1197
Tetraalkylammonium Salt Effects on Model Peptides
Effects of Tetraailkylammonium Salts on the Activity Coefficient of N-Acetyl Ethyl nylalanine, Norleucine, and Norvaline Pradip K. Nandi’ Department of Physicai Chemistry, indian Association for the Cultivation of Science, Calcutta 32, India (Received Juiy 2, 1973; iSevised Manuscript Received October 72, 7973)
Activity coefficients of N-acetyl ethyl esters of phenylalanine, norleucine, and norvaline have been determined in tetraalkylammonium bromide salt solutions. The compounds generally show salting in in these salt solutions the magnitude of which increases with the increase in the size of the alkyl groups in the salt. The molar free energies, AFt,, of transfer of the compounds from water to 1 M salt solution have been calculated. These values indicate that the contributions of the nonpolar and polar portion of the ester molecules toward the transfer process in these salt solutions are not additive in nature. The enthalpies, AH,and entropies, A S , which were calculated from the variation of the activity coefficients with temperature showed the importance of hydrophobic interaction in explaining the observed salting behavior of the compounds. An attempt has been made to apply these results to explain the mechanism of protein denaturation in tetraalkylammonium salt solution.
Tetraalkylam monium salts in aqueous solution occupy an interesting position in solution physical chemistry. Nearly 50 papers most of which have appeared during last 25 years reveal their uniqueness compared to simple salts as electrolytes in solvont water. The recent papers of Wen and Hung and Wirth and LoSurdo have documented the relevant references regarding physicochemical properties of these salts in aqueous solution and also their interaction with other solutes in water.2 The unusual physicochemical properties, compared to ordinary electrolytes, which arise mainly due to the presence of nonpolar groups in these salts have drawn considerable attention to study their effects on the structural stability and reactivity of biological macromolecules. The effectiveness of these salts in denaturing proteins and DNA and affecting the activities of several enzymes, uiz., fumarase, lactate dehydrogenase, etc., has heen d e m o n ~ t r a t e d In . ~ general ~ the effectiveness in causing above changes in the structure and reactivity increases froini tetramethyl to tetrabutyl derivative. The manner in which these salts effect the structure and activity of the macromolecules is not certain. It is still undecided whether an indirect effect uiz. a change in the water “sitmcture” caused by the tetraalkylammonium ions or a “direct binding” between these salts and macromolecules is respornsible for the observed instability of the ordered structure of native protein molecule^.^,^ Our recent studies !have indicated how the groups which are mainly involved in the denaturation process of protein molecule would behave in concentrated salt solutions.1° This informaiticm was obtained from the study of the activity coefficients of model compounds which are representative of tht diffeiw~tgroups present in protein. Here we report similar studies of a few model compounds in tetraalkylammonium bromide solution (R4NBr). The compounds chosen are acetylamino acid ethyl esters (ARE), CI13(=ONHGEI(R)COOCzHs,R being phenyl alanyl, norleucyl, and riorvalyl groups. Since temperature variation is often helpful to understand mechanism of reactions, the effect of temperature on the activity coefficient has been utilized to interpret the mechanism of in-
teraction of R4NBr salt with the model peptide and hence protein. Experimental Section Material and Methods. [14C]N-Acetyl-~-norvalineethyl ester (AnVE) and [14C]N-acetyl-~ -norleucine ethyl ester (AnLE) were prepared by the method of Wolf and Nieman.12 Details of their preparation and characteristics have been reported elsewhere.ll N-Acetyl-L-phenylalanine ethyl ester (APE) obtained from Cyclo Chemical Corp. was recrystallized from water. A solubility phase curve showed the absence of any impurity. Experiments with nearly sevenfold change in the excess solid phase gave the same solubility values within hl%. Ammonium bromide used as Fishers Analytical Grade reagent. Tetramethyl- and tetraethylammonium bromides (MedNBr and EtrNBr) were crystallized twice from methanol. Tetra-n-propyl- and tetra-n-butylammonium bromides (Pr4NBr and Bu4NBr) were twice crystallized from carbon tetrachloride. Glass distilled water was used throughout. N-Dibutyl ether was distilled twice, boiling point 141-142” (lit. 142”).11 AR grade petroleum ether was used. Radioactivity measurements were obtained with a Packard Model 3375 