2050
J. Phys. Chem. 1980, 84,2050-2053
Thermodynamic Studies of Protein-Small Molecule Interaction. 3. Solute-Solute Hydrophobic Interaction of Phycocyanin with Alcohols Chang-Hwei Chen Physical Chemistry Laboratories, Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201 (Received: January 30, 1980)
A variety of alcohols having known free-energypairwise interaction coefficient (AA), were interacted with the protein phycocyanin to further examine the solutesolute hydrophobic interaction between hydrophobic species and protein. These alcohols have the same order of abilities for solute-solute hydrophobic interaction with protein and self-pairwiseinteraction between alcohol molecules: cyclohexanol > butanol > propanol > ethanol, methanol, ethylene glycol. This finding suggests that, the stronger the tendency of an alcohol molecule for pairwise interaction between its molecules, the greater its ability for solute-solute hydrophobic interaction with protein. Comparison of thermodynamic parameters for the interactions of cyclohexanol and quaternary ammonium salts with phycocyanin suggests that solute-solute hydrophobic interaction, rather than ionic interactions, is the predominant force in binding of quaternary ammonium salts to the protein. The shape and the size of a molecule are important in its solute-solute hydrophobic interaction with protein. A thermodynamic characteristic of solute-solute hydrophobic interaction is presented.
Introduction Quaternary ammonium salts, including tetraalkylammonium and azoniaspiroalkane salts, were previously112 used to investigate the effects of alkyl chain length and geometric configuration in alkyl-substituted quaternary ammonium salts on their interactions with protein. A change in hydrophobicity due to the changes in alkyl chain length and molecular shape is responsible for a change in the solute-solute hydrophobic interaction analogous to the previously reported3* solute-solvent hydrophobic hydration. Formation of cyclic groups in these ions considerably weakens their ability for hydrophobic interaction with protein. We also found that an enthalpy-entropy compensation relationship exists in the solute-solute hydrophobic interaction and that the protein phycocyanin (PC) is a good probe for examination of such interactions. Studies of quaternary ammonium ions interacting with PC provide quantitative information on solute-solute hydrophobic interaction between these hydrophobic species and the protein. However, the chemistry in these systems is complicated owing to the presence of a positive charge on nitrogen, which is capable of participating in ionic interactions that can affect the hydrophobic interaction of quaternary ammonium ions. It is therefore logical to investigate, by using PC as a probe, solute-solute hydrophobic interaction in systems with no ionic interactions involved,as, for example, in protein-alcohol systems. Such studies are important to an understanding of proteinquaternary ammonium salt interactions. A homologous series of aqueous alcohol solutions (methanol, ethanol, propanol, and butanol) has been shown to affect a particular property of a protein, such as the thermal transition of ribonuclease7 and collagen8and the helix-coil transition of poly-L-ornithine and poly-L-glutamic acid.g The enhancement of observed effects with increasing hydrocarbon chain length of added organic material is usually interpreted as evidence for hydrophobic interactions. The concentration of alcohol used in this type of study is at least 10% by weight. At such alcohol concentrations, the pH and the dielectric constant of the protein solution are not unaffected. The freezing temperatures of dilute aqueous solutions of a variety of alcohols-including methanol, ethanol, 20022-3654/80/2084-2050$0 1.OO/O
propanol, butanol, cyclohexanol, ethylene glycol, and glycerol-were recently measured,1° and the free-energy pairwise interaction coefficient (AA],, a quantity representing the ability for self-association of a solute A with A in aqueous solution, was computed for each alcohol. The values of (AAJ,are directly related to the abilities of the solutes for hydrophobic interaction. Of these alcohols cyclohexanol has the greatest tendency for interaction between its molecules in aqueous solution, since it has the greatest negative value of (AAJ,. In extension of our previous studies,'I2 we have now examined the solute-solute hydrophobic interaction of PC with cyclohexanol, butanol, propanol, ethanol, methanol, and ethylene glycol. We were interested in direct interaction between alcohol and PC a t lower alcohol concentrations, where the pH and the dielectric constant of the protein solution are essentialy unaffected. This work should assist in an understanding of molecular interactions between quaternary ammonium salts and protein and also of the nature of solute-solute hydrophobic interaction in general.
