A Distinct Proof on Interplay between Trehalose and Guanidinium

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A Distinct Proof on the Interplay Between Trehalose and GdnHCl for the Stability of Stem Bromelain Anjeeta Rani, and Pannuru Venkatesu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b05766 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on August 2, 2016

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The Journal of Physical Chemistry

A Distinct Proof on Interplay between Trehalose and GdnHCl for the Stability of Stem Bromelain Anjeeta Rani and Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi – 110 007, India

ABSTRACT Guanidinium chloride (GdnHCl), a potential denaturant, is well known to denature a number of proteins in vitro as well as in vivo studies. Its deleterious action on stem bromelain (BM) is quite prominent resulting decrease in protein structure and stability. The counteraction of this adverse effect of GdnHCl by the use of osmolytes is scarcely studied and the mechanism is still illusive not exclusive. For the first time, to test elegant and simple counteraction hypothesis as a general mechanism, we utilized fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS) to study the counteraction of GdnHCl-induced denaturation of BM by the trehalose. It is revealed from the investigation of the results that trehalose is efficiently counteracting GdnHCl undesirable impacts on BM stability at molar ratio 1:1 of trehalose and GdnHCl. On contrary, proteolytic activity of BM is increased only for the counteraction study of BM at very high concentrations of GdnHCl, however, still less than BM in buffer. The mutual exclusion of both trehalose and GdnHCl may stand for the counteraction of denaturation of BM resulting in a compact conformation with less solvent exposed surface area, increased secondary and tertiary structures. In addition, a decrease in BM-solvent interactions may also be contributing to some extent as there is little binding of trehalose replacing some water molecules and reducing binding of GdnHCl.

_____________________________ Corresponding author: e-mail:[email protected]; [email protected]; Tel: +91-11-27666646-142; Fax: +91-11-2766 6605 1 ACS Paragon Plus Environment


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INTRODUCTION Proteins are the dynamic entities present in the environment comprised of solvents, crowding agents, osmolytes and small-molecular and macromolecular ligands. Therefore, their adopted conformations are not only depending upon the sequence of the amino acid residues, but also on the solvent environment.1,2 The osmolytes have a great impact on the protein stability, structure and function in aqueous solution. They maintain a high level regulatory responsiveness, adequate protein’s catalytic rates and a precise balance between flexibility and stability of the protein structure.1-3 For that reason, it is suggested that osmolytes might have great importance for maintaining life development and evolution. Some organisms accumulate some of the naturally occurring osmolytes such as trimethylamine-N-oxide (TMAO), betaine, polyols, trehalose, sucrose etc. and also urea like classical denaturant.4 A key paradigm in the biology of adaptation is that the denaturant influences the protein stability and function by increasing the fluctuations of the native state, whereas osmolyte affects the same in the opposite direction.5 In fact, for the species accumulating both osmolytes as well as denaturants, an efficient synergy is required because a serious threat will be posed for the survival of the organisms containing high amount of denaturants in their cells if there is failure or partial failure of counteraction in the terms of stability and functional activity of the protein. In this context, Venkatesu et al.6 revealed that TMAO, betaine, and sarcosine strongly counteract the deleterious actions of urea on α-chymotrypsin. Many studies have found polyols protecting against urea denaturation of the protein.7-16 According to Kumar et al.,7 trehalose acts as potential stabilizer to counterbalance urea-induced unfolding of α-chymotrypsin. Simpson and Kauzmann8 observed that sucrose counteracts the urea-induced denaturation of ovalbumin. Among the sugars and polyols, trehalose has received special attention owing to the particular efficiency in protecting the function and structure of proteins against the chemical denaturation.11,12,17 Trehalose, a non reducing disaccharide, is widely distributed among the living organisms. It is accumulated in large quantities by diverse organisms during a variety of stressful conditions.13 This osmolyte is fundamentally different from the polyol and amino acid classes of osmolytes in terms of its effect on the protein. It has also been shown that only trehalose protects yeast cystolic enzymes against thermal inactivation whereas other carbohydrates fail to do the same.14 2 ACS Paragon Plus Environment

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Therefore, in present study, the potential of trehalose as a protein stabilizer has been further extended by the counteraction study against the denaturation of stem bromelain (BM) caused by guanidinium chloride (GdnHCl). Despite a considerable amount of work done in the field of counteraction in the last five decades,5-12,14-16 a very few reports are available to establish that trehalose engenders protection of proteins from GdnHCl which is generally used for protein folding/unfolding experiments.11,12,15,16 A majority of the available literature is focussed on refolding studies and there is hardly any report mentioning counteraction by trehalose to offset GdnHCl-induced denaturation. Nevertheless, it was quite surprise that there is even no answer to first lingering question aroused in counteraction studies i.e. whether trehalose is interfering in the action of GdnHCl by direct interaction or doing its work independently. Still, the elucidation of the molecular mechanism of counteraction is one of the major challenges in the field of the protein chemistry. Rather than this special case, there is no consensus regarding the answer of this paradox for all other counteraction studies also and all the proposed mechanisms are still elusive and little far from conclusive. This dearth of knowledge provokes us to explore the counteraction ability of trehalose in opposition to GdnHCl action on proteins. With respect to the various issues such as protein folding, stability and function and major practical interests in biochemistry, biotechnology and evolutionary biology specially bottomed on medicinal and agricultural grounds, any mechanism which offers a generalized protection of proteins against denaturation is of basic importance. The system chosen for present study is a protease, stem bromelain (BM). The wide applications of BM in numerous fields of chemical and pharmaceutical industries compel us to study about stability of BM in a variety of media.18,19 Even though BM has a long story of comprehensive studies of its biological aspects but its stability in presence of various osmolytes/denaturants and the mechanism by which it get stabilized by the different cosolvents still remain underground. According to recent report by our laboratory,20 BM starts to denature to a high extent in the presence of GdnHCl and gets highly stabilized in trehalose. However, the counteraction of the GdnHCl-induced denaturation of BM by the use of the trehalose has not been studied yet. In this report, we demonstrate the effectiveness of trehalose for protection of enzymes against the structural damage promoted by GdnHCl to preserve the function of the enzyme by employing fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering 3 ACS Paragon Plus Environment


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(DLS) and Fourier transform infrared spectroscopy (FTIR). Our findings expose novel properties of trehalose with broad implications for protein folding/unfolding studies. To the best of our knowledge, for the first time, the counteraction by trehalose against GdnHCl-induced denaturation of protein has been presented with the help of a combination of molecular techniques with the spectroscopic techniques. A study on interplay between a potential stabilizer and a well known denaturant may lead us to novel approaches for rational design of effective protein stabilizers from future perspective point of view.

