J. Chern. SOC., Faraday Trans. I , 1982, 78, 2275-2278 Osmotic Coefficients of Urea + Guanidinium Chloride Mixtures in Water at 298.15 K BY TERENCE H. LILLEY* AND DENNIS R. TESTER Chemistry Department, The University, Sheffield S3 7HF Received 2nd November, 198 1 The osmotic coefficients of urea + guanidinium chloride mixtures in aqueous solutions have been determined at 298.15 K using the isopiestic vapour-pressure technique. A comparison is made between the results obtained and the corresponding results for other urea+ salt aqueous solution mixtures. There is a correspondence between the urea + salt interactions and the tendency for the salts to destabilise protein structures. It has been known for some time that aqueous solutions containing either urea or guanidinium chloride (GuCl) can induce the denaturation of pr0teins.l More recent work2 has shown that solutions containing either or both of these solutes have a marked effect on the rates of denaturation and renaturation.The present work was stimulated by the observation3 that mixed solutions of urea + GuCl are considerably less efficacious in their effect on the rate of denaturation of lysozyme than solutions containing only one of these solutes. The thermodynamic properties of aqueous urea solutions have been investigated in some considerable detail and there are possibly more precise thermodynamic data available for the urea+water system than for any other system containing a non-electrolyte in water.4 It is only relatively recently that attention has been turned towards the properties of aqueous GuCl ~olutions.~-~ In the present paper we present an investigation of the osmotic coefficient behaviour of binary-solute aqueous mixtures containing urea and GuCl.The results obtained indicate rather pronounced interactions between the two solutes. EXPERIMENTAL AND RESULTS The apparatus and experimental procedure have been described previously.8 The urea was of ultrapure quality and the GuCl was of analytical reagent grade and was twice recrystallised from water. Both substances were intensively dried before use. The experimental results obtained, presented as the molalities of the solutes in isopiestic equilibrium, are given in table 1. In view of the precision to which the osmotic coefficients of urea solutions are known, solutions containing this solute were used as the reference solute in the isopiestic experiments. DISCUSSION It is convenient8 when treating systems such as that considered here to define a term (1) Nm4) = mref 4ref-2mg 4E-mn 4; where, in the present instance, mref is the molality of the urea reference solution in a given experimental run and 4ref is the osmotic coefficient of this reference solution.22752276 OSMOTIC COEFFICIENTS OF UREA+GUC~ TABLE 1 .-EXPERIMENTAL RESULTS : MOLALITIES OF ISOPIESTIC SOLUTIONS rn/mol kg-I m/mol kg-l urea GuCl - A(m4) urea GuCl - A(m4) 13.0462 11.9080 10.7220 8.1091 6.7483 5.3910 2.7056 1.3244 0 8.6956 7.891 5 7.1074 5.3719 4.4763 3.5663 1.8121 0.8913 0 0 0.8420 1.6599 3.3576 4.1947 4.9945 6.5039 7.2487 7.9260 0 0.5580 1.1003 2.2242 2.7824 3.3225 4.3562 4.8749 5.3500 - 0.5389 0.8 172 1.0523 1.0550 0.9809 0.6505 0.3816 - - 0.3314 0.5408 0.7121 0.7146 0.67 13 0.4448 0.248 1 - 6.957 1 6.3602 5.6785 4.3099 3.6022 2.8929 1.4489 0.7528 0 3.3874 3.4353 3.0534 2.2983 1.9178 1.5385 0.7719 0.4019 0 ~~ ~~ 0 0.4122 0.871 1 1.7550 2.1947 2.627 1 3.48 11 3.8735 4.2934 0 0.2227 0.4684 0.9362 1.1684 1.3972 1.8547 2.0679 2.2975 - 0.2477 0.421 1 0.5672 0.5686 0.5338 0.3552 0.2040 - 0.1078 0.1907 0.2583 0.2606 0.2438 0.1597 0.0889 TABLE 2.-cOEFFICIENTS AND THEIR 95 % CONFIDENCE LIMITSa FOR THE REPRFSENTATION OF A(m4) BY EQN (2) A,l/mol-l kg A21/mo1-2 kg2 A,2/mo1-2 kg2 A,,/m01-~ kg3 A,,/molP kg3 A,,/m01-~ kg3 A 14/m01-4 kg4 ~ , , / m ~ l - ~ kg4 - 0.1907 (0.0 127) 2.4341 (0.5623) x lop2 3.7332 (0.3737) x -2.3293 (0.3145) x -2.821 1 (0.7091) x - 1.9235 (0.9351) x lop3 6.2514 (4.3590) x 1.1544 (0.7815) x lop4 a The 95% confidence limits are shown in parentheses.The molalities of GuCl and urea in the binary solute mixtures are denoted by mg and m,, respectively, and 4: and 4; are the osmotic coefficients of GuCl and urea in single-solute-containing solutions at the molalities mg and m,. The osmotic coefficients of urea solutions were obtained from stoke^'^ critical compilation. The osmotic