Protein Denaturation by Ionic Liquids and the Hofmeister Series

Downloaded by STANFORD UNIV GREEN LIBR on September 18, 2012 | http://pubs.acs.org Publication Date (Web): December 18, 2009 | doi: 10.1021/bk-2009-1030.ch007

Chapter 7

Protein Denaturation by Ionic Liquids and the Hofmeister Series Diana Constantinescu, Christian Herrmann, and Hermann Weingärtner* Department of Physical Chemistry, Faculty of Chemistry and Biochemistry, Ruhr-University of Bochum, D-44780 Bochum, Germany

We have used differential scanning calorimetry (DSC) for studying how ionic liquids affect the thermal stability of proteins. Most 1,3-dialkylimidazolium, 1,1-dialkyl pyrrolidinium and tetraalkylammonium salts decrease the thermal stability, but some protic ionic liquids stabilise the native state. The effects can be systematised by the Hofmeister ion series. Depending on the nature of the ions, ionic liquids can also suppress the formation of misfolded proteins and the irreversible deactivation of proteins at high temperatures.

Introduction The unique physical and chemical properties of ionic liquids open fascinating prospects for chemical and technological processes (1-3). Ionic liquids are also of interest in biochemistry, for example in biocatalysis, biopreservation, or drug transportation (1,4,5). In such applications, native proteins are often exposed to high ionic liquid concentrations. How an ionic liquid affects the enzymatic function of a protein can vary sharply according to its ionic composition (1,5-14). The high specificity of salt effects was first noted by Hofmeister (15), who ranked salts according to their effect on the precipitation of hen egg white proteins. Essentially the same ranking is observed for other enzyme properties such as the thermal and functional stability and for many phenomena in other fields such as colloid or surface chemistry (11,17-21). Ionic liquids open options for steering such © 2009 American Chemical Society In Ionic Liquids: From Knowledge to Application; Plechkova, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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108 processes because their variability allows to impart the solvent properties of interest. For example, ionic liquids can be hydrophobic or hydrophilic. The subclass of protic ionic liquids composed of Brønsted acids and Brønsted bases (22,23) involves some salts which offer prospects as highly hydrophilic media for biomolecular processes (6,7). Here, we use differential scanning calorimetry (DSC) to study how ionic liquids affect the thermal stability of native proteins. In the simplest case these processes follow the Lumry-Eyring scheme (24) for the reaction N ⇔ U → I between the native state (A), unfolded state (U), and irreversibly deactivated state (I). The unfolding/refolding equilibrium N ⇔ U is completely reversible, so that after cooling the enzymatic activity is regained. Prolongated heating may, however, result in irreversible processes U → I, which lead to a loss of the enzymatic function. Such irreversible deactivation plagues many processes in enzyme technology. The mechanisms of irreversible deactivation are highly specific to the protein and to solvent conditions such as pH and buffer. They also depend on experimental conditions such as temperature and incubation time. Rather than exploring these mechanisms, we focus here on possibilities to improve the thermal and functional stability by adding suitably chosen ionic liquids.

Protein Unfolding Most of the experiments reported here were conducted with bovine ribonuclease A (RNase A), generally at a protein concentration of 0.36 mM in buffered solutions (25 mM phosphate buffer), with the pH value adjusted to the desired value. RNase A is a relatively small, monomeric enzyme, which is widely used in studies of protein denaturation (25-27). Figure 1 shows the DSC profile of a single scan of a freshly prepared solution of ribonuclease A (RNase A) at pH 7.0 and two profiles of solutions containing choline dihydrogenphosphate ([chol][H2PO4]) at concentrations C = 1 M and 2 M, respectively. In the freshly prepared, ionic liquid-free solution thermal denaturation sets on near 50 oC and is essentially complete at 75 oC, with a midpoint temperature of the transition ('melting temperature') of Tm = 63.5 oC.