MATERIALS AND METHODS Materials. Bromelain (BM) E. C. 3.4.22.32 lot No. B4882 from Ananas comosus, trehalose, guanidinium chloride (GdnHCl) and deuterium oxide (D2O), were purchased from Sigma Aldrich, USA. A highly pure anhydrous sodium phosphate monobasic, sodium phosphate dibasic dehydrate, casein (Hammarsten), trichloroacetic acid (TCA), sodium acetate and acetic acid were purchased from Sisco Research Lab (SRL), India. All chemicals were highly pure and of analytical grade. Sample Preparations. The sodium phosphate buffer (0.2 M) at pH 7, prepared by using distilled deionized water with resistivity of 18.3 Ω cm, was used for preparation of all enzyme samples. The enzyme samples were prepared at 0.5 mg/mL concentration of BM and filtered with 0.22 µm disposal filter (Millipore, Millex-GS) through syringe before performing experiments for all measurements. For DLS measurement, protein concentration was kept 1 mg/mL. Mettler Toledo balance with a precision of ±0.0001 g was used for gravimetric measurements. In order to obtain complete equilibrium, all samples were incubated for 1 hour at 25 oC. The stability of BM was studied in absence and presence of mixtures of trehalose and GdnHCl at molar ratio of 1:1, 1:2 and mixtures of 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 M GdnHCl at a fixed concentration of 0.5 M trehalose for all experiments. There is little variation in pH of the sample on addition of GdnHCl, therefore, all samples pH were adjusted to 7 accordingly. Steady State Fluorescence Measurements. Steady state fluorescence study was performed at 25 oC by using Cary Eclipse spectrofluorimeter (Varian optical spectroscopy instruments, Mulgrave, Victoria, Australia) equipped with an intense xenon flash lamp as the light source equipped with a Peltier- type temperature controller with a precision of ±0.05 oC, using the excitation wavelength at 295 nm with a slit width of the excitation and emission at

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5 nm and 10 nm, respectively. The excitation wavelength of 295 nm is used in order to prevent tyrosine residues from transfer the radiation energy to the tryptophan (Trp) residues. Thermal Equilibrium Unfolding Study. Equilibrium unfolding studies of the BM in the absence and presence of various mixtures of trehalose and GdnHCl, were conducted over a temperature range 15 to 95 oC (heating rate 5 oC/min), using Trp fluorescence as a probe in same Cary Eclipse spectrofluorimeter. The excitation wavelength and emission wavelength was 295 and 310 nm, respectively. All unfolding transitions were analyzed by assuming the two state unfolding mechanism as shown in eq 1.21 N⇌U

(1)

Structural Characterization of BM Using Circular Dichroism. To obtain a deep insight into the structural characteristics of the BM, far-UV CD spectroscopy studies were performed for BM in the absence and presence of various mixtures of trehalose and GdnHCl. The BM concentration was 0.5 mg/mL. Due to the absorption by GdnHCl in far-UV region (≤ 215 nm), a proper subtraction of GdnHCl absorption spectra at its each concentration was carried out for all samples CD spectra. Trehalose absorption spectra were also subtracted from CD spectra of all samples containing trehalose to obtain good results. PiStar-180 spectrophotometer (Applied Photophysics, U.K.), which is equipped with a peltier system for temperature control with an accuracy of ±0.1

o

C and calibrated using (1S)-(+)-10-

camphorsulfonic acid (Aldrich, Milwaukee, WI), which exhibits a 34.5 M cm-1 molar extinction coefficient at 285 nm and 2.36 M cm-1 molar ellipticity (Θ) at 295 nm, was used to record all the CD spectra. The CD spectra are monitored in cuvette with pathlength of 0.1 cm using scan speed 50 nm/min, a response time of 1 s and 1 nm bandwidth and results were expressed as mean residual ellipticity (MRE) in degrees cm2 dmol-1 and are averaged of three scans. Fourier Transform Infrared Spectroscopy (FTIR) Characterization. To unveil the counteraction of the GdnHCl-induced denaturation of BM by trehalose, we examined the changes in amide I band position which may participate in the interactions with solvent as well as co-solvent molecules. Infrared spectra of BM in the absence and presence of various molar ratios of mixtures of trehalose and GdnHCl were recorded in the range of 1600 cm-1 to 1700 cm-1 by using a thermo scientific FTIR spectrometer. Samples were held in an IR cells with ZnSe windows by using 50 µm path length spacers. 256 scans were performed at 2 cm-1 resolution and averaged. All samples were prepared in D2O buffer maintained at pD ~ 7.0 5 ACS Paragon Plus Environment


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with a fixed concentration of the enzyme and equilibrated for 4-5 hrs at 25 oC to facilitate HD exchange in the protein sample. Omnic software was used to analyse FTIR spectra. A subtraction of buffer (D2O, pD 7.0) was carried out from each spectra by using omnic software to compare the changes in the BM in the absence and presence of various mixtures of trehalose and GdnHCl Dynamic

Light

Scattering

Measurement.

The

dynamic

light

scattering

measurements were performed by Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) at a scattering angle of 900. The light source, He-Ne laser is of 4 mW, operating at a fixed wavelength, 633 nm and emitting vertically polarized light. Instrument was provided with a thermostatic sample chamber for maintaining temperature equal to 25 oC in an airtight quartz cell in which sample was loaded. The detection range of this instrument is from 0.1 nm to 10 µm. The samples were spun at 10000 rpm for 10 min and filtered through 0.1 µm pore size milipore syringe filter. The refractive index, deielectric constant and viscosity values were taken into account for all samples. Each measurement consisted of three subsequent individual runs and all results are averaged of three concordant reading. This instrument measured the translational diffusion coefficient from the time dependent fluctuations in the scattering intensity of light by using the instrumental software from which the value of hydrodynamic diameter (dH) was attained by means of the Stokes-Einstein equation:

