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Proline Is a Protein-Compatible Hydrotrope? V. Srinivas and D. Balasubramanian” Centre for Cellular & Molecular Biology, Hyderabad 500 007, India Received September 9, 1994. In Final Form: April 7, 1995@ Hydrotropes are a class of compounds that, at high concentrations,enhance the solubilityof hydrophobic substances in water. Hydrotropy operates beyond a particular concentration known as the minimal hydrotrope concentration (MHC),above which the hydrotrope molecules begin to self-aggregateand form loose noncovalentmolecular assemblies. In this context,we have studied the self-aggregationand hydrotropic behavior of the naturally occurring amino acid L-proline and its derivative 4-hydroxyproline. The solubilizing ability of proline is comparable to that of conventional hydrotropes, while hydroxyproline does not display significant hydrotropic ability, presumably due to the hindrance offered by the hydroxyl group toward self-aggregation. However, the microenvironmental features offered by the proline molecular assembly are found t o be somewhat different from those displayed by the conventional hydrotropes and detergents. Proline is known to be a compatible solute in water-stressed cells. Proline delays the thermal unfolding of the protein a-chymotrypsin by approximately 10 “C, indicating its stabilizing ability on protein structure and conformation and suggesting its ability to act as a “protein-compatible”hydrotrope.
Introduction Hydrotropes are compounds that, a t high concentrations, enhance the solubility of a variety of hydrophobic compounds in water.’ Examples of hydrotropes are sodium salicylate, various benzene sulfonates, and the hydroxybenzenes such as catechol and resorcinoL2 An appreciation of hydrotropy may be had from the fact that 2 M sodium xylenesulfonate enhances the solubility of nitrobenzene in water 50-fold3and 2 M resorcinol increases the solubility of riboflavin 300-fold in water.4 Hydrotropy operates a t high concentrations and is reversible, since dilution with water leads to the precipitation of the solute. Arecent review provides a summary of the current status of the field.5 The mechanism of hydrotropy is becoming increasingly clear. In earlier paper^,^^^ we showed that it differs from simple phase mixing, or the cosolvency process, and also from salting-in action. Complex formation between the hydrotrope and the solubilized molecule has been sugg e ~ t e d ,but ~ , ~this might apply only to specific cases and not to all hydrotropes. We showed it to be a collective molecular effect that operates above a characteristic concentration ofthe hydrotrope in water. Above this value, which is termed the minimal hydrotrope concentration or MHC, the hydrotrope molecules self-aggregate to produce a loose noncovalent assembly which offers a microenvironment of lowered polarity that aids the solubilization of the hydrophobic solute. While this is reminiscent of surfactant self-assemblies, hydrotropic aggregates differ
* Address correspondence to this author.
’List of Abbreviations: ANS, 8-anilino-l-napthalenesulfonate;
DPH, diphenylhexatriene; FDA, fluorescein diacetate; HPLC, highperformance liquid chromatography; T,, melting temperature; MHC, minimal hydrotrope concentration; P, poise; NaBMGS, sodium butylmonoglycolsulfate; NaCS, sodium cumenesulfonate; NaPTS, sodium p-toluenesulfonate; NaS, sodium salicylate. Abstract published in Advance A C S Abstracts, J u n e 15, 1995. (l)Neuberg, C. Biochem. 2. 1916,76,107. (2)McKee, R. H. Ind. Chem. Ind. Ed. 1946,38,382. (3) Booth, H. S.; Everson, H. E. Ind. Eng. Chem. Ind. Ed. 1948,40, 1491. (4) Saleh, A. M.; El-Khordagui, L. K. Int. J. Pharm. 1985,24,231. (5) Balasubramanian,D.; Friberg, J. InSurfaceand Colloid Science; Matijevic, E., Ed.; Plenum Press, New York, 1993; Vol. 15, p 197. (6)Balasubramanian,D.;Srinivas, V.; Gaikar, V. G.; Sharma, M. M. J. Phys. Chem. 1989,93,3865. (7) Srinivas, V.; Sundaram, C. S.; Balasubramanian, D. Indian J. Chem. 1991,30E,147. ( 8 ) Poochikian, G. K.; Cradock, J. C. J. Pharm. Sei. 1979,68, 728. (9)Ueda, S. Chem. Pharm. Bull. 1966,14,22. @