In Ionic Liquids: From Knowledge to Application; Plechkova, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


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Figure 1. Base line-corrected DSC profiles of RNase A (pH 7.0) in an ionic liquid-free solution and in 1 M and 2 M solutions of [chol][H2PO4], respectively. The DSC profile of the ionic liquid-free solution is symmetric and can be described by a two-state approximation. The two-state model assumes a transition reaction N ⇔ U without intermediates. For its physical foundations we refer to a review by Chan et al. (28). For RNase A the adequacy of the twostate approach is well established (25). Upon addition of [chol][H2PO4], the symmetric shape of the DSC profile is retained, but the denaturation transition is displaced to higher temperatures, thus indicating the thermal stabilisation of the native state of RNase A. Figure 2 shows the dependence of the melting temperature Tm on the [chol][H2PO4] concentration up to C = 4 M, above which the solubility of RNase A became too low for DSC experiments. At C = 4 M, the solvent conditions already resemble those in the neat ionic liquid (6,7,29). Tm is increased up to about 20 oC relative to the ionic liquid-free solution. Similar effects were observed by us when adding [chol][H2PO4] to solutions of alcohol dehydrogenase and myoglobin. Fujita et al. reported an even larger stabilisation of cytochrome c by [chol][H2PO4] (6,7). The stabilising effect of [chol][H2PO4] contrasts to an enhanced unfolding induced by most aprotic ionic liquids, for example comprising 1,3-dialkylimidazolium, 1,1-dialkylpyrrolidinium and tetraalkylammonium ions (10). Figure 2 illustrates this behaviour for 1-ethyl-3-methylimidazolium This ionic liquid is liquid at room dicyanamide ([C2mim][N(CN)2]). temperature, and is hydrophilic enough to be completely miscible with water, but again, the solubility of RNase A prevented experiments at ionic liquid concentrations above C = 4 M. Tm decreases monotonously, displacing the denaturation transition at C = 4 M to room temperature. Density data (30) imply for anhydrous [C2mim][N(CN)2] C = 5.98 M. The tentative extrapolation to this concentration (Figure 2) predicts Tm ≅ 20 oC.



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Figure 2. Dependence of the melting temperature Tm of RNase A (pH 7.0) on the concentration of added [chol][H2PO4] and [C2mim][N(CN)2]. The curve for [C2mim][N(CN)2] is tentatively extrapolated to the neat ionic liquid (C = 5.98 M). Our observation contrasts with reports (5,12-14) on a surprisingly high thermal and functional stability of some proteins in neat aprotic ionic liquids similar to [C2mim][N(CN)2], where in part, protein functions were found to be retained well above 100 oC. A turnover to protein stabilisation in anhydrous [C2mim][N(CN)2] cannot be inferred from our data, and would imply a sharp break in the concentration dependence of Tm above C = 4 M. In anhydrous ionic liquids (or ionic liquids with little water) proteins are little soluble, and the reported effects may refer to dispersed states (5), which renders comparison with our data meaningless.

The Hofmeister Series Salt effects on properties of biomolecular solutes often obey the Hofmeister ion series (15-20). It is therefore apt to systematise our observations by ranking the cations and anions in the Hofmeister series. Usually, ion series reported in the literature are limited to inorganic salts. An exception is the work by von Hippel and Wong (16,17) on effects of guanidinium and tetraalkylammonium salts on the thermal stability of RNase A (17) and of other native biomolecules as diverse as collagen, DNA and myosin (16). We have recently extended their work by considering Hofmeister effects on the melting temperature of RNase A induced by some major cations and anions of ionic liquids (10). Figure 3 shows our results for chlorides combined with Li+, Na+, choline, 1-ethyl-3methylimidazolium ([C2mim]+), 1-butyl-3-methylimidazolium ([C4mim]+) and guanidinium ([gua]+), respectively.