=

(2)

where k is the Boltzmann’s constant (1.3806503 × 10-23 m2 kg s-2 K-1 ), T is absolute temperature (K), is viscosity (mPa s), and D is diffusion coefficient (m2 s-1 ). The data were further analyzed by plotting using origin 9 software (OriginLab Corp., Northampton, MA, USA) Activity Measurements of BM. BM activity was assayed using UV-vis spectrophotometer (UV-1800 Simadzu Spectrophotometer). The denatured casein solution (0.5 mL of 0.5 %) was used as a substrate which was incubated for 10 min at 25 oC with BM in buffer (pH~7.0) at 0.5 mg/mL concentration pretreated with the various concentrations of TMAO. Trichloroacetic acid (TCA), 1 mL of 110 mM, was used to stop the reaction after 10 min. The precipitates of the undigested casein were removed by the centrifugating the samples. The absorbance values of a reagent blank were spectrophotometrically measured at 275 nm with which reaction product was correlated. From the standard curve of absorbance 6 ACS Paragon Plus Environment

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of known quantities of the tyrosine (Tyr), activity (units per mL) of BM samples can be determined using the following equation:

=

µ ! " #

3

where V is total volume of assay in mL, v1 is volume of enzyme used in mL, v2 is volume of sample in mL used for UV measurements.

RESULTS Fluorescence Spectral Analysis of Counteracting Effects of Trehalose on GdnHCl-induced Conformational Changes in BM. Intrinsic Trp fluorescence can be used as a probe for detection of the conformational changes in the BM. There are five Trp present in BM. Out of five Trp, three are buried and two are exposed to the surface. The wavelength maxima (λmax) of native BM in buffer at pH 7.0 was observed around 347 nm (black spectra in Figure 1). The denaturation process can be analyzed from the changes in λmax as well as in fluorescence intensity (Imax), both resulting from the change in the polarity of environment of Trp. All detailed explanation for λmax changes of BM for trehalose and GdnHCl are explained in Ref. [19]. Here, we have shown again results for GdnHCl for sake of clarity (data taken from Ref. 19). From our previous study19, it is obvious that there is significant red shift in λmax (347 nm to 352 nm) as well as large decrease in the Imax at higher concentrations of GdnHCl as compared to BM in buffer which emphasizes on the strong denaturing effect of GdnHCl on conformation of BM (only shift in λmax is shown in Figure 1a and fluorescence intensity can be seen in Figure S1a). Here, Figure 1b represents the effect of different concentrations of GdnHCl on native state of BM in presence of 0.5 M trehalose. On contrary to Figure 1a, there is insignificant blue shift in λmax (~1 nm) for 0.5 and 1.0 M of GdnHCl in presence of 0.5 M trehalose as can be clearly observed in Figure 1b. However, at 1.5, 2.0, 3.0 and 4.0 M of GdnHCl in presence of 0.5 M trehalose as can be seen in Figure 1b, the results were almost similar to that monitored for BM in presence of GdnHCl only (a red shift from 347 nm to 352 nm). It may emphasize that this low concentration of trehalose is not effective in offsetting the changes occurring in the Trp environment of BM in presence of higher concentrations of GdnHCl. In addition to this, Trp fluorescence spectra of BM in presence of different combinations of 1:1 and 1:2 ratios of trehalose with GdnHCl are displayed in Figures 1c and 7 ACS Paragon Plus Environment


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1d, respectively. As shown in the Figures 1c and 1d, there is a blue shift in λmax of BM at all studied concentrations of 1:1 and 1:2 ratio of trehalose and GdnHCl with respect to BM in buffer as compared to that in the varying concentrations of GdnHCl in Figures 1a (blue shift is more apparent from inset Figures 1c and 1d). The degree of blue shift is more pronounced in 1:1 (347 nm to 344 nm) as compared to 1:2 cases (347 nm to 345 nm). The viewed blue shift in λmax is an indication of the conformational changes which causes access of Trp towards more hydrophobic environment i.e. core of BM. These results powerfully accentuate on the compaction of BM native state and, hence, more stabilized BM is formed than BM in buffer. For the more transparency, Figures 1e and 1f illustrate the changes in the Trp fluorescence of BM in absence and presence of 2.0 M GdnHCl and the mixtures of 2.0 M GdnHCl with 0.5, 1.0 and 2.0 M trehalose, where 2.0 M GdnHCl is found to be effective for the disrupting the BM. The detected blue shift in λmax of BM (Figure 1e) is accompanied with an increase in the Imax (Figure 1f) for all mixtures as compared to that in 2.0 M GdnHCl. Again, it may be described here that there is internalization of Trp towards less polar environment which results in blue shift as well as decreased quenching. All these observations depict strong counteraction by trehalose against GdnHCl, especially, in case of the mixture at 1:1 of trehalose with GdnHCl which is effortlessly remarkable in Figures 1e and 1f. In this case, a highly noteworthy blue shift in λmax (from 347 to 344 nm) as compared to BM in buffer and increased Imax can be clearly seen. Although all concentrations of trehalose counteracted the conformational changes in Trp environment of BM induced by 2.0 M GdnHCl, observed extent of counteraction at 0.5 M trehalose is very less wherein still an insignificant red shift in λmax by ~1 nm and also highly decreased Imax with respect to BM in buffer (λmax is clearly observable in inset Figure 1e and Imax in Figure 1f). Thermal Stability Analysis of Counteraction of GdnHCl-induced BM Denaturation by Trehalose. From normalized transition curves in Figure 2a, it is quite noticeable that Tm value is decreasing with increasing concentration of GdnHCl in comparison to BM in buffer (Tm ~ 65.3 oC), except at very lower concentration (0.5 M) where Tm is slightly higher than control. It put emphasis on decreased thermal stability in presence of GdnHCl. In Figure 2b, increasing concentrations of trehalose cause increase in Tm values, thereby, increasing thermal stability. Whereas, all transition curves for trehalose and GdnHCl at 1:1 molar ratio are found to be almost overlapped with that for BM in buffer


