ULTRASONIC ABSORPTION MEASUREMENTS IN GLYCINE, DIGLYCINE, AND TRIQLYCINE negative enthalpy resulting from interaction and penetration would exceed the positive enthalpy resulting from the separation of the oil molecules. The decrease
2227
in AH" of SDS a t the oil-water interface compared with the value a t the air-water surface supports the probability of this situation.
Ultrasonic Absorption Measurements in Aqueous Solutions of
Glycine, Diglycine, and Triglycinel by Gordon G. Hammes and C. Nick Pace2 Depurtment of Chemistry, Cornell University, Ithaca, New York 14860 (Received December 14, 1967)
Ultrasonic absorption and velocity measurements have been made on aqueous solutions of glycine, diglycine, and triglycine at 4" over the frequency range 10-175 MHa. Similar measurements were also made on solutions of diglycine in water and in aqueous urea, guanidine hydrochloride, and NaCl a t 10'. I n all cases, a single relaxation process was observed, and the relaxation times were virtually identical. Detailed consideration of various possible mechanisms suggests that the relaxation process is associated with solute-solvent interactions. The solvation apparently involves both the charged groups and the portion of the molecules between the charged groups. Since all of the solutes are mainly in their solvated form, the reciprocal relaxation time (-4 X lo8sec-l) is essentially equal to the rate constant for solvation, which is apparently identical for all three compounds. The differences in the amplitudes of the ultrasonic relaxation are consistent with the interpretation that glycine is more solvated than diglycine, which is more solvated than triglycine. The addition of urea, guanidine hydrochloride, or NaCl results in a decrease of the amplitude of the relaxation effect. However, the relaxation times are not significantly different from those found in water, indicating that the rate constant for solvation in these solutions is similar to that in water.
Introduction
Experimental Section
Water plays an important and unique role in determining the structure and stability of many biological macromolecules. The presence of water is known to give rise to the hydrophobic forces3 which are of prime importance in stabilizing the native, globular structure of protein^,^ the helical structure of nucleic acids,s and probably the structure of lipids in biological membranes. In addition, water plays an important role in determining the stability of hydrogen bonding in these molecules.6 Studies of ultrasonic absorption in aqueous solutions of a variety of different molecules ranging from polymers such as poly-L-glutamic acid7 and polyethylene glycolS-loto simple molecules such as amines1' and ethersl1rl2have shown that this technique is useful for investigating water-solute interactions. In this work ultrasonic absorption studies of glycine, diglycine, and triglycine in water and in aqueous solutions of urea, guanidine hydrochloride, and NaCl are reported. These compounds serve as simple models for the polypeptide chain of proteins. Aqueous urea and guanidine hydrochloride are well known protein denaturants, and solubility studies have shown that one mechanism of importance in this regard is their ability to increase the solubility of peptide groups.13~14
Diglycine and triglycine, Mann Assayed grade, were obtained from R'lann Research Laboratories Inc. Glycine and guanidine hydrochloride were obtained from the J. T. Baker Chemical Co. The guanidine hydrochloride was purified using the procedure of Nozaki and Tanford.16 Urea, A grade, was obtained (1) This work was supported by a grant from the National Institutes of Health (GM13292), (2) National Institutes of Health Postdoctoral Fellow, 1966-1968. (3) W. Kauzmann, Advan. Protein Chem., 14, 1 (1959). (4) C. Tanford, J . Amer. Chem. SOC., 84, 4240 (1962). (5) T. T. Herskovitz, Biochemistry, 2, 235 (1963). (6) H. A. Scheraga, The Proteins, 1, 478 (1963). (7) J. J. Burke, G. G. Hammes, and T . B. Lewis, J . Chem. Phys., 42, 3520 (1965). (8) G. G. Hammes and T. B. Lewis, J . Phys. Chem., 70, 1610 (1966). (9) G. G. Hammes and P. R. Schimmel, J . Amer. Chem. Soc., 89, 442 (1967). (10) G. G. Hammes and J. C. Swann, Biochemistry, 6, 1591 (1967). (11) J. H. Andreae, P. D. Edmonds, and J. F. McKellar, Acustica, IS, 74 (1965). (12) G. G. Hammes and W. Knoche, J . Chem. Phys., 45, 4041 (1966). (13) D. R. Robinson and W. P. Jencks, J . Amer. Chem. Sac., 87, 2462 (1965). (14) Y . Nozaki and C. Tanford, J . BWZ. Chem., 238, 4074 (1963).
