期刊
ANALYTICAL CHEMISTRY
卷 87, 期 13, 页码 6681-6687出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.5b00831
关键词
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资金
- NSERC [298351-2010]
- Canada Research Chair program
- Alberta Ingenuity-Health Solutions
- Canada Foundation for Innovation
- EU [OPPK-CZ.2.16/3.1.00/24023]
The pitcher secretions of the Nepenthes genus of carnivorous plants contain a proteolytic activity that is very useful for hydrogen/deuterium exchange mass spectrometry (HX-MS). Our efforts to reconstitute pitcher fluid activity using recombinant nepenthesin I (one of two known aspartic proteases in the fluid) revealed a partial cleavage profile and reduced enzymatic stability in certain FIX-MS applications. We produced and characterized recombinant nepenthesin II to determine if it complemented nepenthesin I in FIX-MS applications. Nepenthesin II shares many properties with nepenthesin I, such as fast digestion at reduced temperature and pH, and broad cleavage specificity, but in addition, it cleaves C-terminal to tryptophan. Neither enzyme reproduces the C-terminal proline cleavage we observed in the natural extract. Nepenthesin II is considerably more resistant to chemical denaturants and reducing agents than nepenthesin I, and it possesses a stability profile that is similar to that of pepsin. Higher stability combined with the slightly broader cleavage specificity makes nepenthesin II a useful alternative to pepsin and a more complete replacement for pitcher fluid in HX-MS applications.
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