liquid scintillation spectrometer at 4”. Samples (0.1 ml) were added to the vials containing 10 ml of scintillation solution, the composition of which has already been described. The samples were counted for sufficient time to accumulate 20,000 counts in most cases reducing the standard errors of counting to 0.5%.10J1 The solutes were equilibrated with solvents in 12-ml capacity tube sealed with Teflon-lined screw caps, in both distribution and solubility measurements. The tubes were submerged in a water bath in a rotating rack, and mixing was accomplished by rotating tubes end-over-end and a t 25-30 rpm. Temperatures were maintained at 0 i 0.1, 25.0 f 0.05, and 40.0 k 0.1”. Measurements of solubility with AFE at three temperatures and distribution of AnVE and AnLE between aqueous and reference phase at 25 and 40” were carried out by The Journal of Physical Chemistry, Vol. 78, No. 12, 1974
Pradip K. Nandi
$198
methods described hi detail elsewhere.lOJ1 Equilibrations for solubility experiments were carried out for 10 days a t O", 3 days a t 25", and 2 days a t 40". The equilibration time for distribution measurements were 1 hr both a t 25 and 40" for AiVE and AnLE. That the above periods of equilibration were adequate for the equilibrium to be attained were d~!termi~ed as described previously. Samples were removed for counting radioactivity from both phases of each tube to measure the concentration. The c o ~ c ~ n ~ r a of t ~ APE o n was determined spectrophotometrically ixl a IWQ Il Zeiss spectrophotometer, from the diffeyence of abriorption a t 257.5 (Amax) and 300 nm where the abscirbancle was small. Samples of the saturated solution were diluted by factors of 6 to 30 with water for absorbance m~~as~~reinents. This reduces the salt concentration to levels when? no significant effects of salts on the uv spectra woulld be expected. The s o ~ u ~ i lofi ~APE , ~ in water a t 25" and distribution coefficients of AnVE and AnLE between water and reference phases at 25 and 4Q"have been reported elsewhere.1l The solubility values of APE a t 0 and 40" are 2.59 f 0.06 and 6.62 af 0.10 g/l., respectively. Solubility and distribution ~ o e ~ f ~were c i ewithin ~ ~ ~1 ~5 and f3%, respectively, of their mean values. We have assumed that these ranges of ~ x p e ~ i m e n ~"rrox t a ~ apply to determinations in salt solutiom as well. The degree of mutual solutions of the phases in the distribution e ~ . p e ~ ~ has ~ e nbeen ~ s carried out as follows. Aqueous solutions of RsNRr salts were shaken with reference phases. 4liquot of the reference phase was then shaken with f~~rc!shwater, arid this water phase was treated with silver nitrate solution. The absence of any recognizable amount 01' precipitate was considered to show the absence of any dotcctahle mutual solubility of phases which could (effectow ~ ~ s t ~ i lexperiments. ~ut~o~
Results Equations 1 and 2 have been used to determine activity cceffinients from solubility and distribution, respectively. Activity coefficionts of the compounds in water have been assumed to be equal to one; f," is
2.0
Br 1.0
0.8
5
p j a6 t
\
ii
IL W
8
cz
0.1
c
Y
0.2
0.1
I___L
0
1.0
2.0 MOLARITY
Figure 1 . Effects of different salts on the activity coefficients of acetylphenylalanine ethyl ester at 25".
1.0
0.8
5
0.6
w V
E
:: 0.L
0 V
>-
c-
f
0
0.2
f," == (clr "c,"(c,0/c,".") (2) the activity coefficient of the nonelectrolyte, i, in salt solution; C,o and e,. are the molar concentrations of i in water and salt solution; Clr-O and C,r.s are concentrations in the reference phases corresponding to water and to salt solution, respectively 1 3 ~ ~ Figure 1 shows a semilogarithmic plot of the activity coefficient of APE as a function of salt concentration a t 25". The salting out of the compound in NH4Br solution changes to salting in with the introduction of alkyl groups and the magnitude of the effect increases with the increase in the number of alkyl groups in the salt. Log f (i) increases linearly in N&Br solution, (ii) decreases linearly in Me4NBr and Pr4NBr solutions, and (iii) curves upward in Et41VBr and slightly curves downward in Bu4NBr solutions. An1.E shows similar solubility behavior in NM.IBI, EtsNBr, and Bu4NBr (Me4NBr and PrrNBr effects were not carried out) and AnVE shows similar ber (where it has only been studied) solution. Effect of BuJJESr at 25" on the three solutes is shown in Figure 2. Temperature has a marked effect on the activity coefficients of the compounds in R4NBr solution. The decrease The Journal of F'hy6,ical Chemistry, Voi. 78, No. 72, 1974
Figure 2. Effect of butylammonium bromide on the activity coefficients of acetylphenylalanine, acetylnorleucine, and acetylnorvaline ethyl esters at 25'.