Volume YO, Number 6 June 1968
2228
GORDON G. HAMMES AND C. NICKPACE
from Calbiochem. The sodium chloride and sodium Table I : Ultrasonic Parameters for Solutions acetate were reagent grade chemicals. For all soluof Glycine, Diglycine, and Triglycinea tions the pH was adjusted to the desired value using either HC1 or NaOH. l@A/m, Concn,b Concn,c sec/m 1017~, 1Osl', The ultrasonic absorption coefficients, a, and velocim m om uecr/cm uec ties, v, were determined as previously de~cribed.~,'~ Glycine, 4' Values of a / f z (f is the frequency of the ultrasonic 2.04 1.3 54 2.5 wave) could be determined to about 1 2 % , and values of v could be determined to better than i1%. Diglycine, 4"
Results Ultrasonic absorption data for glycine, diglycine, and triglycine in water at their isoelectric pH (5.97, 5.67, and 5.58, respectively, a t 25")17 a t 4" are shown in Figure 1, where a / f 2 is plotted as a function of frequency. Similar data were obtained for diglycine in water and in aqueous solutions of urea, guanidine hydrochloride, and NaCl a t 10". I n all cases studied, the data are consistent with the occurrence of a single relaxation process in the frequency range investigated (10-175 MHz); Le., the frequency dependence of a / f 2can be described by the equation'* AT = 1 + w2T2
+B
where w = 2qf, A and B are constants, and r is the relaxation time. Values of A , B, and T were obtained from the experimental data using the template technique described by Piercy and Subrahmanyam. lS These values are listed in Table I along with the ultrasonic velocities, v, for the same systems. The uncertainty in A , B, and r is approximately =k15, '3, and lo%, respectively, for the higher concentrations of diglycine. The error in A and r is somewhat larger for the other solutions. The solid curves in Figure 1 were calculated with eq 1 using the values of A , B, and given in Table I. All of the data are adequately represented by eq 1, although the changes in a/f2are so small for glycine and triglycine that the occurrence of
1.19m 0.99 m 0.81 m
0.60m Diplycine
70 J
4*-
60
FREQUENCYIMHzI
Figure 1. Plots of a / f 2as a function of frequency for aqueous solutions of glycine, diglycine, and triglycine a t their isoelectric pH a t 4". The error bars correspond to a &2'% error in a/f2. The solid lines are theoretical relaxation curves plotted according to eq 1 using the parameters in Table I. The Journal of Physical Chemistry
1.19 0.99 0.81 0.60
*..
0.24
...
1.47 1.32 1.19 1.00 0.84 0.72 0.51
...
1.32 1.32 1.32
*.. *.. *..
... ...
... ... ... ...
6.9 6.5 5.9 6.2
lo-su, cm/aec
1.536
61 58 55 52.5
2.7 2.7 2.7 2.7
1.525 1.519 1.505 1.479
Triglycine, 4' 8.9 49.5
2.5
1.454
Diglycine, 4.6 4.7 4.4 4.5 4.6 4.7 4.7
2.5 2.6 2.5 2.7 2.5 2.6 2.7
1.565 1.560 1 554 1.539 1.526 1.524 1.503
10" 51
49.5 48 45 43 41.5 39
I
Diglycine in Guanidine Hydrochloride, 10" 1.25 3.1 45 2.5 1.622 3.17 2.3 41.5 2.4 1.679 6.25 1.4 41 2.7 1.739
1.00 1.00
4.85 12.51
Diglycine in Urea, 10" 3.1 40 2.1 43
2.8 3.0
1.651 1.745
0.99
1.00
Diglycine in NaC1, 10' 3.1 43.5
2.4
1.608
a All measurements were carried out in water a t the isoelectric pH. * Glycine, diglycine, or triglycine. Guanidine hydrochloride, urea, or NaCl.
only a single relaxation process cannot be established with certainty. More extensive studies of solutions of glycine and triglycine were not feasible because the amplitude of the relaxation process is very small, even for almost saturated solutions. Reliable data over a range of concentrations, temperatures, and solvents could only be obtained with diglycine solutions. Even in this case, studies of the process were limited because of the small amplitude of the relaxation process a t higher temperatures (>loo). I n addition, studies of diglycine in guanidine hydrochloride solutions could not be ex(15) Y.Noaaki and C. Tanford, J. Amer. Chem. SOC, 89,742 (1967). (16) T. B. Lewis, Ph.D. Thesis, Massachusetts Institute of Technology, Cambridge, Mass., 1965. (17) E. J. Cohn and J. T. Edsall, "Proteins, Amino Acids, and Peptides," Reinhold Publishing Corp., New York, N. Y.,1943. (18) K.F. Herafeld and T. A. Litovita, "Absorption and Dispersion of Ultrasonic Waves," Academic Press Inc., New York, N. Y.,1959. (19) J. E.Piercy and S. V. Subrahmanyam, J. Chem. Phys., 42,4011 (1966).
