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The use of microcalorimetric methods in the study of the protective effects of chemical chaperons
Bohunská, Miroslava ; Pekař, Miloslav (referee) ; Krouská, Jitka (advisor)
This bachelor thesis deals with the study of protective substances against denaturation processes, called chemical chaperones. The theoretical part describes the general characteristics of proteins, description of selected chaperones and methods of differential scanning calorimetry. In the experimental part, the protective effects of four potential protective agents - trehalose, guanidine hydrochloride, 3-hydroxybutyrate and hydroxyectoine - were investigated on the lysozyme model protein. The protective effects of the individual substances were examined by differential scanning calorimetry (DSC), which determined the denaturation temperature of lysozyme in the presence of preservatives. Of all the chemical chaperones examined, the highest protective effect was observed with 3-hydroxybutyrate, which shifted the denaturation temperature to higher levels, and guanidine hydrochloride, which on the other hand lowered the denaturation temperature. At the same time, a reversible denaturation process was found in some substances, which was the most intense in GuHCl.
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Study on protective effect of 3-hydroxybutyrate and its derivatives
Černá, Klára ; Smilek, Jiří (referee) ; Obruča, Stanislav (advisor)
The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.
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The use of microcalorimetric methods in the study of the protective effects of chemical chaperons
Bohunská, Miroslava ; Pekař, Miloslav (referee) ; Krouská, Jitka (advisor)
This bachelor thesis deals with the study of protective substances against denaturation processes, called chemical chaperones. The theoretical part describes the general characteristics of proteins, description of selected chaperones and methods of differential scanning calorimetry. In the experimental part, the protective effects of four potential protective agents - trehalose, guanidine hydrochloride, 3-hydroxybutyrate and hydroxyectoine - were investigated on the lysozyme model protein. The protective effects of the individual substances were examined by differential scanning calorimetry (DSC), which determined the denaturation temperature of lysozyme in the presence of preservatives. Of all the chemical chaperones examined, the highest protective effect was observed with 3-hydroxybutyrate, which shifted the denaturation temperature to higher levels, and guanidine hydrochloride, which on the other hand lowered the denaturation temperature. At the same time, a reversible denaturation process was found in some substances, which was the most intense in GuHCl.
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Study on protective effect of 3-hydroxybutyrate and its derivatives
Černá, Klára ; Smilek, Jiří (referee) ; Obruča, Stanislav (advisor)
The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.
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