Hydration as a Major Factor in Preferential Solvent-Protein Interactions Tsutomu Arakawa Alliance Protein Laboratories, 3957 Corte Cancion, Thousand Oaks, California 91360
CRYSTAL GROWTH & DESIGN 2002 VOL. 2, NO. 6 549-551
Received July 9, 2002
ABSTRACT: It has been well-documented that polar groups on the surface of proteins have water molecules tightly bound to them; that is to say, proteins are hydrated in aqueous solution. If this hydration is maintained in a concentrated solution of a cosolute, a difference in the concentration of the cosolute develops between the bulk solution and the vicinity of the protein, resulting in preferential hydration such that excess water accumulates near the protein under dialysis equilibrium. This preferential hydration leads to cosolute-induced stabilization and saltingout of the protein in concentrated solutions of the cosolute. Preferential binding of the cosolute occurs when it binds to the protein at a level at which the concentration of the cosolute in the vicinity of the protein exceeds the concentration of the cosolute in bulk solution. When binding of the cosolute reaches saturation, a constant level of hydration (physically bound water) can lead to preferential hydration of the protein at higher concentrations of the cosolute. Introduction Concentrated cosolutes are used to precipitate or crystallize proteins from aqueous solution, as well as to stabilize such proteins. In concentrated solutions of a cosolute, the absolute binding of the cosolute and water with the protein determine, under dialysis equilibrium, whether preferential interaction of the cosolute or preferential hydration of the protein will occur, thus affecting the solubility and stability of the protein. Preferential interaction measurements have been carried out to delineate the interactions of proteins with denaturants such as urea and guanidine HCl, with stabilizers such as sugars, polyols, amino acids, amines, and certain salts and with salting-out agents such as ammonium sulfate and poly(ethylene glycol).1-11 Under conditions of dialysis equilibrium, preferential interaction is a function of the difference in concentration of a cosolute between the vicinity of the protein surface and the bulk solvent. This property is not a measure of the absolute (physical) binding of the cosolute, nor does it reveal the mechanism of binding. Preferential hydration (or deficiency of cosolutes in the vicinity of a protein surface) has been observed in concentrated solutions of protein-stabilizing cosolutes.5-9,12 A number of mechanisms for this phenomenon have been proposed, including an increase in the surface tension of water caused by the cosolutes,5,13,14 steric exclusion,9 solvophobicity,10 and osmophobicity.15 Here, I propose the importance of water binding, or hydration, in preferential hydration. Results and Discussion It is well-established that protein molecules have tightly associated water molecules, as is evident from the following reported observations. (i) Enzymatically active protein in the form of a dried powder regains its activity when hydrated at a level of 0.2-0.4 g of water per gram of protein.16 Physical * To whom correspondence should be addressed. Tel: 805-388-1074. Fax: 805-388-7252. E-mail:
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properties such as the heat capacity of the powder protein change at this hydration level.17 (ii) NMR studies have shown the presence of nonfreezable water at a level of 0.3-0.4 g per gram of protein.18 This phenomenon has been attributed to the tight association of water molecules to the protein such that freezing does not occur, which is in contrast to water molecules present in bulk solvent where freezing does take place. The nonfreezing phenomenon occurs even in the presence of urea.19 (iii) Structure-stabilizing cosolutes have manifested a preferential hydration of 0.2-0.4 g of water per gram of protein in measurements of preferential interaction.20 This phenomenon is best explained by assuming that there is 0.2-0.4 g of water bound to the protein, with no binding of cosolute nor penetration of the cosolute into the hydration layer (as will be described in greater detail further below). (iv) A peculiar observation has been made with regard to the preferential interaction of urea and 2-chloroethanol with proteins in concentrated solution.1,2,21 Preferential binding of these two denaturants occurs in middle concentrations of cosolute, but at extremely high concentrations, such as 80% 2-chloroethanol1,2 or 8 M urea,21 preferential hydration is observed. This result can be best explained if protein hydration is maintained in the presence of the cosolutes. (v) Preferential interaction data for urea and guanidine HCl at high concentrations, such as 6 M guanidine HCl and 8 M urea,3,4 have been analyzed for their absolute bindings. Using an assumption of approximately 0.3 g of water per gram of protein, the amount of bound guanidine HCl or urea correlates with binding results calculated from model compound studies. The above observations suggest that protein hydration can be maintained at a level of 0.2-0.4 g of water per gram of protein in the presence of cosolutes, regardless of whether such cosolutes are stabilizers or denaturants. Moreover, this property plays a significant role in the preferential interaction of various cosolutes in
10.1021/cg0200255 CCC: $22.00 © 2002 American Chemical Society Published on Web 09/17/2002
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Figure 1. Plot of interactions vs cosolute concentration. A1 is set at 0.3 g/g, while A3 is set at 0.
