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Utilization of light scattering techniques in study on protein denaturation
Köbölová, Klaudia ; Kalina, Michal (referee) ; Sedláček, Petr (advisor)
This bachelor thesis is focused on verifying the use of light scattering techniques in the study of protein denaturation. The theoretical part of the thesis describes proteins, their general properties, the process of denaturation and it provides an insight into light scattering. In the experimental part of the study I investigated the denaturation effects of four selected potential denaturing agents – urea, guanidine hydrochloride, sodium dodecyl sulfate, copper (II) sulfate – on two model enzymes: lipase and lysosyme. The denaturation effects of these denaturants were studied with the help of the dynamic light scattering method (DLS). In order to describe the change in particle size during denaturation, I used Z-Average, Volume mean and intensity distribution which were determined by this method.
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Preparation and characterization of quatsomes
Havlíčková, Anna ; Szabová, Jana (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with preparing and characterizing quatsomes and similar vesicular systems. The preparation consisted of weighing an equimolar amount of sterol and surfactant, hydration with deionized water, and subsequent sonication with an ultrasonic probe with an energy of 5, 10 and 15 kJ. In this work, two types of quatsomes were prepared, the first contained equimolar amounts of cholesterol and cetyltrimethylammonium bromide (CTAB), and the second consisted of cholesterol carbethopendecinium bromide (Septonex). The optimal dispersion energy of 10 kJ was selected for both types based on turbidity measurements, visual observation and dynamic and electrophoretic light scattering analysis. Furthermore, the observed properties were found to be constant from day 7 after sonication. In addition to quatsomes, similar vesicles were prepared, which contained a surfactant in the form of a sulfate salt. Once again, two types were prepared, the first containing sodium dodecyl sulfate (SDS) and cholesterol, the second sodium tetradecyl sulfate (STS) and cholesterol. Based on the same measurements as for quatsomes, optimal dispersion energy of 10 kJ was selected. Furthermore, mixtures with different ratios of cholesterol and a given surfactant were prepared, in which the effect of cholesterol addition was monitored. The analysis of these mixtures was performed by the same methods as the analysis of quatsomes.
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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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Preparation and characterization of quatsomes
Havlíčková, Anna ; Szabová, Jana (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with preparing and characterizing quatsomes and similar vesicular systems. The preparation consisted of weighing an equimolar amount of sterol and surfactant, hydration with deionized water, and subsequent sonication with an ultrasonic probe with an energy of 5, 10 and 15 kJ. In this work, two types of quatsomes were prepared, the first contained equimolar amounts of cholesterol and cetyltrimethylammonium bromide (CTAB), and the second consisted of cholesterol carbethopendecinium bromide (Septonex). The optimal dispersion energy of 10 kJ was selected for both types based on turbidity measurements, visual observation and dynamic and electrophoretic light scattering analysis. Furthermore, the observed properties were found to be constant from day 7 after sonication. In addition to quatsomes, similar vesicles were prepared, which contained a surfactant in the form of a sulfate salt. Once again, two types were prepared, the first containing sodium dodecyl sulfate (SDS) and cholesterol, the second sodium tetradecyl sulfate (STS) and cholesterol. Based on the same measurements as for quatsomes, optimal dispersion energy of 10 kJ was selected. Furthermore, mixtures with different ratios of cholesterol and a given surfactant were prepared, in which the effect of cholesterol addition was monitored. The analysis of these mixtures was performed by the same methods as the analysis of quatsomes.
|
|
|
Utilization of light scattering techniques in study on protein denaturation
Köbölová, Klaudia ; Kalina, Michal (referee) ; Sedláček, Petr (advisor)
This bachelor thesis is focused on verifying the use of light scattering techniques in the study of protein denaturation. The theoretical part of the thesis describes proteins, their general properties, the process of denaturation and it provides an insight into light scattering. In the experimental part of the study I investigated the denaturation effects of four selected potential denaturing agents – urea, guanidine hydrochloride, sodium dodecyl sulfate, copper (II) sulfate – on two model enzymes: lipase and lysosyme. The denaturation effects of these denaturants were studied with the help of the dynamic light scattering method (DLS). In order to describe the change in particle size during denaturation, I used Z-Average, Volume mean and intensity distribution which were determined by this method.
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|
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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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