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from micelles in exhibiting a higher and often more selective ability to solubilize hydrophobic solutes. For example, the hydrotropic actions of o-, m-andp-hydroxybenzoates, as well as 2,4-, 2,5-, and 2,6-dihydroxybenzoates, are markedly different.8 Such subtle isomeric influences indicate that structural and geometric features of these molecules modulate their self-assembly and h y d r ~ t r o p y .Our ~ study has shown that even a short chain aliphatic sulfate hydrotrope such as sodium butylmonoglycolsulfate (Na BMGS) self-Bggregates a t concentrations higher than 0.7 M in water and displays efficient solubilizing ability beyond this MHC. These studies show t h a t hydrotropy is a collective molecular phenomenon, brought about by the self-assembly of weakly amphiphilic molecules in water, and that is readily modulated by factors such as polarity, steric features, and related factors. Intermolecular packing and interactions that occur in the crystals would be expected to start manifesting themselves a t high concentration in solution, a situation that appears to be realized in some hydrotropes in water.’JO It is in this light that we focus attention on the amino acid proline here. There are several properties of the proline molecule that are striking in this context. Firstly its solubility in water is remarkably highll -as much a s 7 M at ambient temperatures. Secondly, its crystal structure, determined by Kayushina and Vainshtein,12 suggests an orderly packing or layering of the pyrrolidine rings; one could expect to see such intermolecular interactions begin to manifest at high concentrations in aqueous solution. Indeed, such a suggestion has been made earlier by Schobert and Tschesche.13 Should this occur, proline might be expected to display hydrotropic action. Thirdly, proline belongs to the class of “compatible solutes’’ in cell biology that helps cells cope with osmotic stress, and it does so in a noncolligative fashion.14 (Some other compatible solutes are choline and glycine betaine.) Knowledge of the physical chemistry of proline would be of value in understanding its role as a compatible solute. (10) Rath, H. Tenside 1965,2,1. (11)The Merck Index, 11th ed.; Budavari, S., Ed.; Merck & Co., Rahway, NJ, 1989; p 1236,no. 7790. (12)Kayushina, R. L.; Vainshtein, B. K. Kristallografia 1965,10, 834. (13)Schobert, B.;Tschesche, H. Biochim. Biophys. Acta 1978,541, 270. (14)Schobert, B. Nuturwiss 1977,64,386.
0 1995 American Chemical Society
Proline Is a Protein-Compatible Hydrotrope
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Figure 1. Enhancement in the solubility of FDA in aqueous proline solutions at room temperature, as measured by the optical density (OD) values at 488 nm.
Figure 2. Variation in the interfacial tension of the H20:CC14 system at room temperature with concentration of proline and hydroxyproline.
We have investigated the hydrotropic behavior of L-proline in aqueous solution and also compared its action with those of other compatible solutes on the one hand and of some hydrotropes that have been studied earlier on the other. Our results reveal that, unlike the latter, proline offers the solubilized molecule a microenvironmental polarity that is not too different from that of water. This property of proline helps it to sequester biopolymers such as proteins in conformations that are not too different from their native functional form and thus to earn proline the title "protein-compatible".
