SAM KATEAND JANE E. MILLER
277
ium EEects of Urea and Guanidine Hydrochloride on the Volume
mer31 by Protonation of Carboxylate Groups*
~~~g~~
am ICatz" and Jane E. Miller Department of Biochemistry, West Virginia University Medical Center, Morgantown, West Virginia 26506 (Received January 81, 1973) Publicalhn costs assisted by the National Institutes of Health
The presence of urea or guanidine in the system has a depressant effect on the magnitude of the volume changes produced by the protonation of the salts of amino acids, their derivatives, and mono- and dicarboxylic acids. The magnitude of the volume changes produced by protonatioh iJ a function of the composition and structure of the organic acid, whereas the relative decrease of this volume effect varies with the concentration and type of denaturant present. The use of 4,6, and 8 M urea as solvent causes about 10, 15, and 20% reduction of the volume effect produced in water, respectively; 4 and 6 M guanidine hydrochloride effects a reduction of about 40 and 50% relative to that in water. This depressant effect is explicable in terms of the reduction of the activity of water, the water structure breaking action of the solutes, and the dimunition of the electrostriction effect by increasing the dielectric constant of the medium. Guanidine hydrochtoride, a uniunivalent electrolyte, incorporates an additional factor, namely a salt effect which depresses the magnitude of these volume changes.
~ ~ ~ r ~ ~ u c t ~ o ~ The effect of varying the concentration of urea and guanidine hydrochloride on the volume changes, AT', produced by the protofiation of carboxylate groups incorporated in organic acids, amino acids, and their clerivatives 1s the subject of this study. This detailed tabulation will supplement the existing data2 and also provide information necessary to establish the relationship between the magnitude of the volume change, the type of carboxylic acid, and of the medium employed. The influence of varying the concentrations and ratjo of organrc acid to base was also investigated. This information is esseiitinl to elucidate the anomalously low toiurne changes produced by the titration of carbox"late groups incorporated in proteins in 8 M urea and the high values determined in 6 M guanidine hydrochloride, These data may help clarify the role of these denaturants with. respect t o their influence on water structure, Le. , whether they function as structure 6irnalcers"or "brealiers ' ' * +
The volume changes were determined with Teflonsheathed Linderstr#m-Lang dilatometers7 which could be read to 0.01 pl. The methodology, source, and purification of reagents hai7e been described in detaiL2 To wtabhsh concentration effects the following procedure was employed. (j) The 1: 1 protocol: 5 ml of 0.20 M carboxylate containing compound was mixed with 5 ml of 0.1000 M WCI; after mixing, there was a 1:1 ratio of organic acid and organic base. (ii) The 3: 1 protocol: 8 ml of 0.20 M carboxylate containing compound was mixed with 4 ml of 0.1000 N HC1. On occasion the 1%
Journal of Ph&cul
ChemistTy, Vol. 76, N o . 1 9 , 197.8
volumes but not the ratio of solutes were changed to ensure that the experimental volume changes were between 2 and 6 p1. Since the emphasis of this study was to determine relative volumes changes, fewer duplicate experiments were performed than in the initial study in this series.2 Correction was made for unbound protons;* the magnitude of the correction for the specific compound and medium is stated in the appropriate tables. The experiments were performed at 30.8 f 0.001". The range about the mean was 10.25 ml/ mol.
Results The values for the protonation of carboxylate groups of amino acids and their derivatives in water, urea, and
guanidine hydrochloride are summarized in Table I. The mixing protocols were designed to yield, after mixing, systems which had 0.05 LW organic salt and 0.05 M organic acid (1:1 mixing protocol) and 0,15 M organic salt and 0.05 M organic acid ( 3 : 1mixing protocol). This approach permitted us to assess effects of salt concentration, mass action, reliability of data, and the concentration of products. I n water, the mean value for (1) This research was supported in part by U. S. Public Health Service, National Heart and Lung Institute Grant NE 12955. ( 2 ) S. Katz and J. E. Miller, J . Phys. Chem., 7.5, 1120 (1971). (3) S. Kate and J. E. Miller, Biochemistry, IO, 3569 (1971). (4) H. S. Frank and M. W. Evans, J . Chem Phys,, 13, 50 (1945). (5) A. Holtaer and M. F. Emerson, J . Phgs. Chem., 73, 26 (1969). (6) W. A. Hargraves and G. C . Kresheck, ibid., 73, 3249 (1969). (7) K. Linderstr@m-Lang and H. Lanz, C. R. Trav. Lab. Carlsberg, 21, 315 (1938). (8) W. Kauzmann, A. Bodansaky, and J. Rasper, J . Amer. Chem. SOC.,84, 1777 (1962).
