SINGLE TURNOVER MECHANISM OF A TRYPSIN-REACTOR WITH HIGH ENZYME CONCENTRATION

A small column containing 2 mM CH-Sepharose il B-immobilized trypsin w its connected to a flow injection device equipped for potentiometric m easurements (0.01-2 mM protons) and for post-column analysis by spectr ophotometry and capillary electrophoresis (CE). The device was engaged with N alpha-benzoyl-L-arginine pNO(2)-anilide (BAPNA), beta-lactoglo bulin (beta-Lac) and peptides of V8-protease predigested beta-Lac. At a given flow rate, the reaction with BAPNA or beta-Lac (below 2 mM) pr oduced about 1 proton per substrate molecule in each sample (linear re lation to substrate amount); with peptides (below 22 mM), the reaction did not exceed 0.17 acid equivalents per substrate molecule (hyperbol ic dependence). Final experiments demonstrated that the: reactor gave a correct estimate of available lysine in peptides of beta-Lac modifie d with 5-nitrosalicylaldehyde. The: data could be predicted by a kinet ic model describing the reactor performance in 'single turnover' condi tions. The interplay between resident time and the non-catalytic amoun t of trypsin prevented each enzyme molecule from recycling as well as each substrate molecule (containing one or more cleavage sites) from e ncountering the enzyme more than once. In conclusion, both from the ex perimental and the theoretical point of view, this work permitted the analysis of trypsin behaviour in some extreme working conditions and i ndicates how to modulate the performance of an endoprotease-based reac tor. A brief discussion on potential applications in protein mapping a nd tagging and ill the quantitative analysis of protein bioavailabilit y by means of a biosensorial strategy is also described. (C) 1998 Else vier Science B.V. All rights reserved.