ULTRASONIC ABSORPTION MEASUREMENTS IN GLYCINE, DIGLYCINE, AND TRIGLYCINE tended to higher guanidine concentrations (>7 m) because a solid solution forms.
Discussion The data in Table I show that the relaxation times for glycine, diglycine, and triglycine do not differ significantly. This and the fact that the compounds are structurally related suggests that the chemical mechanism responsible for the observed relaxation process is probably similar in all three cases. Indeed, it seems likely that any mechanism giving rise to a relaxation process for glycine would also be present for di- and triglycine; the converse, of course, would not be true. The number of possible mechanisms which might give rise to the relaxation process is fairly small. One obvious possible mechanism is an acid-base reaction involving either the amino or carboxyl groups. The relaxation processes observed with nmr spectroscopy for the protolysis of glycine are considerably slower than observed in this work.20 Moreover, for the diglycine-water system, the relaxation times are independent of concentration and the values of a / f z determined a t 35 and 135 MHz were unchanged at p H values 0.5 higher and lower than the isoelectric pH. These results rule out the possibility of a simple hydrolysis mechanism. Intermolecular processes such as dimerization (or other forms of aggregation) or intermolecular proton transfer can also be excluded by the concentration independence of the relaxation times. The possibility that intramolecular proton transfer between the carboxyl and amino group is responsible for the relaxation process can be ruled out on the following basis. The amplitude parameter, A , is given to a good approximation byz1
where AV and AH are the volume change and enthalpy change for the reaction, p is the thermal expansion coefficient of the solution, p is the solution density, cp is the constant-pressure specific heat of the solution, and r is a known function of the equilibrium constant of the reaction and the total solute concentration. Since the value of p for the solutions is essentially zero a t 4 O , the enthalpy term will make a negligibly small contribution to the amplitude of the relaxation process. The equilibrium constants for the conversion of the zwitterion to the neutral molecule have been estimated by Cohn and Edsall17 and can be used to evaluate I?. (For such a reaction, r = C0/[2 K (l/K)], where K is the equilibrium constant and COis the solute concentration.) Equation 2 and the experimental values of A can then be used to calculate AV for the three compounds a t 4'. The values obtained are A160, rt140, and *150 ml/mol for glycine, diglycine, and triglycine, respectively. These values are unreasonably
+ +
2229
large for the mechanism under consideration and indicate that intramolecular proton transfer is not responsible for the observed chemical relaxation. Rotational isomerization is also an unlikely explanation for the relaxation process, especially with glycine. The only mechanism which appears to be consistent with all of the data is one involving solute-water interactions. This mechanism can be written as
+
G, 3.9320 G,(HzO), (3) where G represents glycine and n is 1, 2, or 3. The value of z cannot be specified. If, as discussed above, the process is similar in all three compounds, then the hydration process is not simply a water-peptide group interaction, since, of course, glycine does not contain a peptide group. This is consistent with the failure to observe a relaxation process for aqueous solutions of urea9 and 2-pyrid0ne.~~It has been predicted theorer e t i ~ a l l yand ~ ~ confirmed ~~~ experimentallyz6 that the relaxation of the ionic atmosphere of simple 1 : l electrolytes is much too small and usually too rapid to be observed with ultrasonics. Of specific interest with regard to glycine, diglycine, and triglycine, is the absence of a relaxation process for either a 6 rn NH4Cl'O or a 4.45 m sodium acetate solution. I n the first sec2/cm is found a t case, a value of a / f 2of 21.6 X 10' between 10 and 165 MHz,10 and in the second case, a value of 64.5 X l O - l 7 secz/cm is found a t 10" between 25 and 145 MHz. This suggests that the relaxation process is not simply a hydration reaction involving the electrostricted water around the charged groups. Thus the relaxation process must be associated with a unique property of the zwitterion structure. Apparently solvation of the portion of the molecules between the charged groups as well as solvation of the charged groups is involved. The increase in the magnitude of the amplitude of the relaxation process, A/m, shown in Table I, suggests either that the hydration process is characterized by larger values of AV or smaller values of K in going from glycine, to diglycine, to triglycine. If AV is assumed constant in all three cases, then this would imply that glycine is more solvated than diglycine, which is more solvated than triglycine. Since in all cases the equilibrium would be expected to lie heavily in favor of the solvated species, the similarity in the relaxation times (20) M. Sheinblatt and H. S. Gutowsky, J . Amer. Chem. SOC.,86, 4814 (1964). (21) M. Eigen and L. de Maeyer in "Technique of Organic Chemistry," Vol. 8, Part 2, 5. L. Friess, E. S. Lewis, and A. Weissberger, Ed., Interscience Publishers Inc., New York, N. Y., 1863. (22) G. G. Hammes and H. 0. Spivey, J . Amer. Chem. SOC.,88, 1621 (1966). (23) M. Eigen, Discussions Faraday Soc., 24, 25 (1957). (24) J. Stuehr and E. Yeager in "Physical Acoustics," Vol. 11, Part A, W. P. Mason, Ed., Academic Press Inc., New York, N. Y., 1965. (25) G. Kurtae and K. Tamm, Acustica, 3, 33 (1953).