Experimental Section Phycocyanin from the blue-green alga Phormidium luridum was purified as described previous1y.l The final protein solution contained a mixture of trimer (S3)and hexamer (S,) in pH 6.0, I = 0.1 sodium phosphate buffer. Cyclohexanol, propanol, and methanol were obtained from Fischer Scientific Co. Butanol was obtained from J. T. Baker Chemical Co., ethylene glycol from Aldrich Chemical Co., and ethanol from IMC Chemical Group Inc. These alcohols were reagent grade and were used without further purification. Aqueous alcohol solutions were prepared in the same buffer. A batch-type solution microcalorimeter was used in these studies. Its instrumentation and the schemes for the calorimetric measurements have been described.l The calibration constant was carefully checked. All experiments were carried out at 25 "C. One compartment of the reaction cell was filled with 0.30 mL of PC solution (27 mg/mL) and the other compartment with 0.30 mL of the desired concentration of alcohol solution. Thermal equilibrium was maintained for at least 2 h before ther0 1980 American Chemical Society
Solute-Solute Hydrophobic Interaction
The Journal of Physical Chemistry, Vol. 84, No. 16, 1980 2051
TABLE I : Values of {:AA}gand the Effect of Alcohols on Aggregates of Phycocyanin
(AA}g,a solute none cyclohexanol but an ol
Jkgmol"'!
a
%of S,
%of S,
pHof medium
54 42
5.95 5.95
54 46 54 52 54 54 54
5.96 5.97 5.96 5.97 5.96 5.96 5.95
0 -684
0.20
46 58
-466
0.20 0.25 0.20 0.30 0.40 0.40 0.40
46 54 46 48 46 46 46
propanol ethanol methanol ethylene glycol
C,, mol/L
-125 -52 15
TABLE 11: Enthalpy of Binding (AHB)of Cyclohexanol t o Phycocyanin
CX,
mochemical measurements. A stable base line indicated that the distillation of alcohol was not observed during thermal equilibrium. Sedimentation velocity experiments were performed in a Spinco Model E ultracentrifuge with schlieren optics. The procedures have been described.l Enlargements of schlieren peaks on the negative plate were traced with a microcomparator. Th.e relative areas under the peaks were then determined with a Science Accessories Corp. graph/pen. The formulas for analyses of calorimetric and sedimentation data have been reported.'J
Results Either 0.20 M cyclohexanol, 0.25 M butanol, or 0.30 M propanol could dissociate the higher aggregate of PC, leaving more S3and less s6 (Table I). Ethanol, methanol, or ethylene glycol at 0.40 M, however, could not dissociate s 6 into Sa,so the relative amounts of these units remained unchanged. The known pairwise free-energy interaction coefficients of these alcoholslO are also listed in Table I for comparison. The observed heat of mixing (AQmiX)in these calorimetric studies represents the sum of the heat of interaction between PC and alcohol (AQint) and the heats of dilution of PC and alcohol. The quantity AQintcan be obtained from AQmiX corrected for dilution effect. Values of AQint for interaction between PC and cyclohexanol (0.14-0.20 M) are given in Tablle 11. In the absence of alcohol, s6 and S3,which are the only species present, equilibrate in solution. The macromolecular dissociation clonstant (Kd) for s6 dissociating into S3at zero alcohol coincentrations is then
Kd
2s3
(1)
Kd = (C3)'/c6 (2) where c6 and C3 are the concentrations of s6 and Sa,respectively. In the presence of an appropriate concentration of alcohol, there is a decrease of s6 and an increase of S3. The change in S3 and s6 as a function of cyclohexanol concentration is shown in Table 11. A two-step mechanism for the interaction between the small molecule and PC, as previously proposed,l is adopted here. No binding of small molecule with s6 is suggested (this reasonable assumption has been discussed previously).l These two steps, the dissociation of s6 into S3 and the binding of the small molecule with S3, can be expressed in eq 3 and 4,
A",
s 6
S3 + mX
-
2s3
M E
S3Xm
(3)
(4)
,a
mol/ % of % of S, S, L
K,
moP/L
mol
mol
mJ
0.200 0.190 0.180 0.170 0.160 0.150 0.140 0
2.40 2.26 2.14 2.01 1.79 1.69 1.59
0.54 0.49 0.45 0.40 0.32 0.27 0.22
1.56 1.43 1.30 1.20 0.91 0.80 0.69
6.72 5.96 5.11 4.51 6.38 3.12 3.92
58 57 56 55 53 52 51 46
42 43 44 45 47 48 49 54
~ n , , b n3,,c
kcal/ mol of S, 54 53 50 48 50
1.1P~~
= (C,)2/C,, where C , = 27 mg/mL X 0.5 X (% of S,)/(9 X l o 4 ) and C, = 27 mg/mL X 0.5 X (% of S,)/(18 X lo4). Mol wt of S, = 9 X l o 4 ;mol w t of S, = 18 x 10". 6n, = n,(,)- n,(o , where n, = C, X 0.60 mL. nsx = n3 X (% of binding{ = n3 X [I - (Kd/Kapp)"*], where n3 = C, x 0.60 mL. Kd at C , = 0 . a
From ref 10.
