Protein environment and structure in neat and hydrated deep eutectic solvents
Sammanfattning: The use of deep eutectic solvents (DESs) has a great potential in the pharmaceutical field due to its many advantages such as biocompatibility, low toxicity and biodegradability. Studies have also shown that proteins can be successfully incorporated into DESs. However, protein stability and structure in these solvents need to be investigated for this technology to be applicable. When adding the protein into the deep eutectic solvent, the sought outcome is not to disrupt the native protein conformation, but to stabilize the protein in the solvent. A factor that makes this difficult is that proteins can change their structure when the local environment of the amino acid residues of the protein is altered. In this thesis, the two proteins bovine serum albumin and lysozyme were studied in three different DESs; choline chloride:glycerol (ChCl:Gly), choline chloride:urea (ChCl:U) and choline acetate:glycerol (ChAc:Gly), all in the molar ratio 1:2. In order to evaluate protein concentration, monitor changes in the secondary structure and how the environment affects the protein, UV-Vis spectrometry, FT-IR and fluorescence spectroscopy have been utilized. Hydrated ChCl:Gly with incorporated proteins has also been studied to understand how the proteins respond to the addition of water in the DES. Finally, proteins in ChCl:Gly were back-extracted into phosphate buffer to see how the proteins behave after being exposed to DES and then transferred back into a neat aqueous environment. From the UV-Vis measurements, the obtained data indicate that the conformation of BSA and lysozyme seems to be best retained in ChCl:Gly. The FT-IR measurements show that the secondary structure is altered for the proteins when they are in DESs. Moreover, the data from the fluorescence measurements suggests that the native conformation of BSA and lysozyme, the environment or the interactions with the environment most likely is altered when the proteins are introduced to the different DESs. Furthermore, when adding water to ChCl:Gly, the protein conformations are better retained compared to when the proteins are in pure ChCl:Gly. After extraction of proteins from ChCl:Gly to phosphate buffer, it is observed that the proteins regain most of their native conformation. Consequently, deep eutectic solvents might be applicable in drug formulation. However, more studies have to be performed (preferably in combination with other techniques enabling further conformational studies) to obtain complementing information.
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