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Figure 3. Melting temperatures Tm of RNase A (pH 7.0) as a function of the concentration of added ionic liquids with Cl− as a common anion. From the salts shown in Figure 3 only NaCl exerts a stabilising effect. The organic chlorides act as denaturating agents. Note that [chol]Cl destabilises RNase A, so that the stabilisation by [chol][H2PO4] reported above must result from the [H2PO4]− anion, perhaps in conjunction with cation−anion synergetic effects. Results for other ion families are documented in reference (10). The concentration dependence of Tm may be nonlinear, and adjacent curves may intersect at high concentrations. A well-defined ion ranking has to resort to the limiting slope of the concentration dependence of Tm. A comprehensive survey of our data for RNase A (10) in conjunction with data reported by von Hippel and Wong (17) yields the cation series Cs+ > Rb+ > K+ > Na+ ≈ [NMe4]+ > Li+ > [chol]+ > [NEt4]+ ≈ [C2mim]+ ≈ [gua]+ > [C4mpyr]+ > [C4mim]+ ≈ [NPr4]+ > [C6mim]+ ≈ [NBu4]+. where [C6mim]+ stands for 1-hexyl-3-methylimidazolium, [C4mpyr]+ for 1-butyl-1-methylpyrrolidinium, and [NR4]+ for tetrahedral tetraalkylammonium ions with methyl (Me), ethyl (Et), propyl (Pr), and butyl (Bu) residues. The stronger the hydrophobicity of the cation, the stronger is the thermal destabilisation. Protein denaturation studies often resort to guanidinium salts such as [gua]Cl or [gua][SCN] as denaturating agents (31), but among the hydrophobic cations considered [gua]+ adopts only an intermediate position. For the most widely used anions of ionic liquids, the Hofmeister series reads [SO4]2− > [H2PO4]− > [O2CMe]− > Cl− > [EtOSO3]− > [BF4]− ≈ Br− > [OTf]− > [SCN]− ≈ [N(CN)2]− >> [NTf2]− where [O2CMe]− stands for ethanoate, [OTf]− for trifluoromethylsulfonate, [EtOSO3]− for ethylsulfate, and [NTf2]− for bis(trifluoromethylsulfonyl)amide. Because most anions are not interrelated by homologous series, the interpretation of the Hofmeister anion series is not straightforward, but it seems that weakly hydrated and hydrophobic anions exert a destabilising effect.



112 Reported Hofmeister anion series usually quote [SCN]− as the most destabilising anion. However, among the widely used anions of ionic liquids, [N(CN)2]− is a similarly strong denaturant as [SCN]−, and the destabilising effect of hydrophobic [NTf2]− is even larger. The interpretation of Hofmeister effects at the molecular level is complex, and it is unlikely that the observations can be rationalised by a single or a few molecular properties. Traditional explanations use the concept of kosmotropic (structure-making) and chaotropic (structure-breaking) ions, which differently affect protein hydration (18,19,32). Even for simple ions there is, however, little consensus on what these properties actually entail (21). It is therefore not surprising that the extension to more complex ions is difficult to rationalise in terms of the chaotrope/kosmotrope terminology. Even without a detailed molecular understanding, such ion series seem, however, sufficiently general to provide a valuable phenomenological guide for assessing salt effects on enzyme stability (16,18) and enzymatic functions (11,18,19). It is important to recall that our rankings rely on the limiting slopes of Tm, whereas many applications resort to ionic liquid-rich solutions. However, in the cases studied here, Tm behaves monotonously, and intersections of adjacent curves are rare. Thus, the quoted series may also form a qualitative guide for assessing the behaviour of concentrated ionic liquids. Extrapolation to neat ionic liquids is, however, difficult.

Irreversible Protein Deactivation Refolding of the protein to the native state is often not complete. Whereas the denaturation equilibrium itself is reversible, additional processes may cause an irreversible loss of the enzymatic function. Depending on sample-specific conditions such as the pH and experimental conditions such as the incubation time at high temperatures, the irreversible deactivation can be founded in a variety of processes. Zale and Klibanov (27) have shown that in RNase A the most important processes are the hydrolysis of amide side chain functions at the aspartic residues, disulfide interchange and the formation of incorrectly folded and kinetically trapped structures. Note that the amyloidal aggregation of incorrectly folded proteins is a well-known phenomenon with regard to some pathological diseases such as Alzheimer's disease (33). For RNase A the pathways for irreversible deactivation (27) depend on the properties of the sample, such as protein concentration, buffer and pH. In addition, pathways for deactivation depend on conditions imposed by the experiments such as the scan rate and maximum temperature of the DSC scan and the conditions imposed by cooling. Rather than considering these details of the deactivation process, we focus here on the effect induced by added ionic liquids. In the DSC profile deactivation reveals itself by a decrease of the area under the endothermic DSC peak. This area reflects the enthalpy of unfolding, ΔuH, which is proportional to the number of proteins participating in the denaturation equilibrium. In repeated DSC cycles, the number of deactivated molecules will accumulate.



