic perchlorate which has received considerable attention is ammonium Another nonmetallic perchlorate of interest is nitronium perchlorate, whose decomposition between 70 and 112" has been studied by Cordes' and at 65" by Marshall and Lewis.6 The kinetic analysis performed by Cordes fitted the MampeP theory of solid-state decompositions remarkably well. As a result of this kinetic analysis he postulated that the rate-controlling step in the decomposition was the transference of the anionic electron to the nitronium ion with subsequent gas phase reactions to produce NOz, Clz, CIOz, NO&l, and Oz. This can be represented by ClOa- +c104' e-
+ NOZ+
+ eNOZ
with the subsequent gas phase reactions being
----)
CIOz
0 2
+ ClOz +NO3 + OC1 OC1 + NO2 +NO&l Clz + 20c1
NOz
--t
0 2
The isothermal decompositions of Marshall and Lewis have been interpreted such that nitronium perchlorate (NOzC104) decomposes into nitrosonium perchlorate (NOCI04) and oxygen with subsequent decomposition of the nitrosonium perchlorate. (1) Supported by the U. 8. Army Missile Command, Huntsville, Ala., Contract No. DA-01-021-AMC-l2596(Z). (2) (a) R. D. Stewart, "Perchlorates," J. C. Schumacer, Ed., American Chemical Society Monograph, Reinhold Publishing Corp., New York,N. Y.,1960,p46; (b) L. L. Bircumshaw and T. R. Phillips, J . C h a . Soc., 4741 (1957). (3) A. K. Galway and P. W. M. Jacobs, Proc. Roy. Soc. (London), A254, 455 (1960). (4) H. F. Cordes, J . Phys. Chem., 67, 1693 (1963). (5) M. D. Marshall and L. L. Lewis, Advan. Chem. Ser., 54, 82 (1966). (6) K.L. Mampel, Z . P h y d k . Chsm., A167, 235 (1940).
Volume 71, Number l d
November 1967
