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Characterization and stabilization of pancreatin
Wurstová, Agáta ; Němcová, Andrea (referee) ; Obruča, Stanislav (advisor)
This work focuses on a study of enzyme mixture pancreatin, its characterization and subsequent encapsulation into liposomes. As a reference proteins bovine serum albumin and trypsin were used. Characterization of pancreatin consisted of two parts. The first part focuses on optimization of methods for the concentration determination by absorption spectrophotometry using basic methods for identifying proteins (Biuret method, Hartree-Lowry method and Bradford method). Moreover, UV spectrums of the protein were measured. As a method for identification of protein´s molecular weight, SDS-PAGE was used. To identify components of pancreatin, LPLC was employed in two modifications, ion-exchange chromatography and size exclusion chromatography. The second part is dedicated to the characterization of pancreatin as enzyme in terms of pH and temperature optimum for the enzyme activities of protease (pH 9, 8 and 50 °C), amylase (pH 7 and 40 °C) and lipase (pH 7 and 50 °C). The last part of this work aimed at an encapsulation of pancreatin into liposomes and DLS analysis of distribution of particles and their zeta potential. Liposomes did not spontaneously release encapsulated enzyme. To confirm that proteins were successfully entrapped into liposomes, their structure was disrupted by application of phospholipase D. In conclusion, liposomes can be utilized as delivery systems for native enzymes.
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Synthesis of core/shell quantum dots for diagnostic
Mihajlovic, A.
In this paper, synthesis of colloidal core and core/shell quantum dots (QDs) was described. First, CdTe QDs capped with glutathione, thioglycolic or mercaptopropionic acid were prepared in aqueous phase, and used for synthesis of colloidal core/shell CdTe/ZnS QDs. Core/shell QDs were used for conjugation with bovine serum albumin (BSA) or immunoglobulin G (IgG) via different crosslinkers (CDI, EDC/NHS, EDC). QDs as well as QDs-protein/antibody conjugates were characterized via UV-Vis spectroscopy and capillary electrophoresis (CE). Based on UV-Vis spectroscopy results it was found that, with increasing concentration of BSA, fluorescence intensity of QDs decreased. CE confirmed formation of QDs-BSA and QDs-IgG conjugates.
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