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(Figure 2c). This inspection of the Figure 2c depicts that Tm values for all these mixtures may be more or less similar to the Tm value of BM in buffer. As can also be clearly seen in Figure 2d, transition curve for BM in mixture of trehalose and GdnHCl at 1:1 ratio is approaching towards that for 2.0 M trehalose as compared to that for BM in buffer as well as in 2.0 M GdnHCl. Tm value for BM in this mixture is ~ 67 oC which quite higher with respect to that in presence of 2.0 M GdnHCl only. This may again put stress on the counteraction ability of trehalose on the denaturing effects of GdnHCl on the thermal stability of BM. All these results are more apparent from Figure 3 which describes the variation of Tm in presence of GdnHCl, trehalose and mixtures of trehalose and GdnHCl at different concentrations. It is clear that GdnHCl adversely effects thermal stability of BM. However, all values of Tm for BM in mixture of trehalose and GdnHCl at 1:1 ratio are higher than control which explicitly elucidates that trehalose is appreciably attenuating the deleterious effect of GdnHCl on BM thermal stability. On the other hand, in comparison, trehalose is effective to a smaller extent in counteracting GdnHCl effects on BM when trehalose is present at 1:2 ratio with respect to GdnHCl. In Figure 3, all Tm values for all sample of 1:2 ratio are slightly lesser or closer to that in control. Circular Dichroism Analysis of Trehalose Effect on GdnHCl-induced Changes in BM Secondary Structure. To ascertain further the counteracting effect of trehalose, far-UV CD-spectral analysis is performed. Secondary structure of BM is identified with mainly two negative bands, at 208 and 222 nm where band at 208 is more intense than 222 nm (shown black in Figure 4) suggesting that BM is belonging to the typical α +β class of enzyme which is in accordance with the results obtained by Reyna et al.22 As can be seen undoubtedly in Figure 4a, there is considerable amount of decrease in the secondary structure of BM as a function of increasing GdnHCl concentrations which is emphasized due to the decreased negative ellipticities with increase in the concentrations of GdnHCl. At 0.5 M GdnHCl, anti parallel β-structures (positive band at ~205 nm and negative band at ~215 nm) are formed as a compensation of helical structures, however, overall secondary structure is less than control. For further increase in concentration of GdnHCl, βturn structures starts to increase. The β-turn structure predominates at 1 M. At higher concentrations, a drastic transition is monitored where arousal of different disordered



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structures (β-turn, 3п-helix as well as other disordered structures) can be visibly observed in place of α and β structures. In case of trehalose (Figure 4b), the negative ellipticities of BM for all concentration of trehalose are either more than control or almost equal. Now, there is less disruption of secondary structure of BM by GdnHCl if there is presence of 0.5 M trehalose; however, this effect is not significantly observable at higher concentration of GdnHCl as is clear from ellipticities for negative bands (Figure 4c). From Figures 4d and 4e, it can be accentuated that a substantial amount of native like secondary structure is present on addition of GdnHCl in BM solution in presence of trehalose. In these cases, there is no doubt that there is significant amount of β-turns present, however, there is no existence of other disordered structures such as 3п -helix or poly (pro) ӀӀ helix type as is clear from Figures 4d and 4e. In our quite recent report,20 trehalose is behaving as a good stabilizer for BM. In mixture of trehalose and GdnHCl, presence of increased secondary structure in BM as compared to that in GdnHCl only is strongly emphasizing on counteracting nature of trehalose against GdnHCl-induced denaturation of BM. On comparing Figures 4d and 4e, again it can be declared that trehalose is more effective in attenuation of the denaturing action of the GdnHCl on secondary structure of BM when it is present in 1:1 ratio with GdnHCl. Combining the close examination of both λmax and ellipticities [Θ], it may be stressed here that there is undeniably change in the conformation of BM under the influence of mixtures of trehalose and GdnHCl which may be attributed to the change in the intramolecular Hbonding. FTIR Characterization of Counteraction Effect of GdnHCl-induced Changes in BM by Trehalose. The amide I band between 1600 and 1700 cm-1 is mainly due to the contribution of C=O stretching vibration of the backbone chain which is highly sensitive to the secondary structure of the enzyme. Any conformational change in the protein as a consequence of the disturbances in the secondary structures is best substantiated by monitoring the shift of amide I band.23 This band for BM in D2O is broad and located around 1653 cm-1 (black spectra in Figure 5). Besides the maxima of amide I band at 1653 cm-1, a shoulder of this band can be clearly observed near 1640 cm-1 in Figure 5. The simultaneous occurrence of these two bands suggests the existence of both α-helix and β-sheet in the native BM.24 This outcome is well supported by our other results from CD spectroscopy study and also in consistent with the report by Reyna et al.22 We have presented here only two 10 ACS Paragon Plus Environment

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concentrations for both GdnHCl and trehalose i.e. 0.5 and 2.0 M in all FTIR Figures 5a-d for sake of more simplicity in interpretation of the results. From Figure 5a, it is obvious from shifting of amide I band that the secondary structures have undergone some changes as a function of increasing concentration of GdnHCl. At very low concentration of GdnHCl, there is negligible change in the band frequency that is apparent from Figure 5a where a shift from 1653 cm-1 (BM in D2O) to 1654 cm-1 (BM in 0.5 M GdnHCl). Therefore, it can be accentuated that at lower concentration of GdnHCl, BM is almost possessing native like secondary structures. However, at higher concentration (2.0 M), there is drastic change in the BM structure as can be seen from the Figure 5a. There is red shift in band frequency from 1653 cm-1 to 1644 cm-1 which may be attributed to the increase in H-bonding of peptide backbone with solvent molecules. Furthermore, it may strongly emphasize on a considerable amount of change of the native secondary structures to the unordered structures (1644 cm-1 is the characteristic of unordered structures/random coils) which is highly exposed to the solvent. It can be stated from these results that there is denaturation of BM native structure in presence of high concentration of GdnHCl. In addition, there is simultaneously arousal of a new small peak around 1623 cm-1 which is again demonstrating the denaturation of BM at higher concentration of the GdnHCl since it highlights increased exposed β-strands at these conditions. On the other hand, FTIR spectra of BM in presence of 0.5 and 2.0 M trehalose show that BM is possessing native like characteristics at these conditions (Figure 5b). The bands for these concentrations of trehalose are positioned nearby the band at 1653 cm-1 for BM in D2O i.e. at 1654 cm-1 for 0.5 M and at 1655 cm-1 for 2.0 M trehalose. This underlines that trehalose is stabilizer of BM native state and may lead to form slightly more compact structure in comparison to BM in D2O. Furthermore, the amide I band for BM shifts to 1655 cm-1 in the presence of mixture of both trehalose and GdnHCl at ratio 1:1 as compared to that in presence of 0.5 M GdnHCl (at 1654 cm-1) and 2.0 M GdnHCl (at 1644 cm-1) which is shown in respective Figures 5c and 5d. In contrast to the band for BM in 2.0 M GdnHCl in the Figure 5d, the immense blue shift by 11 cm-1 in the amide I band on addition of 2.0 M GdnHCl to BM solution in presence of 2.0 M trehalose, illustrates the formation of more compact native structure owing to the increased secondary structures with less exposure of peptide backbone to the solvent. The 11 ACS Paragon Plus Environment