113 Figure 4 shows as a typical example the DSC profile of RNase A at pH 5.5 in the third DSC cycle (maximum temperature 110 oC, scan rate 60 K h-1 after shock-freezing to −80 oC). Under these conditions the behaviour is no more two-state. The endothermic peak shows a low-temperature shoulder, which indicates that some fraction of RNase A undergoes a reversible reaction to a less stable form. This second form may be a misfolded structure or a reversibly unfolding aggregate. Compared to the initial scan, the area under the total endothermic DSC peak is largely reduced, indicating a large fraction of deactivated RNase A. In addition, there is a strong peak above 100 oC due to an exothermic reaction. While these phenomena have not yet been explored by us in detail, in the present context the main observation is the ability of ionic liquids to drastically affect these phenomena. As an example, we show in Figure 4 the DSC of a 1 M solution of [chol][H2PO4], all other conditions being identical to those imposed to the ionic liquid-free solution. Addition of [chol][H2PO4] suppresses the exothermic peak, and increases the fraction of properly refolded RNase A. For further illustration we consider in Figure 5 the denaturation of RNase A at pH 5.5 and 7.0 at milder conditions than in Figure 4. The upper temperature of the DSC scan was set to 90 oC. Cooling was performed to room temperature. At pH 5.5 unfolding is now largely reversible. The area of the second scan differs by less than 10% from that in the initial scan. Addition of [chol][H2PO4] leaves the profiles unaffected (not shown). If the pH is increased to 7.0, denaturation is almost irreversible. The area in the second scan (dashed line) is less than 5% of that in the initial scan (solid line). [chol][H2PO4] restores, however, the reversibility. The thermal stabilisation of RNase A by [chol][H2PO4] is accompanied by a drastic increase in reversibility of the denaturation process.

Figure 4. DSC profiles of the third DSC cycle of RNase A (pH 5.5) in the ionic liquid-free solution and with 1 M [chol][H2PO4].



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Figure 5. DSC profiles of the initial scans (solid lines) and the first rescans (dashed lines) for RNase A in ionic liquid-free samples at pH 5.5 and 7.0, and in samples containing 1 M and 2 M [chol][H2PO4] at pH 7.0.

To further study the effect of ionic liquids on enzyme stability, we have considered the development of the DSC profiles of RNase A (0.36 mM, 10 mM phosphate buffer, pH 5.5) in successive DSC scans to 110 oC after shockfreezing to −80 oC. Figure 6 shows the fraction of deactivated proteins deduced from the area of the endothermic peak for ionic liquid-free RNase A and for solutions of three prototypical ionic liquids at C = 1 M. The fraction of deactivated proteins largely increases in progressive scans. Addition of ionic liquids can suppress or enhance deactivation. It appears that some basic trends in the Hofmeister series are also relevant for protein deactivation. The thermal destabilisation of RNase A by [C4mim]Cl is accompanied by an increase in the fraction of deactivated proteins. The weakly denaturating ionic liquid [chol]Cl slightly suppresses the deactivation of RNase A. The thermal stabilisation of proteins by [chol][H2PO4] is accompanied by strong suppression of protein deactivation.



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Figure 6. Fraction of irreversibly deactivated RNase A (pH 5.5) after successive scans for the ionic liquid-free (IL-free) solution and with 1 M , [chol]Cl, and [C4mim]Cl, respectively.

Finally, we mention similar observations by us for myoglobin, insulin and yeast alcohol dehydrogenase (ADH) and by Byrne et al. (9) for hen egg white lysozyme. Figure 7 shows results for ADH (protein concentration 0.012 mM, 25 mM phosphate buffer, pH 7.6). ADH enables the biocatalytic transformation of alcohols to aldehydes and ketones. It aggregates at comparatively mild conditions, here already during the first DSC cycle (19). [chol][H2PO4] suppresses the exothermic peak and increases the fraction of properly refolded ADH.

Figure 7. DSC profiles of 0.012 mM yeast ADH (pH 7.6, 25 mM phosphate buffer) without ionic liquid and with 0.5 M [chol][H2PO4].


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Conclusions


By creating a hydrophilic environment, some protic ionic liquids as cosolvents to water can lead to thermal stabilisation of proteins. By contrast, the most widely used hydrophobic ionic liquids exert a destabilising effect. In addition, ionic liquids may largely affect the irreversible deactivation of denaturated proteins induced at high temperatures. Carefully selected ionic liquids can suppress this deactivation. The factors controlling the effect of ionic liquids on protein deactivation seem to be intrinsically connected with those controlling the thermal stability of native proteins.

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Protein Denaturation by Ionic Liquids and the Hofmeister Series

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