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Single turnover mechanism of a trypsin-reactor with high enzyme concentration.

Articolo
Data di Pubblicazione:
1998
Abstract:
A small column containing 2 mM CH-Sepharose 4B-immobilized trypsin was connected to a flow injection device equipped for potentiometric measurements (0.01–2 mM protons) and for post-column analysis by spectrophotometry and capillary electrophoresis (CE). The device was engaged with Na-benzoyl-L-arginine pNO2-anilide (BAPNA), beta-lactoglobulin (b-Lac) and peptides of V8-protease predigested b-Lac. At a given flow rate, the reaction with BAPNA or b-Lac (below 2 mM) produced about 1 proton per substrate molecule in each sample (linear relation to substrate amount); with peptides (below 22 mM), the reaction did not exceed 0.17 acid equivalents per substrate molecule (hyperbolic dependence). Final experiments demonstrated that the reactor gave a correct estimate of available lysine in peptides of b-Lac modified with 5-nitrosalicylaldehyde. The data could be predicted by a kinetic model describing the reactor performance in ‘single turnover’ conditions. The interplay between resident time and the non-catalytic amount of trypsin prevented each enzyme molecule from recycling as well as each substrate molecule (containing one or more cleavage sites) from encountering the enzyme more than once. In conclusion, both from the experimental and the theoretical point of view, this work permitted the analysis of trypsin behaviour in some extreme working conditions and indicates how to modulate the performance of an endoprotease-based reactor. A brief discussion on potential applications in protein mapping and tagging and in the quantitative analysis of protein bioavailability by means of a biosensorial strategy is also described.
Tipologia CRIS:
03A-Articolo su Rivista
Elenco autori:
FERMI P; BIFFI R; CONTI V; RAMONI R; GROLLI S; P. ACCORNERO; BIGNETTI E
Autori di Ateneo:
ACCORNERO Paolo
Link alla scheda completa:
https://iris.unito.it/handle/2318/3232
Pubblicato in:
JOURNAL OF BIOTECHNOLOGY
Journal
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