beyond. Experiments using pyrene as the solubilizate gave similar results and showed that proline brings about a 10-fold increase in pyrene solubility in water. We have also investigated the solubilizing ability of proline in the case of the steroids progesterone and estradiol and found that it caused a 4-fold enhancement in their solubilities in water at 25 "C. These results show that proline enhances the solubility of diverse hydrophobic compounds severalfold in water. In this behavior, proline is as efficient as the hydrotropes sodium salicylate (NaS), p-toluenesulfonate (NaPTS), cumenesulfonate (NaCS), and NaBMGS. However, the solubilization curve (Figure 1)displayed by proline is less steep and somewhat more shallow than those of NaS and NaPTS and comparable to that of NaBMGS;6 this might be a reflection of the fact that aromatic hydrotrope molecules might be able to pack better through stacking of the planar benzene rings. Figure 1 suggests that solubilization becomes significant above a proline concentration of about 2.5 M or so (halfway point). Dilution of the solubilization mixtures containing proline beyond this concentration led to the precipitation of FDA. Figure 1also compares the solubilizingability of other compatible solutes toward FDA and shows them to be less effective than proline in this respect. The related molecule 4-hydroxyproline has a limited solubility in water, ca. 2.5 M at 27 "C,but choline and glycine betaine are highly soluble, yet none of these is as efficient as proline. Hydrotropes are believed to self-aggregate. One way of monitoring this self-association is to study the concentration dependent variation of surface tension. Such studies of other hydrotropes show breaks in the surface tension curves at concentrations corresponding to their In this connection, we found that proline MHC is not significantly surface active at the aidwater interface. However it is able to alter the 1iquid:liquid interfacial tension of the water/CC14 interface. The variation of the interfacial tension of this binary liquid system with increasing addition of proline is shown in Figure 2. The decrease in the interfacial tension occurs in a biphasic fashion, trailing off beyond 3 M or so. Proline is only modestly surface active; however, this would yet act as a cohesive force that aids its self-assembly and hydrotropy. It is of interest to compare the behavior of other compatible solutes with t h a t of proline. Figure 2 shows that 4-hydroxyproline is similar to proline in its interfacial activity at a concentration up to its saturation limit of 2.5 M in water. Figure 1 shows that up to this limit, hydroxyproline is not able to solubilize hydrophobic compounds in water; its hydrotropy appears to be limited by its solubility in water. We encountered a similar situation with the other compatible solutes, glycine betaine and choline. These
Experimental Procedure L-Proline and 4-hydroxyproline were obtained from Sigma Chemical Co. and verified by HPLC and amino acid analysis to be of excellent purity. All the other chemicals of the highest available purity were obtained from various commercial sources. Fluorescein diacetate (FDA) was used as the representative hydrophobic molecule for solubilization studies because of its negligible solubility in water, its high optical absorptivity (log 6 = 2.1 at its band maximum near 480 nm) that aids an easy and accurate determination of its concentration, its low chemical reactivity, and its electrical neutrality. Solubilization experiments were done by equilibrating the solubilizate with various concentrations of proline overnight in a constant-temperature shaker. Surface tension measurements were made at room temperature (ca.25 "C)usinga White Fischer platinum ring tensiometer. Interfacial tension was measured using water and carbon tetrachloride as the two liquids. Fluorescence spectra were recorded using a Hitachi model F-4000 spectrofluorimeter. Fluorescence polarization measurements were done using diphenylhexatriene (DPH) as the probe (excited at 365 nm) and the medium microviscosities were estimated using the simplified and the equation applicable for steady state meas~rementsl~ experimentalprotocol describedearlier.16 Heavy atom quenching of fluorescence was followed using perylene as the fluorophore and Cs+ as the quencher and plotted using the Stern-Volmer equation. Thermal denaturation studies on proteins were done by monitoring the changes in their optical density at 280 nm (or in their fluorescence band in the 340-350 nm region) with temperature, in the presence of varying concentrations of the additive, using a Hitachi model 330 spectrophotometer.
Results and Discussion Figure 1shows the enhancement of the solubility of the dye FDA in water, brought about by increasing concentrations of L-proline. Addition of proline up to almost 1 M does not solubilize FDA to any significant degree. Upon further addition of proline, the solubility of FDA gradually increases and reaches a plateau at 4.5 M proline and (15)Shinitzky, M.; Barenholz, Y. Biochim. Biophys. Acta 1978,515, 367.