coefficients of single-solute solutions containing GuCl were found to agree very well with those obtained by Schrier and S ~ h r i e r , ~ and consequently their parametric expression was used to obtain values for the osmotic coefficients of GuCl solutions. The agreement between the present results for GuCl single-solute solutions and those calculable from the results obtained in two recent investigations by Bonner' and by Bates and coworkers6 are acceptable by most standards. We have included theT.H. LILLEY A N D D. R. TESTER 2277 calculated values of A(m4) for the various mixtures in table 1. As is now customaryg the values of A(m4) were fitted to an equation of the form Table 2 gives the values of the coefficients ( A t j ) , along with their 95% confidence limits, which were required to fit the results adequately. In a notional sense, Aij represents a measure of the interaction of i species of GuCl with j species of urea; e.g. A,, corresponds to interactions between two GwCl species with two urea species. There are several manipulations which are possible given these coefficients. For example, by appropriate integration and differentiation of eqn (1) and (2) we obtain the following expression for the logarithm of the ratio of the activity coefficient of GuCl in urea+GuCl mixture ( y g ) to that in a solution containing only GuCl ($) at the same molality as the GuCl in the mixture: The corresponding expression for the logarithm of the activity coefficient ratio for the urea is In (y,/y:) = x D/(i+j- 1) A i j mk mip1.j-1 i-1 (4) Eqn (3) and (4) in association with the coefficients given in table 2 and the activity coefficients of the solutes in solutions containing only one solute component4. may be used to calculate the activity coefficients of urea and GuCl in mixtures. As was mentioned in the Introduction, urea + GuCl solutions denature proteins rather effectively.However, it has also been suggested1" that urea mimics, to some degree, an average peptide group in proteins. Given this assertion it is convenient to calculate the free energy of transfer of urea from water to GuCl solutions under the condition that the urea molality approaches zero. This free energy of transfer (A&?) f I I I I 1 0 2 4 6 8 salt molality/mol kg-' FIG. 1.-Variation of the free energy of transfer of urea (A&) with salt molality.2278 OSMOTIC COEFFICIENTS OF UREA+GuCI will then give an indication of how a peptide group interacts with GuCl in aqueous systems. The expression for the free energy of transfer under the above condition is A&? = RT C (Ail/i>mt.i-1 In fig. 1 we illustrate the dependence of the free energy of transfer of urea on the molality of added salt for GuCl. Included in this figure are the corresponding results for urea in solutions of NaC1,ll LiCPO and CaC1,.lo The results for all four salts indicate an attractive interaction with urea (i.e. the free energies of transfer are negative) and the ranking order in terms of such interactions is NaCl < LiCl < GuCl < CaCl,. This is the same as the order observed' for the destabilising effect of salt solutions on protein structures. In other words urea + salt interactions in aqueous solution appear to follow the Hofmeisterl series. The implication of this is that, given the common features of structure between urea and the peptide bond, the denaturation of proteins by salts is related to how salts interact with peptide bonds. We acknowledge help in various ways from K. G. Davis. See P. H. Von Hippel and T. Scheich, Acc. Chem. Res., 1969,2,257 and F. Franks and D. Eagland, Crit. Rev. Biochem., 1975, 3, 165, for references to early work. C. Tanford, Ado. Protein Chem., 1968, 23, 121. B. Robson and T. H. Lilley, to be submitted for publication. The data have been compiled by R. H. Stokes, Aust. J . Chem., 1967, 20, 2087. M. Y. Schrier and E. E. Schrier, J . Chem. Eng. Data, 1977, 22, 73. J. B. Macaskill, R. A. Robinson and R. G. Bates, J. Chem. Eng. Data, 1977, 22, 411. T. H. Lilley and R. P. Scott, J. Chem. SOC., Faraday Trans. I , 1976, 72, 184. T. H. Lilley and R. P. Scott, J. Chem. SOC., Furaday Trans. 1, 1976, 72, 197. V. E. Bower and R. A. Robinson, J. Phys. Chem., 1963, 67, 1524. ' 0. D. Bonner, J. Chem. Thermodyn., 1976, 8, 1167. lo M. Y. Schrier, A. H. C. Ying, M. E. Ross and E. E. Schrier, J. Phys. Chem., 1977, 81, 674. (PAPER 1 / 1707)