currently , there is a great interest in the multipurpose application of recently revived substances , organic salts possessing relatively low melting points ( also known as  room - temperature ionic liquids ,  rtils , or  plastic ionic crystals  ) . these substances , when mixed with water , are able to form a huge amount of unique liquid or semisolid compositions apt for the novel nanotechnological and bionanotechnological developments [ 14 ] . because of virtually unlimited possibilities for the variation of an intrinsic molecular balance between the charged and uncharged ( hydrophobic ) groups on both positive and negative ionic components , these substances may offer potentially ideal milieu for any kind of reactant species including biomolecules ( e.g. , globular proteins ) , assembled in any kind of ( bio)nanodevice . for the latter case , the major point , actually , implies design of optimal environmental conditions for the improved stability , activity , and refolding ability of  working  elements such as proteins , for example [ 422 ] . furthermore , introduction of protic rtils , thanks to their essential protonic conductivity , offers superior conditions for the proper functioning of bioinspired nanovoltaic cells / devices [ 1 , 3 , 22 ] . as to the stability issue for globular proteins under the impact of different additives , including rtils , it can be adequately approached by the method of differential scanning calorimetry ( dsc ) [ 4 , 5 , 811 , 1317 , 2326 ] . specifically , the dsc technique detects the temperature - induced unfolding ( melting ) process ( associated with a global transition along the protein 's collective conformational coordinate ) as a variation of the protein 's partial heat capacity versus temperature and , in uncomplicated cases , in a model - free way ( directly ) determines the unique  transition temperature  ( temperature of thermal melting ) , tm , the unfolding ( melting ) calorimetric enthalpy , hcal ( i.e. , the heat absorbed through the unfolding transformation around tm ) , and calorimetric entropy , scal [ 2729 ] . numerous papers on dsc studies for the stabilizing / destabilizing impact of various organic additives [ 4 , 5 , 811 , 1317 , 2326 , 30 ] , including rtils [ 4 , 5 , 811 , 1317 ] , on the proteins ' global stability have been published . however , because of a complex , dualistic character of the rtil impact ( due to the involvement of oppositely charged surface - active ions ) , notwithstanding of several serious attempts [ 16 , 17 , 21 ] , no conclusive rationalization of the existing data was drawn so far . obviously , more systematic and careful work with the application of the dsc combined with other methodologies is required . indeed , in many cases of serial dsc studies implying the rtil impact , typically , only the parameter tm , as a measure of stability , has been considered ; the easily calculable parameter hcal has been addressed in very few cases ( see , e.g. , [ 5 , 8 , 9 ] ; for other similar studies involving uncharged additives , see , e.g. , [ 4 , 23 , 26 , 30 ] ) . it is instructive that another easily calculable parameter , scal ( vide infra ) , has never been implicated for this kind of series . an entropic contribution is equally important for the understanding of the protein stability phenomenon , as its enthalpic counterpart [ 29 , 3133 ] . in fact , stability of the native state results from the fine balance between a multitude of the enthalpic and entropic contributing factors ( vide infra ) [ 4 , 27 , 30 , 31 , 33 ] . among other model proteins , in which thermal stability has been extensively explored by the dsc technique , -chymotrypsin ( -ct , ec 3.4.21.1 ; mr = 26,000 ) , a hydrolytic enzyme of biotechnological importance , should be mentioned [ 4 , 5 , 23 , 24 , 26 , 30 ] . however , detailed studies regarding the impact of rtils on -ct , using highly sensitive dsc technique ( implying determination and analysis of all the key parameters , tm , hcal , and scal ) , are still lacking . in this communication , we report preliminary data and principal thermodynamic analysis on the stabilizing impact of biotechnologically important concentrations of a protic ionic salt , choline dihydrogen phosphate , [ ch][dhp ] , on the thermal stability of model protein , -ct . determination of both enthalpic and entropic components contributing to the protein melting process allowed for a conjecturing of fine interfacial effects behind the notable stabilization impact . highly purified and lyophilized -ct was purchased from fluka and [ ch][dhp ] was from iolitech , gmbh , and used without further purification . the -ct samples for the dsc experiments were prepared at concentrations of 1.5  mg / ml , by dissolving in phosphate 10 m buffer solutions ( ph 4.2 ) . microcalorimetric measurements for temperature - induced melting ( denaturation ) of -ct in buffered [ ch][dhp ] aqueous solutions were performed with a dasm-4a adiabatic scanning calorimeter ( biopribor , russia ) integrated with a pci - das1001 ( measurement computing corporation ) interface unit , providing direct pc access . the methodological aspects of the dsc technique and data processing have been described earlier [ 23 , 27 , 28 , 30 ] . figure 1 depicts the zero - baseline - corrected calorimetric curves ( partial heat capacity of protein versus temperature ) for the temperature - induced melting of -ct in the absence and presence of the [ ch][dhp ] additives , 0 , 0.5 , 1.0 , 2.0 , and 3.0  mol / l . accordingly , figures 2 , 3 , and 4 and table 1 depict results of thermodynamic analysis of these melting curves . the values for a melting enthalpy , hcal , were determined as areas under the calorimetric curves of figure 1 [ 23 , 2730 ] : 
 ( 1)hcal=t1t2cp(prot )  dt , 
 where t is the absolute temperature and t1 and t2 are the temperatures that correspond to the start and completion of heat absorption due to the protein 's thermal melting ( t1 < tm < t2 ) . the values of the counterpart parameter , melting entropy , scal , can be determined as follows : 
 ( 2)scal=t1t2cp(prot)t  dt . 