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above result strongly put emphasis on counteraction propensity of trehalose for the GdnHClinduced denaturation of BM. For 0.5 M trehalose with 0.5 M GdnHCl, there is insignificant increase in compactness of BM as compared to BM in D2O. DLS Analysis of Counteraction Effect of Trehalose on Disruption of the BM Structure. Figure 6 illustrates intensity distribution graph of BM in water and presence of varying concentration of GdnHCl and different mixtures of GdnHCl with trehalose. The water was used to prepare all samples to avoid interference caused by the buffer and each sample is maintained at pH~7. The peak for BM in water (shown black in Figure 6) is highly monodisperse with PDI 1 M). In the mixtures of tehalose and GdnHCl at 1:1 molar ratio, the percentage activity is decreased as compared to both BM in buffer as well as that in GdnHCl only except at 0.5 M : 0.5M of trehalose with GdnHCl where activity is more or less similar to that of control. On the other hand, in the case of mixtures of trehalose and GdnHCl at 1:2 ratio, BM possesses more activity in comparison to that in 1:1 ratio and GdnHCl only (except at 0.5 M : 1 M), however, less than control. It may be emphasized here that BM in crowded environment of different molar ratio of trehalose and GdnHCl is becoming more rigid in respect of its active site, therefore, activity of BM is decreased in comparison to control. This decrease in the percentage activity of BM is more prominent in case of 1:1 ratio as there may be more compact and rigid conformation of BM. In case of higher concentration of GdnHCl, there is very loose BM conformation which leads to highly decreased activity. It is quite clear from Figure 6a where dH is increased to a large extent at higher concentrations of GdnHCl. At these concentrations of GdnHCl, 1:2 ratio of trehalose with GdnHCl is somewhat effective in counteracting the adverse effect of GdnHCl on BM activity as there may be slight compacting of BM structure which may bring active site residues in close proximity to work in concerted way in comparison to loose conformation of BM in higher concentrations of GdnHCl.

DISCUSSION From the assessment of the numerous researches over the past decades, it is reasonably unambiguous that the counteracting role of the osmolytes is a key for the survival of the organisms accumulating high amount of denaturants that might result in severe biological damage. Again, our data indicates the existence of critical contribution of the counteraction ability of these protecting solutes against the redundant imbalance in protein stability caused by denaturants. From the observed blue shift in Trp fluorescence spectra (Figure 1c), increase 13 ACS Paragon Plus Environment


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in the negative ellipticity in CD spectra (Figure 4c), shift in amide I band to the higher frequency (Figures 5c and 5d), hydrodynamic radius (Figure 6d) on addition of GdnHCl to BM solution in presence of trehalose at molar ratio 1:1, it can be depicted that there is formation of more compact structure with decreased exposure of Trp and decrease in the dH of BM and increased secondary structures as compared to BM in presence of same concentrations of GdnHCl only. These inferences lead us to one way thinking i.e. trehalose is having amazing propensity to counteract the denaturing effects of GdnHCl on BM stability ensuing a more compact BM. To probe into the mechanism how trehalose does counteraction against GdnHCl, a detailed structural analysis of all components of the complex system comprising of BM, water, trehalose and GdnHCl is initially required. GdnHCl does not H-bond to the backbone of protein and may H-bond with carboxylic groups; however, this may happen at the level of random counter, thus, again ruling out Hbonding with protein.26 Its poor tendency to hydrate leads it to interact with hydrophobic surfaces primarily composed of aliphatic side chains, however, more weakly.26 It may mainly interacts through space demanding stacking interactions against peptide group through cation-ᴨ interactions with exposed aromatic residues of BM.27 Apparently, the simulations studies also point out that guanidinium ion (Gdm+ ) is planar in nature and can interact in a stacking manner with a number of planar amino-acid side chains (Arg, Trp, Gln) of BM. In addition, there are a large number of long range electrostatic interactions of Gdm+ to the charged residues of BM.28-30 Altogether there is enhanced number densities of Gdm+ at certain locations on the protein surface as a result of these interactions. Eventually, these favourable bindings give rise to decrease in the free energy of unfolded state owing to its larger solvent exposed backbone area which, in turn, shifts the equilibrium towards unfolding of BM.11,15,16,31 On the other hand, trehalose is special in respect of its physical properties. Our research group reported20 that except at very low concentrations (0.1 M), trehalose is unfavourably interacting to BM due to high hydration number, steric hindrance, topological constraints and also due to the formation of trehalose clusters resulting from increased trehalose-trehalose interactions at its higher concentrations (0.5-2.0 M). Thus, it implies that there are net repulsive interactions between trehalose and BM, indeed, comprising of a major contribution from that between trehalose and backbone of BM. Moreover, according to Hong et al.32, trehalose has a highly unfavourable interaction with aliphatic carbon of the peptide backbone of the protein. Consequently, it may be stated that trehalose remains preferentially 14 ACS Paragon Plus Environment

