University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Website: @unf

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Classification: Biological fluid -> mucus -> cow -> nasal

Citations 1

"Single Turnover Mechanism Of A Trypsin-reactor With High Enzyme Concentration"
J. Biotechnol. 1998 Volume 60, Issue 1-2 Pages 81-95
Paola Fermi, Riccardo Biffi, Virna Conti, Roberto Ramoni, Stefano Grolli, Paolo Accornero and Enrico Bignetti*

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 N-α-benzoyl-l-arginine pNO2-anilide (BAPNA), β-lactoglobulin (β-Lac) and peptides of V8-protease predigested β-Lac. At a given flow rate, the reaction with BAPNA or β-Lac (below 2 mM) produced about 1 proton per substrate mol. in each sample (linear relation to substrate amount) with peptides (below 22 mM), the reaction did not exceed 0.17 acid equivalent per substrate molecule. (hyperbolic dependence). Final experiments demonstrated that the reactor gave a correct estimate of available lysine in peptides of β-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 behavior 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 quanitative analysis of protein bioavailability by means of a biosensorial strategy is also described.
Enzyme, trypsin Lysine Potentiometry Electrode Spectrophotometry Electrophoresis Reaction order Kinetic Modeling