in the activity coefficients increases with increase in the temperature from 0 to 40" (Figure 3). The curvature of the plot of log f us. salt concentration decreases with increase in temperature. As with other simple electrolytes, the temperature effect on the activity coefficient in NH4Br is not pronounced as in R4NBr solution.ll The activity coefficient, h , of a nonelectrolyte is related to molar salt concentration, C, by the Setchenow eq
K, is the salting constant and is termed as salting out or
Tetraalkylammonium Salt Effects on Model Peptides
1199
TABLE I: Salting Constants of the Amino Acid Ethyl Esters at Three Temperatures"
1.0
08
Compound
Salt
00
25'
40'
~~
0.8
APE 04
AnLE
r
z
w
AnVE
Y
LL
t;
0.2
> t
5y
-0.05 -0.14 -0.16 -0.31
0.07 -0.09 -0.32 --0.39 -0.73 0.10 -0.41 -0.26
0.09 -0.08 -0.31 -0.54 -11,.08 0.18 -0.57 -0.44
Calculated as described in the text.
a
00
NH4Br Me4NBr Et4NBr Pr4NBr BuaNBr NH4Br Bu4NBr Bu4NBr
TABLE 11: Free Energy Transfer AFt, (cal/mol) of the Amino Acid Ethyl Esters from Water to 1 M Salt Solutiona
0.1
0.08
Compound
Salt
APE 4nLE APE AnLEb APE AnLE APE APE AnLE AnVE
NH4Br
O0
0.06 0.04
2.0 M
1.0
[Bu, NBr) Figure 3. Effect of tetrsrbutylammonium bromide on the activity coefficients of acc?tylphenylalanineethyl esters at different temperatures.
salting in constant rmpectively depending upon whether the activity coefficients of the nonelectrolyte increases or decreases in salt solution. The salting constant gives a convenient method for comparison of salt effects on various electrolytes The values of the salting constant in cases of linear plots were obtained by drawing the best straight line through all the points and determining the slope. Inkhe case of cunred plots, salting constants were estimated from initial slopes approximated by passing straight line visually through points below 1M.10 Salting out constants in NH4Br and salting in constants of the esters in R4NBr solutions are compareld in Table I. of' comparison of the effect of R4NBr salts would be to compare the free energy of transfer, AFtr, which can be calculated from the equation A&, = RT In and represents the free energy of transfer of 1 mol of dilute solution of a given compound i, from water into the salt solutions at the same concentration in each solvent. The values of AF,, of the compounds from water to 1 M salt solution are presented in Table 11. AFtr values at 0" for APE in Me4NBr and Et4NBr solution show considerable decrease than the values at 25". There is, however, practically no differences in the values at 25 and 40" in these solutions Temperature dependence of AFt, is more pronounced in salts wiith larger alkyl groups. Discussion The aim of the present work is to study the behavior of compounds which contain a peptide group in tetraalkylammonium salt solution to understand the structural stability of the protein molecule in such solution. The model which has been used here for the denaturation process of proteins is the same as has been used previousEy.10JiJ5 l6 Upon denaturation, the peptide (CHCONH) and nonpolar groups, which are buried in the interior of
Me4Br
250
100
-65
Et4NBr
-140
Pr4NBr BuaNBr
-200
Calculated from equation A F = Values are rounded to the nearest 5. a
00
-560
130 - 110 0 315 80
-
-500 -1200 - 640 - 465
40'
120 135 - 115 0 - 310 - 80 - 770 - 1630 - 945 715
-
RT In f:. From single data at 1 M.
