Comparison of Sucrose and Trehalose for Protein Stabilization Using Differential Scanning Calorimetry

The disaccharide trehalose is generally acknowledged as a superior stabilizer of proteins and other biomolecules in aqueous environments. Despite many theories aiming to explain this, the stabilization mechanism is still far from being fully understood. This study compares the stabilizing properties of trehalose with those of the structurally similar disaccharide sucrose. The stability has been evaluated for the two proteins, lysozyme and myoglobin, at both low and high temperatures by determining the glass transition temperature, Tg, and the denaturation temperature, Tden. The results show that the sucrose-containing samples exhibit higher Tden than the corresponding trehalose-containing samples, particularly at low water contents. The better stabilizing effect of sucrose at high temperatures may be explained by the fact that sucrose, to a greater extent, binds directly to the protein surface compared to trehalose. Both sugars show Tden elevation with an increasing sugar-to-protein ratio, which allows for a more complete sugar shell around the protein molecules. Finally, no synergistic effects were found by combining trehalose and sucrose. Conclusively, the exact mechanism of protein stabilization may vary with the temperature, as influenced by temperature-dependent interactions between the protein, sugar, and water. This variability can make trehalose to a superior stabilizer under some conditions and sucrose under others.

Figures S1 and S2 present representative DSC curves for the three distinct categories.The left panel illustrates the cycles of systems that experience crystallization upon cooling, attributed to the high water content.The central panel depicts the cycles of systems that do not crystallize during cooling but undergo cold crystallization during the heating cycle.
Finally, the right panel displays the systems that exhibit no crystallization events.Tables S1-S7 present a summary of all experimentally obtained data including the wt% of each component in the samples.The change in heat capacity (∆Cp) during the glass transition as well as the onset (T onset g ), inflection (T infl g ) and end (T end g ) points of the glass transition are presented.In addition, the denaturation temperatures (T den ) are listed for the samples that contain protein.Whether the sample undergo crystallization, cold crystallization, or both is also stated.For these samples the onset (T on m ) and melting temperature (T m ) (i.e. the position of the endothermic dip) of the melting process, as well as the melting enthalpy (∆H m ) were recorded, from which the wt% amorphous water in the system (Am.H 2 O) was calculated.

Additional plots glass transition temperature
To perform an extensive analysis to compare the stabilizing effect of sucrose and trehalose, both at lower as well as higher temperatures, the data was plotted both as a function of the protein and sugar content, respectively.Figures S5 and S6 display the glass transition temperature, T g , as a function of the protein content (wt%) for the sucrose and trehalose containing samples, respectively.The black diamonds, in Figure S5, correspond to a sugar:protein weight ratio of 1:3, where the T g is seen to decrease with increasing water content.The blue squares in Figures S5 and S6 represent the samples with a sugar:protein weight ratio of 1:1, which show a T g slightly higher for the trehalose-containing samples.However, it should be mentioned that the homogeneity of the trehalose samples was more difficult to ensure due to a slightly more limited solubility.The red circles correspond to a sugar:protein weight ratio of 3:1.Comparing T g of the two disaccharide systems it can be concluded that there is little to no difference between the two systems.
Figures S7 and S8 present the T g as a function of the sugar content (wt%).For the sugar:protein weight ratios 3:1 and 1:0 (and also 1:1 in the case of sucrose) clear "V-shaped" curves are visible for both disaccharides due to that the left branch of the "V" corresponds to samples that crystallized during cooling, which explains the abrupt increase of T g .
Figure S7: The T g as a function of the sugar content (wt%).The black diamonds, blue squares, red circles, and green triangles represent sucrose:lysozyme weight ratios 1:3, 1:1, 3:1, and 1:0, respectively.In Figures S11 and S12 the T den is displayed as a function of the sugar content (wt%).
Here it is obvious that the T den increases with increasing sugar content and also with increasing sugar:protein ratio.This is reasonable since more sugar molecules per protein molecule can assist in maintaining preferential hydration of the protein.

Figure S1 :
Figure S1: Representative DSC cycles for sucrose containing systems at different sucrose:lysozyme ratios as well as different levels of hydration.

Figure S2 :
Figure S2: Representative DSC cycles for trehalose containing systems at different trehalose:lysozyme ratios as well as different levels of hydration.

Figure S3 :
Figure S3: Demonstration of how a typical glass transition was analysed.The insets show the glass transition onset, inflection, and endpoint.

Figure S4 :
Figure S4: Demonstration of how a typical melting process was analysed.The insets point out the onset and melting temperatures, as well as the enthalpy of the melting process.

Figure S10 :
Figure S10: The T den as a function of the protein content (wt%).The blue squares, and red circles represent trehalose:lysozyme weight ratios 1:1, and 3:1, respectively.

Figure
Figure S12: T den as a function of the sugar wt%.The blue squares, and red circles represent trehalose:lysozyme weight ratios 1:1, and 3:1, respectively.