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excluded from protein surface at all studied concentrations attributing to increase in the free energy of the unfolded state. Accordingly, BM stay more favourably in native state leading to the formation of more compact BM with less exposed surface. A number of simulation studies are available in literature in respect of the changes in water structure in presence of these co-solvents which, in turn, may also be primary cause for their effect on protein stability. In this regard, according to Camilloni et al.,30 it may be declared that GdnHCl is found to be uniformly distributed both in bulk as well as in first hydration layer of BM without perturbing them as a consequence of geometric constraints in GdnHCl which escort far weaker GdnHCl-water interaction than water-water. On the other hand, trehalose has a higher tendency to bind both with water of hydration layer of protein and protein itself, therefore, may disturb water structure. Guo and Friedman also supported the claim that trehalose strongly influences the hydration waters and also integrate into hydrogen bonding networks favouring enhanced protein stability.33 NMR, simulation and ultrasonic studies provide a strong evidence for the manipulation of water layer around protein by trehalose.34-38 Conversely, there is no evidence about consistency of this behaviour of both cosolvents when present together. From the simulation studies by Sandip et al.,39 trehalose cluster size is highly decreased in presence of urea. This may be the case for the mixture of trehalose and GdnHCl where the presence of GdnHCl may interfere indirectly with Hbonding network of trehalose-trehalose molecules. Therefore, there may be no or less trehalose clusters near the surface of the protein at all studied concentrations and still, there is binding of trehalose to some extent to the surface of the protein which may dominant over binding of GdnHCl occurring through comparatively weaker interactions. The binding of trehalose, in turn, results in removal of water molecules from surface of BM i.e. decreased BM–solvent interactions. Thus, trehalose role is highly dominant over the GdnHCl which attributes to stabilization of BM. In addition, according to our last report on BM,20 it is found that efficacy (m-value) of trehalose to increase the free energy of unfolded state is very high as compared to that of GdnHCl to decrease in the free energy of unfolded state which is the case when both are present in BM solution in absence of each other. In present case, the counteracting ability of trehalose for GdnHCl-induced BM denaturation may be as a consequence of destabilization of unfolded state rather than stabilization of native state which is widely 15 ACS Paragon Plus Environment


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argued since last decade.16 Subsequently, even in presence of good amount of GdnHCl, the extent of this preferential exclusion is overriding on BM. The preferential exclusion of trehalose causes equilibrium to shift towards the folded state which may direct GdnHCl not to bind to the BM molecule. Hence, both trehalose and GdnHCl remain highly excluded from the BM resulting in even more stable conformation than BM in buffer. This type of observation is well supported by report of Zhang et al.40 which states mutual exclusion of trehalose and urea from surface of chymotrypsin inhibitor 2. Therefore, both binding of trehalose and preferential exclusion may play together to counteract the deleterious effect of GdnHCl on BM. The overweighed nature of the trehalose above GdnHCl may cause conformational changes accompanied with burial of the nonpolar groups of BM i.e. the movement of Trp towards hydrophobic core attributing to the observed blue shift in λmax of BM in Trp fluorescence (Figures 1b-1e) as compared to both BM in buffer as well as BM in GdnHCl only (Figure 1a). These conformational changes include an increase in secondary structure of BM as compared to that observed for BM in GdnHCl only as there is lack of GdnHCl to bind the surface of BM at 1:1 molar ratio of trehalose and GdnHCl (Figures 2d, 5c and 5d). Alternatively, stabilization of secondary structures and tertiary structure by trehalose may be escorting for the modulation of the influence of the GdnHCl. However, the mutual exclusion of both trehalose and GdnHCl may lead to BM in such a compact form with reduced flexibility especially around active site region which is essentially required for the activity of any enzyme. This may be concluded from the reduced activity of BM under the condition of 1:1 molar ratio of GdnHCl and trehalose in Figure 7. This is the most probable explanation for the reduced activity on the basis of all other observations. There may also be some other reasons behind this reduced activity of BM, for example, loss of active site water molecules required to catalyze the proteolysis which is accompanied with the compaction of BM as a result of stong preferential exclusion of co-solvents or change in pKa value of the active site residues, thereby, inhibition of concerted function of active site residues. In reality, this study provides a distinct interplay between trehalose and GdnHCl towards BM stability and activity. Therefore, it becomes quite important to relate activity to the stability of any enzyme in order to apply for further pharmaceutical applications. Accordingly, a straight forward interpretation can be put here that trehalose being an efficient compatible osmolyte, shows incredible counteracting properties against denaturation of protein structure and stability. The need to balance the beneficial and deleterious effect of 16 ACS Paragon Plus Environment

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any solute is the base of evolution for various counteracting mechanism for the survival. This feature of trehalose may serve as a general tool with such strategy to overcome the potential interferences by some solutes in protein stabilization and provide new parameters for modulating stress tolerance and protein aggregation by denaturants which is the cause of a number of protein related diseases. This emerging understanding of the mechanism of the counteraction opens broad avenues for further inquiry and promising applications. Likewise, further efforts are required to find whether this highly unexplored mechanism of counteraction of GdnHCl effects on protein by trehalose is consistent with other proteins as well. This article may provide thrust for further study by simulation methods to explore these mechanisms by quantifying the contributions of different types of interactions in presence of different proteins.

CONCLUSIONS Motivated by the need to understand the structural basis of denaturant-induced destabilization of proteins and its counteraction by the osmolytes, we have investigated counteraction ability of trehalose against guanidinium chloride (GdnHCl) denaturation of stem bromelain (BM) structure and stability. To the best of our knowledge, for the first time, the detailed study of the counteraction by trehalose put forward by us for GdnHCl-induced protein denaturation by employing the various experimental techniques i.e. fluorescence, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy and dynamic light scattering (DLS). As a result, trehalose is found to remarkably counteract GdnHCl effects on BM at 1:1 molar ratio of trehalose and GdnHCl at which BM possesses a more compact conformation with increased secondary and tertiary structures and also increased thermal stability relative to that in GdnHCl only. These compact BM acquires reduced proteolytic activity. Consequently, we anticipate the conclusions of our work to be reasonably robust. The mutual exclusion of both hydrated trehalose and poorly hydrated or bare GdnHCl is playing a key role in counteraction of adverse effect of GdnHCl on BM. Nevertheless, binding of trehalose to the BM surface by H-bonding may also contribute in this act by directing GdnHCl not to bind the BM surface. This is accompanied with the removal of water molecules from first solvation layer of the BM. Therefore, discouragement of protein-solvent interaction is also present there to support the counteraction by trehalose. The present suggested mechanism might be not distinct, however, may useful for the design of strategies for the stability study for the novel enzymes. The significance of counteraction hypothesis in 17 ACS Paragon Plus Environment


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the biology of adaptation can be further successfully applied to the unnaturally mutated proteins in vitro by the utilization of these mechanisms in order to remove the complications in pharmaceutical industries caused by numerous chronic disorders.

SUPPORTING INFORMATION (SI) Figure S1 and S2 representing non-normalized Trp fluorescence spectra at 25 oC and nonnormalized thermal transition curves of BM in the presence of various mixtures of trehalose and GdnHCl, respectively.

ACKNOWLEDGEMENTS We are gratefully acknowledged to the Council of Scientific Industrial Research (CSIR), New Delhi, through the Grant No. 01 (2713) 13/EMR-II for financial support and A. R. is grateful to University Grants Commission (UGC), New Delhi for providing SRF (Senior Research Fellowship).