AHB,
108X 1 0 8 X
K,
where X is the small molecule (alcohol), m is the molar binding ratio of X to S3, AHd is the molar enthalpy of dissociation of s6 into s3(-17 kcal/mol of S6)y2and AHB (kcal/mol of S3)is the molar enthalpy of binding of X to S3. The molar enthalpy of interaction between PC and X (AHint)is then AHint = A",
~AHB
(5)
Therefore AHBcan be expressed as
where Sn6 = n6(0)- n6(,). The terms n6(0)and n6(,) denote the numbers of moles of n6 in the absence and presence of X, and n3, is the number of moles of S3 binding with X. The dissociation constant ( K ) for S3Xmcan be defined as
-
S3 + mX
(7)
K = (C~)IJ(CX)~/(C~)~
(8)
S3X,
where ( c 3 ) b , and C, are the concentrations of unbound trimer, bound trimer, and X, respectively. The sum and (c3)b is the total concentration of s3in solution of (c3),, (C3). Rearrangement of eq 8 gives 1 + K-l(C,)m = c3/(c3)u
(9)
The apparent macromolecular dissociation constant (Kapp)in the presence of X is defined as Kapp
= (C3)'/c6
(10)
where C3 and c6 are the total observed concentrations of S3and s6 in the presence of X. Since no binding of S6with X is assumed, the unbound trimer and free hexamer equilibrate in solution according to eq 2. Combination of eq 10, 9, and 2 gives Kapp = Kd[l
+ K-l(cx)m]z
(11)
Comparison of eq 9 and 11 gives Equation 12 enables one to compute n3,, which is needed for the calculation of A H B as shown in eq 6. Rearranging eq 11 and taking the logarithm, one has
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The Journal of Physical Chernlstty, Vol. 84, No. 16, 1980
Chen
TABLE 111: Thermodynamic Functions of Binding of Cyclohexanol and Quaternary Ammonium Bromides to Trimers of Phycocyanin
AHBlrn,
(AHB/ (AHB/ mV4, m)/2, kcal/ 1 /, kcal/I / m o l o f X molof X
AHB, kcal/mol kcal/mol of S, A S B , eu m ofX 51 f 2 177 f 8 2.8 18 49+ I 1 7 0 i 22 1.1 45 11 26f 2 93f 6 1.1 24 6 8+3 32t 9 1.2 7 4 -1 f 4 2 + 13 1.6 -0.6 -0.2 -12 f 5 -37f 16 1.0 -12 -6 a Bu = butyl, Pr = propyl, Et = ethyl, and 6:6 Br and 4:4 Br are azoniaspiroalkane salts where 6:6 Br = (CH,),N(CH,),Br and 4:4 Br = (CH,),N(CH,),Br. AGB,
small moleculea cyclohexanol Bu,NBr Pr,NBr 6:6 Br Et, NBr 4:4 Br
Figure 1. Plot of cyclohexanol.
