Proteins transition temperature lowered

The presence of a solvent, especially water, and/or other additives or impurities, often in nonstoichiometric proportions, may modify the physical properties of a solid, often through impurity defects, through changes in crystal habit (shape) or by lowering the glass transition temperature of an amorphous solid. The effects of water on the solid-state stability of proteins and peptides and the removal of water by lyophilization to produce materials of certain crystallinity are of great practical importance although still imperfectly understood. 

As the temperature is lowered further, the viscosity of the unfrozen solution increases dramatically until molecular mobility effectively ceases. This unfrozen solution will contain the protein, as well as some excipients, and (at most) 50 per cent water. As molecular mobility has effectively stopped, chemical reactivity also all but ceases. The consistency of this solution is that of glass, and the temperature at which this is attained is called the glass transition temperature Tg-. For most protein solutions, Tg- values reside between -40 °C and -60 °C. The primary aim of the initial stages of the freeze-drying process is to decrease the product temperature below that of its Tg- value and as quickly as possible in order to minimize the potential negative effects described above. 

To test the hypothesis that the conformational flexibility of the thermophilic enzyme is lower at room temperature than at higher temperatures, Kohen and Klinman measured, by FTIR, the time course of H/D exchange of protein N-H sites in deuterium oxide for the thermophilic alcohol dehydrogenase. Their measurements were made at the optimal host-organism temperature of 65 °C and at 25 °C, below the transition temperature. They also included yeast alcohol dehydrogenase at 25 °C, which is the optimal temperature for its own host organism. 

In a more recent study, Bello 857) has pointed out that this does not necessarily indicate denaturation in this solvent, and, in fact, that high degrees of order in both glycol and glycerol are maintained at least up to 90% polyol. The transition temperature is lowered so that in pure glycol it is near 25°. Under these conditions the protein can be expected to be very sensitive to other parameters such as the concentration of another solvent component. The acid transition 80S) is affected slightly or not at all by aqueous ethylene glycol concentrations up to 4 M, in marked contrast to dioxane, for example. 

Endothermic peaks were observed between 70 and 90°C, but they were not evident after re-scans. This result could be a result of protein denaturation. The area of the endothermic transitions (related AH of protein denaturation) increased with water content. The temperature of protein denaturation diminished at RH 98%, which was coincident with a high amount of freezable water, as shown in Figure 43.1. At low RH values, the temperatures of the endothermic transitions were similar for the different genotypes. Above 88% RH, the protein denaturation temperature for Ollagiie was lower than those for the other varieties. 

In accordance with the reversibility of the colloidal properties of thermally sensitive particles, the adsorption of proteins is also found to be reversible in the same cases. In fact, 90% of adsorbed protein can be desorbed just by lowering the temperature (i.e., from above to below the volume phase transition temperature). The hydration processes of the particles lead to a reduction in adsorption afhnity, which favors the desorption process (Figure 12.25). Furthermore, the desorbed... 

Helmkamp (1980a) studied the effect of the fatty acid composition of the acceptor lipid on the stimulation of phosphatidylinositol transfer from rat liver microsomes to phosphatidylcholine vesicles by bovine brain exchange protein. Acceptor vesicles containing egg phosphatidylcholine or dioleoyl phosphatidylcholine gave approximately the same transfer activity, whereas dielaidoyl phosphatidylcholine or dimyristoyl phosphatidylcholine vesicles produced lower transfer rates. Zborowski and Demel (1982) used the same protein and measured the rate of transfer of phosphatidylinositol from a monolayer to phosphatidylcholine vesicles. Vesicles of egg, dioleoyl, dielaidoyl, and dipalmitoyl phosphatidylcholine, even below its phase transition temperature, all gave equivalent transfer rates. However, a reduced rate was found when dimyristoyl and dilin-oleoyl phosphatidylcholine, and other phosphatidylcholines with two polyunsaturated fatty acids, were used. Table IV shows a comparison of the transfer activities measured in the two assays. The transfer rates are expressed as a percent of the transfer rate obtained with egg phosphatidylcholine acceptor vesicles. 

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