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Non-covalent interactions of tryptophan in protein structure
Sokol, Albert ; Fišer, Radovan (advisor) ; Jurkiewicz, Piotr (referee)
A thorough knowledge of non-covalent amino acid interactions within a protein structure is essential for a complete understanding of its conformation, stability and function. Among all the amino acids that usually make up a protein, tryptophan is distinguished both by its rarity and size of its side chain formed by an indole group. It is able to provide various types of indispensable interactions within the protein and between different polypeptide chains, but also between the protein and a biological membrane. In addition, it is the most commonly used natural fluorophore. Databases of solved protein structures are commonly used to study amino acid interactions and allow more or less complex analyzes of the issue. Thus many non-covalent interactions that may occur between tryptophan and other amino acids have been found. However, most of these analyzes focus on specific interactions and do not follow up the tryptophan's environment as a whole, where all amino acids interact. Some newly developed methods have been used in this Thesis, specifically the occurrence profiles of the individual amino acids around the indole group of tryptophan and the results were compared with an available literature. The amino acid that has the greatest preference for tryptophan turned out to be tryptophan again, and...
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Determination of tryptophan, serotonine and melatonin in plants by using HPLC
Pavlů, Věra ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
This thesis deals with the development and optimization of a method for the determination of tryptophan and its metabolites - serotonin and melatonin - in plant material, in grapevine, during one analysis. It uses a high-pressure liquid chromatography. The theoretical part is about tryptophan, its metabolism and basic properties of its metabolites - serotonin and melatonin. Their occurrence in wine is also discussed. Analytical techniques by which these analytes can be determined are also provided. Then information about modern stationary phases, that are suitable for this species, is included. The experimental part consists of optimization of the method, measurement of calibration dependences and measurement of real samples. It is measured by the method of reverse phase chromatography. As first stationary phase it is used a C18 column with core-shell packing, second is a BEH Phenyl column. The mixture of 10 mM acetate buffer (pH = 4.5) and methanol is used as the mobile phase. For detection UV at wavelength 254 nm is used, then for greater sensitivity mass detectionis is used. The basic conditions for the experiment have been set. At the beginning of the analysis, the mobile phase contains 95 % (v/v) buffer and 5 % (v/v) methanol. Then the methanol content is linearly increased to 80 % (v/v) from...
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Non-covalent interactions of tryptophan in protein structure
Sokol, Albert ; Fišer, Radovan (advisor) ; Jurkiewicz, Piotr (referee)
A thorough knowledge of non-covalent amino acid interactions within a protein structure is essential for a complete understanding of its conformation, stability and function. Among all the amino acids that usually make up a protein, tryptophan is distinguished both by its rarity and size of its side chain formed by an indole group. It is able to provide various types of indispensable interactions within the protein and between different polypeptide chains, but also between the protein and a biological membrane. In addition, it is the most commonly used natural fluorophore. Databases of solved protein structures are commonly used to study amino acid interactions and allow more or less complex analyzes of the issue. Thus many non-covalent interactions that may occur between tryptophan and other amino acids have been found. However, most of these analyzes focus on specific interactions and do not follow up the tryptophan's environment as a whole, where all amino acids interact. Some newly developed methods have been used in this Thesis, specifically the occurrence profiles of the individual amino acids around the indole group of tryptophan and the results were compared with an available literature. The amino acid that has the greatest preference for tryptophan turned out to be tryptophan again, and...
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Determination of tryptamine and tryptophan in biological material by capillary electrophoresis
Šimonová, Alice ; Křížek, Tomáš (advisor) ; Taraba, Lukáš (referee)
The aim of this bachelor thesis was the development of the method for the determination of tryptamine and tryptophan in biological material by capillary electrophoresis. Total length of silica capillary with inner diameter 75 m was 50.0 cm and effective length was 8.5 cm, injection of the sample was on the short end of the capillary. The composition of background electrolyte, injection method and on-line preconcentration techniques were optimized. Background electrolyte was acetic acid of 1,6 mol/l concentration, sample was injected hydrodynamically with pressure of 5 kPa for 5 s and driving voltage was -30 kV. The analytes were detected at wavelength of 220 nm and the inner standard aniline was detected at wavelength of 200 nm. Time of separation was only 2 minutes, which is the main advantage of this method. The limits of detection were 0.002 mmol/l for tryptamine and 0.001 mmol/l for tryptophan, the limits of quantification were 0.006 mmol/l for tryptamine and 0.005 mmol/l for tryptophan. Repeatability of peak areas and migration times of standards of 0,045 mmol/l concentration related to inner standard showed values of relative standard deviation lower than 5 %. The use of optimized method was tested on Nicotiana tabacum leaves and on cultivating medium of oomycete Pythium oligandrum. Key...
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Study of associated anisotropy decay curves of tryptophan fluorescence of proteins
Pultarová, Martina ; Večeř, Jaroslav (advisor) ; Heřman, Petr (referee)
This work focuses on simulation of anisotropy decay curves of a model system where protein P1 with one tryptophan interacts with regulating protein P2 without tryptophan to form a complex P1P2 with simultaneous change of tryptophan fluorescence lifetime of the complex. Under these conditions, the heterogeneous solution of proteins P1 and P1P2 exhibits non- exponential fluorescence anisotropy decay which is deduced also from equations presented in this work. Such curves analytically simulated with and without Poisson noise are the main results of this work. The heterogeneous curves were analyzed using computer programs developed earlier for homogeneous systems. As expected such analysis yields correct values of fluorescence lifetimes but does not recover anisotropy parameters used in simulations. Despite of high intensities of simulated data, anisotropy decay curves of homogeneous solutions are very noisy and can be used only on a relatively short time interval of their decay.
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Chiral separation of tryptophan and its derivatives by HPLC
Voborná, Markéta ; Kalíková, Květa (advisor) ; Vozka, Jiří (referee)
This bachelor thesis is focused on study of retention and enantioseparation of tryptophan and its derivatives by high performance liquid chromatography. The set of chiral analytes contained D,L-tryptophan, 5-hydroxy-D,L-tryptophan, 5-fluoro- D,L-tryptophan, D,L-tryptophanol, D,L-tryptophan methyl ester, D,L-tryptophan butyl ester, D,L-tryptophan octyl ester, D,L-tryptophan benzyl ester and N-BOC-D,L-tryptophan. Four different chiral stationary phases were used, i.e. cyclofructan-based (Larihc CF6-P column), two macrocyclic antibiotics (Chirobiotic V and T columns) and amylose-based (Chiralpak AD-RH column). Reversed phase and polar organic separation modes were used. Enantiomers of 5-fluoro-D,L-tryptophan, 5-hydroxy-D,L-tryptophan and D,L-tryptophan were successfully separated on teicoplanin chiral stationary phase in polar organic mode. The amylose-based chiral stationary phase proved to be the most suitable for enantioseparation of esters of tryptophan. Three of them (D,L-tryptophan methyl ester, D,L-tryptophan butyl ester and D,L-tryptophan benzyl ester) were partially enantioseparated in reversed phase mode. Keywords: HPLC, tryptophan, enantioseparation
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