AUTHOR CONTRIBUTIONS Corresponding Author e-mail: [email protected]; [email protected] Tel: +91-11-27666646-142; Fax: +91-11-2766 6605.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interest.


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(26) Lima, W. K.; Rӧsgen, J.; Englander, S. W. Urea, but not Guanidinium, Destabilizes Proteins by Forming Hydrogen Bonds to the Peptide Group. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 2595-2600. (27) Rodríguez-Sanz, A. A.; Cabaleiro-Lago, E. M.; Rodríguez-Otero, J. Interaction Between the Guanidinium Cation and Aromatic Amino Acids. Phys. Chem. Chem. Phys. 2014, 16, 22499-22512. (28) Mason, P. E.; Brady, J. W.; Neilson, G. W.; Dempsey, C. E. The Interaction of Guanidinium Ions with a Model Peptide. Biophys. J. 2007, 93, L04-L06. (29) Mason, P. E.; Neilson, G. W.; Dempsey, C. E.; Barnes, A. C.; Cruickshank, J. M. The Hydration Structure of Guanidinium and Thiocyanate Ions: Implications for Protein Stability in Aqueous Solution. Proc. Natl. Acad. Sci. USA 2003, 100, 45574561. (30) Camilloni, C.; Rocco, A. G.; Eberini, I.; Gianazza, E.; Broglia, R. A.; Tiana, G. Urea and Guanidinium Chloride Denature Protein L in Different Ways in Molecular Dynamics Simulations. Biophys. J. 2008, 94, 4654-4661. (31) Auton, M.; Bolen, D. W.; Rösgen, J. Structural Thermodynamics of Protein Preferential Solvation: Osmolyte Solvation of Proteins, Aminoacids, and Peptides. Proteins 2008, 73, 802-813. (32) Hong, J.; Gierasch L. M.; Liu, Z. Its Preferential Interactions with Biopolymers Account for Diverse Observed Effects of Trehalose. Biophys. J. 2015, 109, 144-153. (33) Guo, F.; Friedman, J. M. Osmolyte-Induced Perturbations of Hydrogen Bonding Between Hydration Layer Waters: Correlation with Protein Conformational Changes. J. Phys. Chem. B 2009, 113, 16632-16642. (34) Vilen, E. M.; Sandstrom, C. NMR Study on the Interaction of Trehalose with Lactose and Its Effect on the Hydrogen Bond Interaction in Lactose. Molecules 2013, 18, 9735-9754. (35) Lins, R. D.; Pereira, C. S.; Hünenberger, P. H. Trehalose-protein Interaction in Aqueous Solution. Proteins 2004, 55, 177-186. (36) Jain, N. K.; Roy, I. Effect of Trehalose on Protein Structure. Protein Sci. 2009, 18, 24-36. (37) Sonoda, M. T.; Skaf, M. S. Carbohydrate Clustering in Aqueous Solutions and the Dynamics of Confined Water. J. Phys. Chem. B 2007, 111, 11948-11956. (38) Lerbret, A.; Bordat, P.; Affouard, F.; Hédoux, A.; Guinet Y.; Descamps, M. How do Trehalose, Maltose, and Sucrose Influence Some Structural and Dynamical Properties 21 ACS Paragon Plus Environment


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FIGURE CAPTIONS Figure 1. Trp fluorescence spectra of BM 25 0C: (a) in presence of varying concentrations of GdnHCl; 0.5 (red), 1.0 (green), 1.5 (blue), 2.0 (cyan), 3.0 (pink) and 4.0 M (yellow), (b) in presence of mixtures of 0.5 M trehalose with varying concentrations of GdnHCl; 0.5 (red), 1.0 (green), 1.5 (blue), 2.0 (cyan), 3.0 (pink) and 4.0 M (yellow), (c) in presence of the mixtures of trehalose and GdnHCl at molar ratio of 1:1 i.e., 0.5 M : 0.5 M (red), 1.0 M : 1.0 M (green), 1.5 M : 1.5 M (blue) and 2.0 M : 2.0 M (cyan), (d) in presence of the mixtures of trehalose and GdnHCl at molar ratio of 1:2 i.e., 0.5 M : 1.0 M (red), 1.0 M : 2.0 M (green), 1.5 M : 3.0 M (blue) and 2.0 M : 4.0 M (cyan), (e) and (f) in presence of 2 M GdnHCl (red) and mixtures of varying concentrations of trehalose with 2 M GdnHCl; 0.5 (green), 1.0 (blue) and 2.0 M (cyan) possessing molar ratio 1:4, 1:2 and 1:1 of trehalose : GdnHCl, respectively. The black spectra show BM in Buffer in all figures and Figures 1a-e are normalized Trp fluorescence spectra. Figure 1a is taken from ref. [19]. The inset figures are showing wavelength shift in the respective figures. Non-normalized fluorescence spectra are shown in Figure S1. Figure 2. Thermal transition curve of BM: (a) in presence of varying concentrations of GdnHCl; 0.5 (red), 1.0 (green), 1.5 (blue), 2.0 (cyan), 3.0 (pink) and 4.0 M (yellow), (b) in presence of varying concentrations of trehalose; 0.5 (red), 1.0 (green), 1.5 (blue) and 2.0 M (cyan), (c) in presence of mixtures of trehalose and GdnHCl at 1:1 molar ratio; 0.5 M : 0.5 M (red), 1.0 M : 1.0 M (green), 1.5 M : 1.5 M (blue) and 2.0 : 2.0 M (cyan) and (d) in presence of 2 M GdnHCl (red), 2 M trehalose (green) and mixture at 1:1 ratio of 2 M trehalose with 2 M GdnHCl (blue). All Tm values were calculated by following Ref. [20]. Non-normalized thermal transition curves are shown in Figure S2. BM in Buffer (control) is shown as black curve. Figure 3. Variation of Tm values as a function of different concentrations of GdnHCl (Red), different concentrations of trehalose (green) and mixtures of trehalose and GdnHCl at molar ratio 1:1 (blue) and 1:2 (cyan). BM in Buffer (control) is shown as black line. Tm values for BM in varying concentrations of GdnHCl and trehalose are taken from Ref. [19]. Figure 4. Far-UV CD analysis of BM at 25 0C: (a) in presence of varying concentrations of GdnHCl; 0.5 (red), 1.0 (green), 1.5 (blue), 2.0 (cyan), 3.0 (pink) and 4.0 M (yellow), (b) in presence of varying concentrations of trehalose; 0.5 (red), 1.0 (green), 1.5 (blue) and 2.0 M