 under the assumption that a two - state model for the protein melting process is operative , that is , ( a ) at tm concentrations of the native and melted ( unfolded ) protein are equal and ( b ) virtually the entire entropy change takes place at this temperature ( therefore , gm = hcal  tmsm = 0 ) , ( 2 ) is simplified as follows : 
 ( 3)sm=hcaltm . however , one may question whether ( 1 ) to ( 3 ) and related analysis ( vide infra ) are valid for the case of -ct , known as a protein in which thermal unfolding process , in general , deviates from a simple two - state pattern ( see , e.g. , [ 24 , 30 ] ) . rather detailed analysis of this issue is given in our previous paper   ( see also [ 17 , 34 ] ) . in brief , in the course of a thermal melting of most globular proteins of modest dimensions , including -ct , the native and  normally  unfolded states can be considered as yet dominantly populated states against the relatively minor , thermally induced molten globule ( predenatured ) [ 25 , 34 ] ,  intermediate ( metastable transitional ) [ 16 , 34 ] , and misfolded / self - aggregated ( postdenatured ) states [ 16 , 29 , 35 ] . importantly , the dsc curves retained symmetrical , nearly gaussian shape ( half widths were within 6.7  0.2  k throughout ) upon the addition of 0.5 to 3 mol / l [ ch][dhp ] ( the fact that favors the abovementioned outline , yet , as a crude approximation ) , whilst their maximum position ( tm ) was gradually shifted to higher temperatures ( for ca . 11  k , in overall ) , pointing to the notable stabilizing impact of [ ch][dhp ] ( table 1 ) . the values of scal ( attained for selected cases ) and sm , as determined through ( 2 ) and ( 3 ) , coincided within the limits of experimental error , that is , an additional support for the practical applicability of ( 3 ) . the slope for the first change of tm versus the [ ch][dhp ] ) content ( from 0 to 1  mol / l [ ch][dhp ] ) was a little bit larger than one for the rest of the concentration range ( figure 2 ) . this fact could be ascribable to the experimental scatter of experimental points if there is no remarkable and drastic mutually compensating change of both hcal and sm resulting only in a modest shift of tm ( figures 2to 4 , table 1 ) . next , in a concentration range from 1 to 3  mol / l [ ch][dhp ] , parameter hcal increased gradually very little , whereas parameter sm remained unchanged within the limits of experimental error . this occurrence allowed parameter tm to increase further steadily ( figure 2 ) ( vide infra ) . 16  k increase in the melting temperature of another model protein , ribonuclease a , upon the adding of 3 mol / l [ ch][dhp ] ( overall , 20  k increase upon the adding of 4  mol / l [ ch][dhp ] ) . the shift values for tm and a trend for the dependence of tm on the [ ch][dhp ] content are very similar to ours , having the distinct slope break at 0.5  mol / l ( see figure 2 of ) . however , as can be judged based on the incomplete figure 1 of , the increase in tm was accompanied by the substantial increase in hcal . unfortunately , the whole trend for hcal ( and , even more so , for sm ) is not reported in . in another recent work , .   reported on ca . 11  k increase in a melting temperature of the third model protein , lysozyme , upon the adding of 2.5 the overall effect is absolutely similar to ours ( implying the interpolated value of tm  74c at 2.5 mol / l [ ch][dhp ] for our system ) ; however , the overall dependence of tm on the [ ch][dhp ] content for lysozyme , in contrast to our case ( figure 1 ) , exhibited well - pronounced minimum at 0.5 again , the authors of   did not report the values for hcal and scal ( sm ) at any concentration of the additive . the lacking information would allow one to see the variable balance between hcal and sm behind the resultant behavior of tm in these studies , hence , to compare the complete data for all three model proteins and gain better insight into atomistic mechanisms of stabilization . nonetheless , the first analysis of our results together with comparable published data [ 11 , 15 ] permits presuming that the mechanisms behind the initial drop in values of both hcal and sm and further slight increase with and virtual independence of tm , respectively , are obviously different . when going from zero to 0.5  1  mol / l additive concentration , seemingly , strong association between the [ ch][dhp ] components and proteins ' charged groups takes place that should be rather specific to the protein entity and its ionization state ( ph ) ; note different patterns of tm for -ct and ribonuclease a on the one hand and lysozyme on the other hand ( note also different hcal patterns for -ct and ribonuclease a , mentioned above ) . around this point of