kcal/mol of S, -2.2 + 0.4 -1.8 f 0.4 -1.6 f 0.3 -1.6 t 0.3 -1.5 + 0.4 -1.0 f 0.2
-In C X - In [(K,,/Kd)1'2 - I]vs. - In C, for PC binding with
The plot of - In [(Kapp/Kd)llZ - 11 vs. - In C, for X = cyclohexanol is shown in Figure 1. Values of m and In K can be determined from the slope and the intercept. The free energy change (AGB) and the entropy change (ASB) in the binding of X to S3can then be calculated from K and AHBby using eq 14 and 15, where KB is the assoAGB = -RT In KB (14) ASB = (AHB- AGB)/T (15) ciation constant for S3X, binding and is equal to 1/K. Values of m, A",,AGB, and ASB for binding of cyclohexanol to S3are presented in Table 111.
Discussion Alcohol-PC Interactions. The free-energy pairwise interaction coefficient (AA) is a measure of self-interaction of a solute in aqueous sofution. The higher the negative value of (AA), for a solute, the stronger its tendency for pairwise interaction between its molecules and the greater its ability for hydrophobic interaction. The ability for pairwise interactions of these alcohols is in the order: cyclohexanol > butanol > propanol > ethanol > methanol > ethylene glycol.1o Pairwise enthalpy of interaction (AAJh is 240 for butanol, 134 for propanol, 59 for ethanol, and 59 cal kg/mo12 for methano1.l' It is important to notice that (AA), and (AAJ, for these alcohols are opposite in sign. Solute-solute hydrophobic interaction between a small molecule and PC will weaken the S3-S3interaction needed to form and stabilize s6.1'2Recent X-ray diffraction studies12have shown that S3is a stable subunit and have confirmed that contact of two S3units results in the formation of Sg. Our measurements of the relative amounts of S3and s6 (Table I) demonstrate that the ability of these
alcohols for solute-solute hydrophobic interaction is in the order: cyclohexanol > butanol > propanol > ethanol, methanol, ethylene glycol. Thus a small molecule which has a strong tendency for pairwise interaction between its molecules will have a correspondingly strong ability for solute-solute hydrophobic interaction with protein. The uncertainty in interpretation of the effects caused by changes in the pH and the dielectric constant of the medium can be neglected by studying the interaction of the protein with dilute aqueous alcohol solutions. We found the pH of the medium essentially unchanged by the concentrations of alcohols listed in Table I. The dielectric constants at 20 0C14916are the following: cyclohexanol, 15.5; butanol, 17.90; propanol, 20.81; ethanol, 25.00; methanol, 32.35; and ethylene glycol, 38.66. Dielectric constants of aqueous solutions of propanol, ethanol, methanol, and ethylene glycol have been determined,14and those of cyclohexanol and butanol solutions can be estimated from the dielectric increment based on Kirkwood's theory.18 In the presence of 0.20 M cyclohexanol the dielectric constant of water is decreased by only 1.4 from 80.37 at 20 "C. For useful thermodynamic analyses a concentration (C,) range of 0.14-0.20 M is needed for cyclohexanol. Higher concentration ranges are needed for the other alcohols: 0.25-0.50 M for butanol, 0.30-0.60 M for propanol, and >0.80 M for ethanol, methanol, and ethylene glycol. In our earlier studies the concentration (C,) of quaternary ammonium salts was 0.10 M or less. For relevant comparison of PC-alcohol with PC-quaternary ammonium salt systems, the concentration range of alcohol used in calorimetry after mixing should be close to 0.10 M. For this reason, thermodynamic functions for binding of alcohol with PC are reported and evaluated here only for cyclohexanol. Comparison of Cyclohexanol-PC and Quaternary Ammonium Bromide-PC Interactions. Large positive values of AH and A S are usually associated with hydrophobic i n t e r a c t i o n ~ . ~The ~ ~ J value ~ of AHBfor cyclohexanol is comparable with that for tetrabutylammonium salt Bu4NBr(Table 111). As for Bu4NBr,the large positive AHB can be explained as a result of strong hydrophobic interaction of cyclohexanol with PC, resulting in an endothermic release of water molecules upon protein-small molecule binding. Part of this large positive AHBmay well be associated with a change in protein conformation. The value of ASB for cyclohexanol presented in Table I11 is also comparable with that for Bu4NBr. A strong perturbation of the water structure surrounding the protein occurs as cyclohexanol binds to PC. The solute-aolute hydrophobic interaction between cyclohexanol and PC, resulting in a release of solvent, should give a positive contribution to the entropy. Part of this large positive change in entropy may well be associated with the conformational changes resulting from the binding of ligand to protein and from
The Journal of Physical Chemistry, Vol. 84, No. 16,
Solute-Solute Hydrophobic Interaction
40
I
P
Pr4NBr
I
-100
0
100
I 200
A S (eu) Figure 2. Plot of AH, vs. AS, for binding of cyclohexanol and quaternary ammonium bromides to PC.