Volume 7.9, Number 6 June 1068
2230 suggests that the rate constant for solvation is the same in all three cases, about 4 X lo8sec-'. (The reciprocal relaxation time is equal to the sum of the rate constants for solvation and desolvation.) Since the relaxation times at 4 and 10" are essentially identical, the activation energy for solvation must be small. The rate constants for desolvation cannot be estimated without a knowledge of the equilibrium constants, but they will be less than 4 X lo* sec-', which is considerably slower than the rate of dissociation of a water molecule from substituted amines.26 The values of B shown in Table I correspond to the value of a / f z at high frequencies, i.e., beyond the relaxation discussed above. At 4", the value of a / f 2for H2O is 46 X ~ e c ~ / c m ;B~ 7exceeds this value in all cases. These increased values of B cannot be attributed solely to changes in the classical viscous absorption, since the classical values of a / f 2for the solutions, calculated from the measured viscosities, differ by less than 1.5 X sec2/cm from that of water (cf. ref 18 for a discussion of this calculation). Thus in the presence of solute, either the structure of water is altered and/or additional unrelaxed reactions are occurring. Note that (B - BH,o)/m (m is diglycine molality) is 3.9 X sec2/m cm for glycine, 11.7 X 10-17 secz/m secz/m cm for tricm for diglycine, and 14.6 X glycine, a variation which is similar to that found for A/m. Such effects have previously been interpreted as being due to an increase in structured water (Le., water clusters) due to the hydrophobic groups on the solute.8-10 This is consistent with the observed order of effectiveness of the solutes in increasing B. The addition of urea, guanidine hydrochloride, or NaCl to the diglycine-water system causes a pronounced decrease in the magnitude of the relaxation process, as shown in Figure 2. However, the relaxation time is virtually unaffected, the only significant change being a slight increase in 7 in 12.51 m urea. The small effect of these compounds on 7 and the fact that a single relaxation is still observed suggests that they exert their influence on the amplitude parameter, A/m, by participating in the same solvating reaction as H20. The decrease in A/m could result from an increase in the over-all equilibrium constant for the interaction, from a decrease in the over-all AV, or from a contribution from AH which might become significant in these solutions. Robinson and Jencks13have shown that the solubility of acetyltetraglycine ethyl ester in HzO is increased by the addition of urea and even more
The Journal of Physical Chemistry
GORDON G. HAMMES AND C. NICKPACE
MOLALITY
Figure 2. A / m (m is diglycine molality) plotted as a function of molality of urea, 0, guanidine hydrochloride, 0,and NaCl, A, for aqueous solutions of diglycine a t 10'.
so by the addition of guanidine hydrochloride. The observed decreases in A/m are consistent with a possible increase in the over-all equilibrium constant for the solvation reaction in urea and guanidine hydrochloride. However, Robinson and Jencks also showed that the solubility of their peptide model was decreased by NaC1, while NaCl is approximately equally as effective in decreasing A/m as guanidine hydrochloride. One possible explanation for this is that for urea and guanidine hydrochloride the value of A/m is decreased by an increase in the equilibrium constant, while for NaCl the decrease is due to the decrease in AV. Alternatively, some type of ion-pair interaction might be the source of the decrease in A/m caused by NaCl and guanidine hydrochloride. I n any event, the lack of a significant change in the relaxation time suggests that the rate constant for solvation is essentially identical in water, aqueous urea, and aqueous guanidine hydrochloride, whereas the rate constant for desolvation may vary somewhat in the three cases. The smooth continuous decrease in A/m with urea and guanidine hydrochloride concentration and the constant relaxation time contrasts markedly with results from ultrasonic studies of polyethylene glycol in ureae and guanidine hydroch1oride;lO in these latter cases the parameter A/m remains essentially constant in guanidine hydrochloride and increases in urea, and a sharp decrease in the relaxation time occurs between 2 and 3 m urea and guanidine hydrochloride. In the polymer solutions, a cooperative change in the water-polymer structure is suggested, while in the present work the number of water molecules involved is apparently too small to involve cooperative effects. (26) E. Grunwald and E. K. Ralph, 111, J . Amer. Chem. Soc., 8 9 , 4405 (1967). (27) J. M. M. Pinkerton, Nature, 160, 128 (1947).