aqueous solution with proteins and the effect of the cosolutes on protein stability and solubility. Preferential interaction is a thermodynamic parameter and differs from absolute binding. Preferential interaction is defined as the difference in the concentration of a cosolute in the vicinity of a protein and its concentration in bulk solvent. This property may be expressed as follows:
(A3 /A1) - g3 where A3 and A1 are the absolute binding values of cosolute and water (hydration), respectively, in grams per gram of protein and g3 is the concentration of the cosolute in bulk solvent in grams per gram of water. Preferential interaction of a cosolute may be expressed as follows:
(∂g3/∂g2) ) A3 - g3A1 where components 1, 2, and 3 in this equation are water, protein, and cosolute, respectively. The constancy of temperature and chemical potentials of water and cosolute is omitted for the sake of simplicity. This relationship shows that with a constant value for the hydration (A1), the second term increases as the bulk concentration (g3) increases. In other words, if there is no cosolute binding, (∂g3/∂g2) is always negative and decreases linearly with g3. This condition, which has been observed for many protein-stabilizing and salting-out cosolutes,5-7,12,14 is shown graphically in Figure 1. Preferential hydration can be expressed as
(∂g1/∂g2) ) -(∂g3/∂g2)/g3 Assuming that there is no cosolute binding, then (∂g1/ ∂g2) will be equal to A1. Preferential hydrations observed for protein-stabilizing cosolutes such as certain salts, amino acids, sugars, polyols, and methylamines are typically in the range of 0.2-0.4 g (of cosolute) per gram of protein, indicating that water binding is the source of excess water (and cosolute deficiency) near the protein surface in concentrated solutions of these cosolutes.
Arakawa
Figure 2. Plot of interactions vs cosolute concentration. A1 is set at 0.3 g/g, while a small binding of cosolute is assumed.
Preferential cosolute binding is observed only when A3 is greater than g3A1. This is due to the fact that with a constant value of A1, cosolute binding (A3) must occur at the level at which the cosolute concentration (A3/A1) in the vicinity of the protein becomes greater than g3. Below are two examples of cosolute binding. In the first example, A3 achieves saturation at low concentrations of cosolute and its value is small. Although, as shown in Figure 2, there is preferential cosolute binding, this binding may be too small to measure by dialysis equilibrium using such techniques as densimetry or refractometry. These measurements become possible at higher g3 values, however. In that case, as also shown in Figure 2, only negative preferential cosolute binding (or preferential hydration) is observed, in agreement with results obtained for cosolutes such as arginine HCl.22 Because the value for cosolute binding (A3) is small, (∂g3/∂g2) crosses over zero at low g3 ) (A3/A1). On the other hand, a small, but high affinity, binding (such as the case in Figure 2) can be measured by a more sensitive technique such as radioactivity or spectroscopic measurements. Because such measurements can be done at lower concentrations of cosolute where g3 is small, preferential binding of the cosolute can be observed. However, if the measurements are extended to a higher concentration of the cosolute, preferential hydration will be observed as shown in Figure 2. The second example, shown in Figure 3, illustrates a case where the cosolute binding value is large. In this instance, (∂g3/∂g2) increases with g3 and crosses over zero when g3 is large. This large cosolute binding results in a value of (∂g3/∂g2) that can be detected by dialysis equilibrium using either densimetry or refractometry. Preferential cosolute binding has been observed with protein denaturants such as guanidine HCl, urea, and 2-chloroethanol. It is striking that with such large values of cosolute binding, preferential hydration (or negative cosolute binding) still occurs at extremely high concentrations of the cosolute. This result is due to the fact that cosolute binding achieves saturation, while g3A1 increases linearly with g3. It means that measurements of preferential interaction at concentrations of cosolute well beyond binding saturation can lead to negative preferential cosolute interactions. This phe-
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weakly to proteins, A3U ) A3N ) 0 and hence A1U must be greater than A1N. This result is expected from the exposure of more peptide groups and polar groups upon unfolding. For solubility, ∆(∂g3/∂g2), which is defined as
∆(∂g3/∂g2) ) (∂g3/∂g2)ppt - (∂g3/∂g2)soln ) (A3ppt - A3soln) - g3(A1ppt - A1soln)
Figure 3. Plot of interactions vs cosolute concentration. A1 is set at 0.3 g/g protein, while a large binding of cosolute is assumed.