(16)Shobha, J.;Balasubramanian, D. Proc. Indian Acad. Sci., Chem. Scz. 1987, 98, 469.
Srinivas and Balasubramanian
2832 Langmuir, Vol. 11, No. 7, 1995
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Figure 3. (a) Variation in the wavelength and intensity (If)of the fluorescence spectral band maximum of the probe A N S as a function of the concentration of proline and hydroxyproline. A N S concentration, 10 pM;excitation wavelength at 365 nm. (b) Quenching of the fluorescence of perylene solubilized in 0.5 M proline, 5 M proline, and 2 M hydroxyproline in water by added CsCl. Pervlene concentration, 1uM: excitation at 435 nm and emission at 470 nm. F, and F are the fluorescenceintensities in the absence &d presence of the quencher.
two compounds do not display significant interfacial activities or any solubilizing ability even a t the highest concentrations. The remarkably high solubility of proline in water and its interfacial activity thus appear to be important factors in its hydrotropic action. Assemblies of hydrotropes have been seen to offer a relatively less polar interior where the solubilizate can be placed. The polarity features of such microenvironments are readily monitored using fluorescence probes such as ANS, the emission wavelength (and intensity) of which shifts significantly with the polarity of the medium,17or pyrene, for which the Ham effect ratio of the first and third vibronic bands in its fluorescence spectrum is altered by a change in the medium polarity.lsJg The results we obtained with proline solutions in water are surprising. The pyrene Ham ratio, 13/11, has a value of 0.6 in water and was found to shift very little even as the proline concentration was increased to 5 M in water, suggesting that the polarity offered by proline even beyond its MHC is not notably different from that ofwater. Sequestration of the solubilizate (FDA or pyrene) appears to occur so that solubilization is effected, but the microenvironment offered to the solubilizate by the proline molecular assembly is not significantly less polar than water. The same result was obtained using A N S as the polarity probe. The emission maximum of 10 pM A N S in water was seen around 522-523 nm, and in 5 M proline it moved to near 510 nm. This very modest blue shift indicates that the polarity of the proline solution is only slightly lower than that of water and is comparable to that of about 20% methanol in water (v/v). Figure 3a shows the variation of A N S fluorescence as the concentration of proline is increased in aqueous solution. Here again, a transition is observed in the emission wavelength values of the probe a t a concentration of around 2 M proline. The emission intensity, another polarity sensitive property, also increases a t high concentrations of proline. The supramolecular assembly of proline does not seem, however, to offer a microenvironment that is as nonpolar as that of any other hydrotrope; we have shown earlier6 that NaBMGS blue-shifts A N S fluorescence to 460 nm, and increases its intensity by almost 40-fold. In contrast, again, even saturated solutions of hydroxyproline do not significantly shift the emission band ofANS (17) Stryer, L.Science 1968,162, 526.
(18)Nakajima, A. J. Mol. Spectrosc. 1976,61, 467. (19) Dong, D. C.; Winnik, M. A. Photochem. Photobiol. 1982,35,17.