the consequent releacie of water from the protein binding site. The enthalpy-entropy compensation phenomenon implies a specific linear relationship between entropy and enthalpy changes in water solutions of proteins and small molecule^.^^ A straight-line plot of A H B vs. A&, as we found for the binding of quaternary ammonium salts with PC, is consistent with this phenomenon. As a result of compensation in enthalpy and entropy changes, only minor changes in free energy occurred. AGp, for the interaction of cyclohexanol with PC (Table 111) also has a small negative value (-2.2 kcal/mol of Sa). Values of AHBand ASB for cyclohexanol lie on the straight-line plot of AH* vs. A& for binding of quaternary ammonium bromides with PC (Figure 2). A contribution by charge-charge interactions is present in the systems of quaternary ammonium salts but not in cyclohexanol. Thus, the results suggest that bindings of both cyclohexanol and quaternary ammonium salts to PC are a consequence of slolutesolute hydrophobic interaction, and the predominant force in binding of quaternary ammonium salts to PC is solute-solute hydrophobic interaction rather than ionic interactions. Examination of A H B and AGB for the interactions of protein with hydrophobic species as listed in Table I11 shows that A", is positive and AGB is negative for cyclohexanol, B u a B r , lPr4NBr,and 6:6 Br. The result that AHBis positive, AGB i s negative, and AHBand AGB are opposite in sign is characteristic of solute-solute hydrophobic interaction. This observation is analogous to the previous findings that pairwise interaction between alcohol molecules has a positive value of pairwise enthalpy of interaction (A&,, a negative value of free-energy pairwise interaction (a),, and opposite signs of {& andI (AA]h-lo'l1 ],
1980 2059
The term (AHB/m)/4 represents the hypothetical binding enthalpy for each of the four alkyl chains in a tetraalkylammonium ion. A value of 11 kcal was found for the interaction of PC with each C4H9chain in BulNBr (Table 111). The higher value of AHB/m for cyclohexanol (18 kcal) suggests that a molecule of cyclohexanol is significantly stronger than a C4H9chain of Bu4NBr in hydrophobic interaction with PC. This finding was expected, since cyclohexanol contains six CH2groups, while a C4H9 chain contains only four. Moreover, the four C4H9chains in Bu4NBr tetrahedrally attach to nitrogen,ls and so probably all four C4H9chains do not interact with PC a t the same time. In addition, each C4H9chain in Bu4NBr is attached to nitrogen, which carries a positive charge that ultimately weakens the ability for solute-solute hydrophobic interaction. The value of 4 kcal for (AHB/m)/2 listed in Table I11 is the hypothetical enthalpy in the binding of each cyclic group of 6:6 Br, an azoniaspiroalkane salt, with PC. This value is less than one-fourth of the AHB/m value for cyclohexanol. Each cyclic group of 6:6 Br contains six CH2 groups attached to nitrogen to form a seven-member ring, whereas the cyclohexanol molecule has a six-member ring. Previous studies have shown that the shape of a solute molecule is important for its interaction with surrounding water molecules, a six-member ring being a favorable one.lg Our results provide evidence that the shape of a small molecule is also important in solute-solute hydrophobic interaction. Of course, the nitrogen in 6:6 Br, as in Br4N+, carries a positive charge, which ultimately weakens the ability of each cyclic group for solute-solute hydrophobic interaction. Acknowledgment. I thank Professor Henry S. Frank and Dr. Donald S. Berns for commenting on this manuscript.
References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19)
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