the native protein molecule, would be exposed to surrounding solvent with consequent increase in their concentration (decrease in the activity coefficient, salting in) in denaturing solvents compared to water. The studies of the model compounds in these solutions are expected to provide information which can be applied to the groups they represent in the protein molecule. Our previous work on model peptides showed the balance between the salting i n of the newly exposed polar group (CHCONH) and the salting out (increase in the activity coefficient) of the simultaneously exposed nonpolar groups would determine the stability of one state or the other of a protein molecule in concentrated salt solution. This study also showed that nonpolar side chains are affected similarly to the hydrocarbons by salt solutions, although the magnitude of the salting out constant was appreciably higher for the hydrocarbons. At present no information is available about the behavior of peptide group in RdNBr solution. We have chosen compounds CH&ONHCH(R)COOCzHs for this purpose. This study, however, would not give us any direct information about the behavior of peptide or ester groups themselves, but the combination of hydrocarbon side chain and polar group in these compounds might be considered as a model for the portion of the polypeptide chain present in protein. The compound where R represents the phenylalanyl group has been studied relatively thoroughly. The similarity of salting behavior of these types of compounds has been shown with compounds where R is norleucyl or norvalyl in a few RaNBr solution. The observed negative values of A&, in general, for the transfer of 1 mol of ARE from water to 1 M R4NBr salt solution show that the transfer process is spontaneous (Table II). Aliphatic and aromatic hydrocarbons show similar behavior in R4NBr solution.2,17The values of A& a t maximum concentrations (shown in parentheses Table III) of R4NBr a t which Wen and Hung studied the alkanes The Journal of Physical Chemistry, VoI. 78, No. 12, 1974
Pradip K. Nandi
1200
TABLE 111:
-.AFt, (cal/mol) for the Transfer &Different Solutes from Water to RaNBrSolution at 25
l___l_--l_-
Salt
______I___
Propme
B u J ~ B ~ 170b
AnVE
Butane
ZOO(Q.52)
115b
AnLE
l'i'O(0.28)
Pr4NBr
EtrNBr MerNBr
%El&
50(0.40)
85 85
60(0.38) 0(0.77)
Benzene
1220" 1680d 935e 1240d 645c 765d 465O 480d
APE
Butane
900(0.77) 11856 440(0.82) 510" 310(0.86)
Benzene
115b
685c(0.31m)
1ZOb
6 5 0 c ( Q40 . m)
1601
410f
85b
365c
320e
llO(0.94) 115e
a Values in the parentheses are the maximum concentrations in molarity and molality (shown) of the salts for which data are available for the alkanes. A F t r values for alkanes at these concentrations are compared with the interpolated values. AFt, values of ARE and banzene at the same concentrations of RhNBr. Reference 2a. Reference 2b. From the salting constant values at 1 M (ref 17). e From the salting constant values at 1 M. From salting constant data at 1 M from J. J. Morrisoii and N B. B. Johnstone, J.Chem. SOC.,3655 (1955).
'
the relevant data are available), since as has been already have been compared with A F t r values a t the same interpointed out, the backbone would be favorably disposed in polated salt concentrations for AnVE and AnLE. In abBu4NBr solution. In fact benzene is 500 cal more favorsence of any similar solubility data of toluene in R4NBr able for the process a t 1 M. These considerations indicate solution, the values of A F t r for benzene obtained from that the contributions of nonpolar and polar components salting constant of Desnoyers, et al., have been compared in these compounds are not additive toward the transfer with AFtr values obtained for APE. In addition, A F t , process in tetraalkylammonium salt solution. values of lbenzene around 1 M from recent studies of Another significant observation in this study is that Wirth and LoSurdo have also been compared with the APE is transferred much more favorably than AnVE and transfer values of APE a t the same interpolated salt conAnLE. That this results primarily due to the difference in centration (A. LoSurdo, personal communication) .I8 the aromatic and aliphatic groups is borne out by the The results show that the transfer of AnVE and AnLE comparison of the transfer values of alkane and benzene at to larger tetraalkylammonium salt solutions is energeticomparable salt concentrations obtained from the studies cally more favorable than transfer of propane and butane, of Wen and Hung, Wirth and LoSurdo. and LoSurdo (perrespectively, whereas in lower tetraalkyl salts (where sonal communication), also presented in the Table AnLE has only been studied) the reverse is true. In conIII.a.18 In addition a AFt, value of -2500 cal/mol for the trast, APE is always energetically less favorable than bentransfer of naphthalene to 1 M Bu4NBr solution suggests zene for the transfer to all R4NBr solution by a considerthat, in general, aromatic molecules or groups are responable amount {