from its aqueous solution value. Here, too, the solubility of hydroxyproline appears to be the limiting factor. Figure 3b shows the efficiency with which the heavy metal ion Cs+ quenches the fluorescence of perylene that is solubilized in aqueous solutions of proline. Proline a t 5 M is able to protect or shield the fluorophore from quenching by Cs+ better than at a concentration of 0.5 M, consistent with the interpretation of a self-aggregate of proline beyond its MHC, which is able to shield or sequester the solubilized fluorophore. Here too, the ineficiency of 2 M hydroxyproline to inhibit the Cs+mediated quenching effectively is indicative ofits inability to form a hydrotropic assembly. Schobert and Tschesche have noted13that the contrasting behavior of proline and its 4-hydroxy derivative leads to the conclusion that aggregation of proline molecules occurs via their hydrophobic pyrrolidine rings. Since the hydrophobicity here is not pronounced, amphiphilicity and surface activity are weaker and self-association should occur in a less efficient and less cooperative fashion than in the case of detergentseZ0The MHC value of proline is thus high and perhaps represents not the onset of a micellelike compact assembly but stepwise oligomerization. That this self-aggregate of proline is loosely organized is apparent from the above fluorescence results. In further investigation of this point, we estimated the microviscosity of the proline assembly by measuring the anisotropy of the fluorescence of diphenylhexatriene (DPH) solubilized therein.15 The microviscosity value measured by DPH in 0.5 M proline was 0.45 P and it increased to 1.14 P in 5 M proline, indicative of a more viscous environment in the latter case. It is notable in this connection that the bulk viscosity of aqueous proline solution increases steeply beyond 2-3 M concentrations.13 These results suggest that proline is able to selfaggregate and sequester or include guest molecules within, a s hydrotropes do. Yet, the microenvironment it offers is more polar, closer to that ofwater than of nonpolar media. This would be of value in its interaction with biopolymers which need to maintain their native, active conformation. In this light, the interaction of proline with proteins is of particular interest, considering its role as a compatible solute and as an “enzyme protector’’ (a term used presumably to suggest that it stabilizes the native structure of globular proteins and does not inhibit enzyme (20) Mukejee, P. J. Pharm. Sci. 1974,63,972.
Proline Is a Protein-Compatible Hydrotrope
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activity21). Hence we studied the effect of proline on the structural properties of some proteins. Figure 4 shows the effect of 5 M proline on the thermal denaturation profiles of the globular protein enzyme a-chymotrypsin in the pH 5 region. Proline is seen to increase the denaturation temperature (T,) from 56 to 66 “C, thus offering the enzyme structural stability and protecting it from thermal denaturation. Results with the enzyme ribonuclease A were also qualitatively similar; the denaturation temperature was slightly increased. In contrast, NaBMGS or NaCS destabilizes the protein somewhat, by decreasing the T, value and precipitating the protein from solution. Proline seems to be a benign solubilizing agent that maintains the stability of these globular proteins in solution. It is interesting to note in this connection that Reddy and co-workers22and Taneja and AhmadZ3have reported that proline stabilizes the (21) Borowitzka, L. J.; Brown, A. D. Arch. Microbiol. 1974, 96, 37.
structure and activity of lactate dehydrogenase and cytochrome C, respectively. Neuberg, who introduced the concept of hydrotropes over 80 years ago, was seeking the biological role of these compounds. Proline, one finds, might be a hydrotrope with such a role. Its hydration is high,12-14which aids compatible solutes to preserve the necessary content of water demanded when cells and molecules are waterstressed. While choline, glycine betaine, and 4-hydroxyproline share this property, proline has an additional feature of relevance; it is able to self-aggregate and provide a host system that can solubilize or sequester guest molecules. The dual property of high hydration and selfassembly makes the microenvironment of the proline system close to or compatible with that of water and thus far more benign than those of conventional hydrotropes. It is this feature that helps proline maintain the native conformation of proteins and keeps them functional. Proline thus qualifies to be termed a “protein-compatible hydrotrope” and we suggest that it would be a more acceptable solubilizing agent in comparison to detergents and other hydrotropes, which tend to denature proteins and inactivate them biologically.
Acknowledgment. We are thankful to Dr. Ch. Mohan Rao for helpful discussions. D.B. thanks the Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India, ofwhich he is an honorary professor. We thank a referee for suggesting the term “protein-compatible hydrotrope.” LA9407267 (22) Chadalavada, S. V. R.; Reddy, B. V. B.; Reddy, A. R. Biochem. Biophys. Res. Commun. 1994,201, 957. (23) Taneja, S.; Ahmad, F. Biochem. J. 1994, 303, 147.