MEDIUM EFFECTS ON VOLUME CHANGES
2779
-
I
I: A V of PaatonsCion of Carboxylate Groups in Amino Acids and Amino Acid Derivatives
~~~~~
---~-
I _
Roaotani 9
@-Alanine Disodium glutamate Disodium glutathionate y-Amino-n-butyric acid Glycylglycine Sodium N-acetylglycinate Mean value Q
Protocol
Ha0
1:1 3: 1 I: 1
7.1
6.40 6.3 7.0 6.7 7.8" 7.5 8.1 8.2
7.4 8.0 7.6 9.3 8.7
3 :1 1: 1 3:1 I: 1
9.4 9.3
3: 1 1:l 3: 1 1:l 3: 1 1:l
9.2 11.0
3:l 1-2% correction for unbound protons.
4M
7.gC 7.9"
9.5
%3%.
11.0
9.2a 9.2
9.1
7.7
8.8
7.6
c
3-4%.
d
A V , ml/mol------------------------.
Urea-BM
6.1. 6.1 6.8 6.5 7.36
7.3 7.7 7.8 7.w 7.6" 8.7b 8.9 7.4 7.4
7
8 M
5.8b
5.9
Guanidine hvdrorhloride 4 ?if 6 K
4.5a 4.2
6.4 6.2 6.93 6.8 7.4 7.4 7.2d
4.75
7.16
5.76 6 9" 6.Gb
8.5b 8.4 7.0 7.0
4,5"
5.66 5,P 5.9" 5.56.26 I
.5,6 5.3
3.66 3.75 3.85 3.4b 4.P 4.7c 4.P
4.4h 5.D 4.7c 6.OC 5.4c
4.7 4.4
4-5%.
I___
AV was about 9 mllmol with the range being about 2 ml/rnol. The presence of an elevated salt concentration, data obtained from the 3: 1 mixing protocol, was about 0.3 mi/rnoi lower than that determined at the lower salt concentration. The use of 4, 6, and 8 M urca depressed these values by about 15, 20, and 25% respectively. The presence of guanidine hydrochloride caused a more proirounced effect with AV being reduced about 40 and 58% in 4 and 6 M guanidine hydrochloride, respectively. The presence of a high salt content lended to reduce the values for AV in water and guanidine hydrochloride. I n water and in 4 and 6 M gmnidine hydrochloride the valucs for AV were about 0.3 ml/mol lowcr for the 3 : l mixing protocol compared to the 1:1 mixing protocol. However, systems containing urea showcd virtually no salt effect in 6 and 8 M urea and a decrease of about 0.1 ml/mol in 4 M urea. Correction for unbound protons was not required when water was the solvent; however, when urea and guanidine hydrochloride were incorporated in the rjystem corrections were needed for organic acids with pK 5 3.6. To illustxate the magnitude of the correction consider glyc~rlglycine~ pK == 3.148. There was 4 4 % unbound protons in 6 and 8 M urea; in the other bysterns the correction for unbound protons was lower (see Table I[). The presence of a high concentration of the salt of the organic acid, 3 : l protocol, reduced the amount of unbound protons in accordance with mass action effects. The correction for unbound protons in guanidine hydrochloride was slightly higher than that required for urca. I n guanidine hydrochloride the correction factor Cor the 1. : P and 3 : 1 systems were virtually the same. 'The AV produced by protonation of several monoand dicarboxylate organic compounds are summarized in Table TI. llcsd of these data pertain to values ob-
tained by the 1: 1 mixing protocol since solubility considerations limited the use of elevated salt concentrations. This dictated the use of a 2:l mixing protocol, ie., the ratio of the salt to acid after mixing was 2:1. Since the emphasis of this study was to determine medium effect on protonation processes, it is of interest to consider the influence of those solutes 011 the dicarboxylic acids which exhibit large volume changes.8 These data reveal that the relative diminution of AV caused by medium effects were relatively independent of the compound structure: Le., the mono- and dicarboxglate organic compounds exhibited similar relative volume decreases. The reduction of AV due to urea was about 12, 15, and 20% in 4, 6, and 8 M urea. I n 4 and 6 M guanidine hydrochloride reductions of 40 and 50% were observed. Salt concentration effects apparently occur; however, the data w e too limited to permit a valid assessment. The relative reduction of volume changes noted for the organic acids is similar to that observed for amino acids indicating that similar medium effects are operational, but the magnitude of the volunie effect is determined by the structure of the carboxylate con taking compound and by 'che medbxn.
Discussion The volume changes produced in water by the protonation of carboxylate groups ranged from 7.7 ml/mol for sodium formate to 19.7 mljmol for disodium maleate. However, the AV for thia process involving simple and polyfunctional amino acids did not exhibit this span of values but gave a mean value for AV of 9 ml/mol with a range of 2 ml/mol. The rationale for the variation of AV for protonation of polyfunctional compounds has been explained in detail by Kauzmann, (9) J. T. Edsall and J. Wyman, "Biophysical Chemistry," Academic Press, New York, N. Y . , 19511, p 452 The Journal of Phvsical Chernistrg, Vol. 78, A'o. 19, 1972
------..-: A V of elre Protonation of Organic Bases ,-----.--------~--
Protocol
E20
4 1M
AV, rd/mol-- ~ Urea------------6 M 8 M
1:1. 2: 1
7.7
6.80. 6.7
6.5" 6.P
~
RcRotsnt
Sodium formate
Sodium acei,a'cs Disodium indonate Disodium maI.ea.te
1:l I. :I. 1:J.
