Thursday, December 04, 2003

Description
The specific cysteine proteinase substrate Z-Phe-Arg-MCA is used as described by Barret and Kirschke (1981). The substrate, Z-Phe-Arg-NMec, which is barely fluorescent, is hydrolyzed to liberate 7-amino-4-methylcoumarin, and this is quantified by its intense fluorescence after the reaction has been stopped with monochloroacetate.

Procedure
Total soluble protein (50靏) from crude leaf extract is diluted with a 0.1% Tween20 solution to a total volume of 500mL. Proteinase reaction buffer (340mM sodium acetate, 60mM acetic acid, 4mM disodium EDTA, and 8mM DTT) (250ml) is added. For temperature equilibration and activation of enzymes, the solution is placed at 30癈 for 1 min. After equilibration, 250mL of a 20mM solution of the cysteine proteinase substrate Z-Phe-Arg-Nmec dissolved in dimethyl sulfoxide is added. After 10 min at 30癈, stopping reagent (100mM sodium monochloroacetate, 30mM sodium acetate, 70 mM acetic acid, pH 4.3) (1mL) is added. The fluorescence of the free 7-amino-4-methylcoumarin is determined by excitation at 370nm and emission at 460nm in a fluorometer.

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