nomenon has in fact been observed with urea and 2-chloroethanol.1,2,21 A similar trend has also been observed for urea and guanidine HCl (Arakawa and Timasheff, unpublished results). Thus, a constant level of hydration in the presence of concentrated cosolute can explain the following: (i) observed preferential hydration for protein stabilizers; (ii) absolute bindings of guanidine HCl and urea; and (iii) observed preferential hydration (or negative preferential cosolute interaction) and crossover from preferential cosolute binding to preferential hydration at extremely high concentrations of urea and 2-chloroethanol. However, there are certain cosolutes whose preferential hydration greatly exceeds A1. In the case of cosolutes such as poly(ethylene glycol) and 2-methyl2,4-pentanediol, proteins not only bind to water molecules but also repel these cosolutes. On the basis of this mechanism, it can be concluded that there will be a wide variety of cosolutes that show preferential hydration or negative preferential cosolute binding. As the bulk cosolute concentration (g3) increases, it will create a greater difference in concentration of the cosolute between the bulk solution and the vicinity of the protein where the hydration layer is formed. In fact, many water soluble compounds have shown negative preferential cosolute binding.5-7,12,14 If preferential interaction can be explained by a constant value of A1, what explains the effects on protein solubility and stability? For cosolutes to stabilize proteins, ∆(∂g3/∂g2), which is defined as
∆(∂g3/∂g2) ) (∂g3/∂g2)U - (∂g3/∂g2)N ) (A3U - A3N) - g3(A1U - A1N) must be negative, where U and N indicate the unfolded and native states, respectively. For cosolutes that bind
must be positive for those cosolutes that induce saltingout. (The notations “ppt” and “soln” indicate proteins in precipitate and solution.) Thus, A1ppt must be smaller than A1soln, provided that A3ppt ) A3soln ) 0. This result is also expected from the contact between protein molecules in the precipitated or crystal state. On the basis of this proposed mechanism, a wide variety of cosolutes are expected to have stabilizing and saltingout effects, which is in agreement with previously reported observations.20 In conclusion, preferential hydration or negative preferential cosolute binding, which have been observed for urea and 2-chloroethanol, can be explained by a constant level of hydration of protein in the presence of these cosolutes. References (1) Timasheff, S. N.; Inoue, H. Biochemistry 1968, 7, 25012513. (2) Inoue, H.; Timasheff, S. N. Biopolymers 1972, 11, 737-743. (3) Lee, J. C.; Timasheff, S. N. Biochemistry 1974, 13, 257265. (4) Prakash, V.; Loucheux, C.; Scheuffle, S.; Gorbunoff, M. J.; Timasheff, S. N. Arch. Biochem. Biophys. 1964, 210, 455464. (5) Arakawa, T.; Timasheff, S. N. Biochemistry 1982, 21, 65366544. (6) Arakawa, T.; Timasheff, S. N. Biochemistry 1982, 21, 65456552. (7) Arakawa, T.; Timasheff, S. N. Arch. Biochem. Biophys. 1983, 224, 169-177. (8) Arakawa, T.; Timasheff, S. N. Biochemistry 1984, 23, 59125923. (9) Arakawa, T.; Timasheff, S. N. Biochemistry 1985, 24, 67566762. (10) Gekko, K.; Timasheff, S. N. Biochemistry 1981, 20, 46674676. (11) Lee, J. C.; Lee, L. L. Y. Biochemistry 1987, 26, 7813-7819. (12) Arakawa, T.; Timasheff, S. N. Biophys. J. 1985, 47, 411414. (13) Lee, J. C.; Frigon, R. P.; Timasheff, S. N. Ann. N. Y. Acad. Sci. 1975, 253, 284-291. (14) Lee, J. C.; Timasheff, S. N. J. Biol. Chem. 1981, 256, 71937201. (15) Bolen, D. W.; Baskakov, I. V. J. Mol. Biol. 2001, 310, 955963. (16) Rupley, J. A.; Gratton, E.; Careri, G. Trends Biochem. Sci. 1983, 3, 18-22. (17) Yang, P.; Rupley, J. A. Biochemistry 1979, 18, 2654-2661. (18) Kuntz, I. D.; Brassfield, T. S.; Law, G. D.; Purcell, G. V. Science 1969, 163, 1327-1331. (19) Kuntz, I. D. J. Am. Chem. Soc. 1971, 93, 514-516. (20) Timasheff, S. N.; Arakawa, T. J. Crystal Growth 1988, 90, 39-46. (21) Muralidhara, B. K.; Prakash, V. Curr. Sci. 1997, 72, 831834. (22) Kita, Y.; Arakawa, T.; Lin, T. Y.; Timasheff, S. N. Biochemistry 1994, 33, 15178-15189.
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