2: x
1-2% correction for unbound protons. _I____
_____
'7.5 10.7 15.8
-
14.1 17.4
9.2 43.5 16.7
17.3
16.6
15.7
3-4oj,.
el in terms of the contribution of the type of substituents, net charge, arid stereochemical considerations. The reduction of AV of protonation due to salt effect is well documented2 Konreacting electrolytes influence this process by ionic strength effect and by altering the reactant's activity coefficient. This factor can reduce the magnitude of AV substantially, e.g,, the AV of neutralization of HC1 by KaOH decreased from 20.3 to 16.7 ml/mol as the concentration of NaCl increased from 0.05 to 2 M . 2 Thus, the reduction of A V observcd for the 5: I mixing protocol compared to the 1:1protocol is to be expected. The magnitude of this attenuation of AI7 is too small to be ascribed t o specific solute-reactant interaction. These data confirm the observatiorr that the magnitude of AV is a function primarily of the composition and structural organization of the compound being The presence of urea or guanidine hyprotonated.* drochloride causes a relative reduction of these volume effects which is siimibr for all compounds tested, and this effect js dezermined by the type and concentration QF the denaturant Hargraves and Kresheck6 explain the medium effect of 6 M urea in terms of the water structure ?xeali.ing; action of urea; this approach preslimably applies to guanidine hydrochloride. These authors pcrstizlate that the positive values found for the AVto term, the Mcrence between the partial molar volume of compounds hi 6 14 urea and in water for carboxylic acids, carboxylates, ctc., was the resultant of the contribution 01tho polar. and nonpolar moieties comprising the rnoieculle. The positive values for this function were interpreted as being evidence that urea functioned as a wafer structure breaker because the introduction of an organic side chain into the urea-water system produced a positive volume eifect reflecting an additivity process. A corollary of this hypothesis is that the values for AT'," ~houldincrease fiith increasing side-chain length; this has been verified experimentally. We propose that in addition to the vlater structure effect attributable to cnaturanls there are other contributing factors. The presence of urea and guanidine hydrochloride in the solvent dec*reaswthe activity of water, 11,l2 thereby reil'he Journal of Physical Chamistru, Vol. 76, N o . 19, 1972
-
6.4b 6.6" 8.8 12.7 16.1
9.5
19.7 19.2
* 2-353,.
"
-___
---
Guanidine hydrochloride 4M BM
5.26
4.7.
5.9a
4.P
6.56 8.9" 11.8 11.5
%,6b
7.2b 9.60 9.3
ducing the amount of water available for ion-water interaction. Another factor is based on electrostatic considerations, namely that the magnitude of the volume changes associated with protonation can be estimated by the Drude-Nernst13 equation. The equation which has been discusse viously2b8states that
The terms are defined as: p, compressibility, V , volume of the medium; D, dielectric constant; eZ, electrical charge, and r , the radius of the sphere irnniersd in the medium. The introduction of urea or guanidine hydrochloride into the solvent increases the dielectric constant of the m e d i ~ m , ' causing ~ ' ~ ~ x reduction of the volume effect since AV varies inversely %?iththe square of the dielectric constant (see ref 2 for discussion). The large depressant action of guanidine hydroehloride reflects the operation of still anokher factor; this is a salt effect associated with the ionization of guanidine hydrochloride, a uni-univalent electrolyte (see Results) Specific solute-reactant interactions may play a role vi7ith respect to these volume changes; hovever, to produce such a concordant relationship for these relative volume depressant effects it would require that the association constants for these int,erartions be similar. This does not appear to be highly probable in view of the disparate character of the carboxylate compounds. The data reported here are in accord with the frame of reference projectcd from the initial paper in this series;2this lends credence Lo the colic usions presented prevjously. (10) H. H. Weber, Biochem. Z , 218, 1 (1930). (11) G. Scatchard, W. J. Kamer, and S. E. Wood, J ~ Amer. . Chem. Soc., 60, 3061 (1938). (12) N.D. Ellerton and P. J. Dunlop, J . Phys. Chem., 70, 1831 (1966). (13) P.Drude and SV. Nernst, Z . Phys. Chem. (Leipzig), 15, 79 (1894). (14) E. C. Cohn and J. T. Edsall, "Proteins, Amino Acids, and Peptides," Reinhold, New York, N. Y . , 1943, p 140. (15) Reference 9, p 323.