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(cyan), (c) in presence of mixtures of 0.5 M trehalose with varying concentrations of GdnHCl; 0.5 (red), 1.0 (green), 1.5 (blue), 2.0 (cyan), 3.0 (pink) and 4.0 M (yellow), (d) in presence of the mixtures of trehalose and GdnHCl at molar ratio of 1:1; 0.5 M : 0.5 M (red), 1.0 M : 1.0 M (green), 1.5 M : 1.5 M (blue) and 2.0 M : 2.0 M (cyan) and (e) in presence of the mixtures of trehalose and GdnHCl at molar ratio of 1:2; 0.5 M : 1.0 M (red), 1.0 M : 2.0 M (green), 1.5 M : 3.0 M (blue) and 2.0 M : 4.0 M (cyan). BM in Buffer is shown as black spectra in all figures. Figure 4a is taken from ref. [19]. Figure 5. FTIR spectra of BM at 25 0C: (a) BM in presence of 0.5 (green) and 2.0 M (pink) GdnHCl, (b) BM in presence of 0.5 (green) and 2.0 M (pink) trehalose, (c) in presence of 0.5 M GdnHCl and mixtures of trehalose with GdnHCl at molar ratio 1:1 containing 0.5 M trehalose and 0.5 M GdnHCl and (d) in presence of 2.0 M GdnHCl and mixtures of trehalose with GdnHCl at molar ratio 1:1 containing 2.0 M trehalose and 2.0 M GdnHCl. BM in D2O is shown black in all figures. Figure 6. The hydrodynamic diameter (dH) of BM at 25 0C: (a) BM in presence of varying concentrations of GdnHCl; 0.5 (red), 1.0 (green), 1.5 (blue), 2.0 (cyan), 3.0 (pink) and 4.0 M (yellow), (b) BM in presence of varying concentrations of trehalose; 0.5 (red), 1.0 (green), 1.5 (blue) and 2.0 M (cyan), (c) in presence of mixtures of 0.5 M trehalose with varying concentrations of GdnHCl; 0.5 (red), 1.0 (green), 1.5 (blue), 2.0 (cyan), 3.0 (pink) and 4.0 M (yellow) and (d) in presence of the mixtures at molar ratio of 1:1 of trehalose and GdnHCl; 0.5 M : 0.5 M (red), 1.0 M : 1.0 M (green), 1.5 M : 1.5 M (blue) and 2.0 M : 2.0 M (cyan). BM in Buffer (control) is shown as black peak. Figure 7. Variation of percentage activity of BM as a function of different concentrations of GdnHCl (Red), different concentrations of trehalose (green) and mixtures of trehalose and GdnHCl at molar ratio 1:1 (blue) and 1:2 (cyan). BM in Buffer (control) is shown as black line. Tm values for BM in varying concentrations of GdnHCl and trehalose are taken from Ref. [19]. Table 1. The hydrodynamic diameters (dH) of BM in water, in presence of varying concentrations of GdnHCl, varying concentrations of trehalose and mixtures of trehalose with GdnHCl at different concentrations.


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1.1

1.1

1.0

1.0

0.9

0.9

0.8

0.8

0.7 320 330 340 350 360 370

380 320

1.000

1.1

330

1.1

340 350 360 370 380 1.000 0.998

0.995

1.0

1.0 0.990

340

345

0.996

340

350

345

350

0.9

0.9

0.8

0.8

0.7 0.7 320 330

340

350

360

370

380 320 330 340 350 360 370 380 110

1.1

1.000

0.996

1.0

100

0.992 340

345

350

355

90

0.9 0.8

80

0.7

70

320 330 340 350 360 370 380

320 330 340 350 360 370 380



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1.0

1.0

0.8

0.8

0.6

Page 26 of 33

0.6

0.4 0.4

0.2 0.2

30 40 50 60 70 80 90 1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2 30 40 50 60 70 80 90

30

40

30

40

50

50

60

60

70

70

80

80

90

90


Page 27 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


68 64 60 56 0

1

2

3

4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 33

0 75

-2

60

-4

45

-6

30

-8

15

-10

0

-12

-15 200

6 4 2 0 -2 -4 -6 -8 -10 -12 200

210

220

230

-14 200

240

210

220

230

240

210

220

230

240

0 -2 -4 -6 -8 -10 210

220

230

-12 200

240

2 0 -2 -4 -6 -8 -10 -12 200

210

220

230

240


Page 29 of 33


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

5

6 5

4

4

3

3

2

2 1

1 2

4

6

8

10

12

14

2

16

5

5

4

4

3

3

2

2

1

1

2

4

6

8

10

12

14

16

2

3

3

4

4

5

5

6

6

7

7


8

Page 31 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


140 120 100 80 60 0

1

2

3

4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 33

Table 1. The hydrodynamic diameters (dH) of BM in water, in presence of varying concentrations of GdnHCl, varying concentrations of trehalose and mixtures of trehalose with GdnHCl at different concentrations.

Various conc.

dH (nm) of

Various conc.

dH (nm) of

Various conc.

dH (nm) of

Various conc. of

dH (nm)

GdnHCl [M] +

GdnHCl

trehalose

GdnHCl

Various conc. of

[M]

[M]

[M] + 0.5 M

trehalose

trehalose

(molar ratio 1:1)

[M]

0

4.6±0.2

0

4.6±0.2

0

4.6 ±0.2

0

4.6±0.2

0.5

4.8±0.2

0.5

4.6±0.3

0.5

4.2±0.1

0.5+0.5

4.2±0.1

1.0

5.6±0.3

1.0

4.2±0.3

1.0

5.1±0.3

1.0+1.0

4.7±0.2

1.5

6.45±0.5

1.5

4.6±0.4

1.5

5.6±0.2

1.5+1.5

4.7±0.2

2.0

6.75±0.5

2.0

4.4±0.3

2.0

6.7±0.2

2.0+2.0

5.0±0.3

3.0

9.8±0.4

3.0

8.5±0.2

4.0

10.7±0.5

4.0

10.2±0.3


Page 33 of 33

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TOC GRAPHIC


A Distinct Proof on Interplay between Trehalose and Guanidinium

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