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Application of acid proteases from nepenthes in hydrogen/deuterium exchange.
Darebná, Petra ; Man, Petr (advisor) ; Stráňava, Martin (referee)
Application of acid proteases from Nepenthes in hydrogen/deuterium exchange Petra Darebná (Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic) Nepenthes are mostly found in Borneo and Sumatra. They are one of a few carnivorous plants which produce its own proteolytic enzymes (nepenthesin I and nepenthesin II), which provide an alternative source of nitrogen and other nutrients in case that these plants grow in a soil which lacks such nutrients. These aspartate proteases are capable of proteolysis at a very low pH of a digestive gastrovascular cavity fluid after catching insects and in a cooperation with other proteins participating in digestion process. Processing of a digestive fluid, isolated from a digestive gastrovascular cavities of carnivorous plants of the Nepenthes genus and its possible application as a tool in a protein study using hydrogen/deuterium exchange were done in this thesis. Isolates of the digestive fluids were purified from coarse-grained impurities by centrifugation, activated by acidification and concentrated by ultrafiltration. The amount of proteins and their protein profile were monitored and an activity of acidic proteases was determined by enzymatic assay. Consequently, using LC-MS/MS and model proteins, the cleavage preferences...
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Preparation and use of acid proteases for digestion in H/D exchange.
Kukla, Jan ; Man, Petr (advisor) ; Pompach, Petr (referee)
- 5 - Abstract Hydrogen/deuterium exchange coupled to mass spectrometry (HX-MS) utilizes the spontaneous exchange of protein backbone amide hydrogens for deuterium atoms from solution to gain information about changes in protein structure. To localize these changes to specific areas of the protein, enzymatic digestion by aspartate proteases is used. The proteases' ability to produce small overlapping peptides and to provide full sequence coverage of the studied protein is essential for pinpointing the protein regions of interest. In this study recombinant proteases nepenthesin I (Nepenthes gracilis) and rhizopuspepsin (Rhizopus chinensis) were prepared and compared to commercially available proteases porcine pepsin A and aspergillopepsin (Aspergillus saitoi). The comparison was performed using various activity assays, where the effects of pH, temperature and denaturing and reducing agents on the activity of the proteases were studied. All four proteases were also immobilized on a polymeric resin POROS and their activity in an online HX-MS digestion setup was tested using myoglobin as a model substrate.
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Preparation and use of acid proteases for digestion in H/D exchange.
Kukla, Jan ; Man, Petr (advisor) ; Pompach, Petr (referee)
- 5 - Abstract Hydrogen/deuterium exchange coupled to mass spectrometry (HX-MS) utilizes the spontaneous exchange of protein backbone amide hydrogens for deuterium atoms from solution to gain information about changes in protein structure. To localize these changes to specific areas of the protein, enzymatic digestion by aspartate proteases is used. The proteases' ability to produce small overlapping peptides and to provide full sequence coverage of the studied protein is essential for pinpointing the protein regions of interest. In this study recombinant proteases nepenthesin I (Nepenthes gracilis) and rhizopuspepsin (Rhizopus chinensis) were prepared and compared to commercially available proteases porcine pepsin A and aspergillopepsin (Aspergillus saitoi). The comparison was performed using various activity assays, where the effects of pH, temperature and denaturing and reducing agents on the activity of the proteases were studied. All four proteases were also immobilized on a polymeric resin POROS and their activity in an online HX-MS digestion setup was tested using myoglobin as a model substrate.
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Application of acid proteases from nepenthes in hydrogen/deuterium exchange.
Darebná, Petra ; Man, Petr (advisor) ; Stráňava, Martin (referee)
Application of acid proteases from Nepenthes in hydrogen/deuterium exchange Petra Darebná (Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic) Nepenthes are mostly found in Borneo and Sumatra. They are one of a few carnivorous plants which produce its own proteolytic enzymes (nepenthesin I and nepenthesin II), which provide an alternative source of nitrogen and other nutrients in case that these plants grow in a soil which lacks such nutrients. These aspartate proteases are capable of proteolysis at a very low pH of a digestive gastrovascular cavity fluid after catching insects and in a cooperation with other proteins participating in digestion process. Processing of a digestive fluid, isolated from a digestive gastrovascular cavities of carnivorous plants of the Nepenthes genus and its possible application as a tool in a protein study using hydrogen/deuterium exchange were done in this thesis. Isolates of the digestive fluids were purified from coarse-grained impurities by centrifugation, activated by acidification and concentrated by ultrafiltration. The amount of proteins and their protein profile were monitored and an activity of acidic proteases was determined by enzymatic assay. Consequently, using LC-MS/MS and model proteins, the cleavage preferences...
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