the additive concentration , the specific interactions , probably , arrive at the saturation level , after which more gradual / monotonic changes of tm ( in all three cases ) and very small / no variation of hcal and sm ( as detected in our case ) can be observed . the small changes in hcal , seemingly , are connected with the formation of additional contacts and some redistribution of existing ones between the protein and the [ ch][dhp ] components . development of the latter process seems to favor proteins ' gradual stabilization via the hcal rather than the sm parameter . according to the analysis of   ( the most systematic one , so far ) , the choline cation is deemed to act as a slightly destabilizing component whereas dihydrogen phosphate anion is known as an efficient stabilizer . in addition , this pattern turned to be dependent on the additive concentration and has to be also essentially ph dependent . hence , around ph 4.2 , where the exposed amino groups are 100% positively charged , the [ dhp ] anions , able to effectively interact with these groups through the coulombic attraction , may eventually replace the existing hydrogen - bond networks originally formed by solvating water and contribute to the protein stabilization . the enthalpic rather entropic nature of the stabilization impact of [ ch][dhp ] above 1  mol / l concentration definitely points to a prevailing role of the hydrogen - bond network strengthening rather than that of additional strengthening of electrostatic interactions ( between any kind of the  point charges  involved ) . at this ph , the surface exposed carboxylate groups of -ct are for only ca . 50% negatively charged ; therefore , respective coulombic and associated hydrogen - bonding interactions may play relatively minor role . further systematic work is in progress aiming at more comprehensive insights at the atomistic level . ( a ) we applied the very sensitive dsc technique to study an impact of 0.5 to 3  mol / l additives of the biotechnologically relevant ionic substance [ ch][dhp ] on the thermal stability of a model globular protein -ct and found the notable stabilizing effect of 11  2  k regarding the thermal unfolding ( melting ) temperature , tm , that varied monotonically with the additive concentration . ( b ) the calorimetric melting enthalpy ( hcal ) and transition entropy ( sm ) parameters were determined simultaneously throughout the series , and the trend analysis indicated a two - phase pattern including the low - concentration ( 0 to 1 mol / l [ ch][dhp ] ) and high - concentration ( 1 to 3  mol / l [ ch][dhp ] ) ) regions , connected to the initial dramatic and the follow - up minor or no changes of hcal and sm , respectively . ( c ) the atomistic mechanism behind the observed overall stabilizing effect , presumably , is guided by the ion pair formation between the protein 's charged groups and the [ ch][dhp ] components , with a prevailing role of restructured hydrogen - bonded networks facilitated mostly by coulombic interaction between the [ dhp ] anions and solvent - exposed positively charged amino groups of -ct . ( d ) a need for the determination of parameters hcal and scal ( sm ) throughout this kind of series , for the proper understanding of atomistic mechanisms behind the stabilizing / destabilizing impact of various additives on proteins , is clearly demonstrated .
an impact of 0.5 to 3  m choline dihydrogen phosphate , [ ch][dhp ] , the biotechnologically relevant ionic substance , on the thermal stability of a model globular protein , -chymotrypsin ( -ct ) , has been studied exploiting the highly sensitive differential scanning calorimetry ( dsc ) technique . 
 the notable overall stabilizing effect of 11  2  k regarding the thermal transition ( melting ) temperature , tm , has been detected . 
 for this kind of series , for the first time , the calorimetric melting enthalpy ( hcal ) and transition entropy ( sm ) parameters have been determined simultaneously throughout . 
 the first analysis indicated a two - phase impact implying ( a ) the initial , dramatic drop in both hcal and sm , obviously connected to specific , direct interaction between the [ ch][dhp ] components and -ct 's charged groups ( within 0 to 1 
 mol / l [ ch][dhp ] ) , leading to the essential rearrangement of the interfacial hydrogen - bonded ( hb ) network ; and ( b ) the follow - up ( within 1 to 3.0 
 mol / l [ ch][dhp ] ) , modest changes in hcal and lack of changes in sm , seemingly connected with a subsequent steady strengthening of already reformed hb network , respectively . 
 these changes , presumably , are primarily facilitated by coulombic interactions between the [ dhp ] anions and solvent - exposed positively charged amino groups of -ct .