38] Pineapple cysteine endopeptidases

Abstract

This chapter focuses on pineapple cysteine endopeptidases. The pineapple plant contains at least four distinct cysteine endopeptidases. The major endopeptidase present in extracts of plant stem is stem bromelain, and fruit bromelain is the major endopeptidase in the fruit. Two additional cysteine endopeptidases, ananain and comosain, are detected only in the stem. Both stem and fruit bromelain are assayed with protein substrates such as casein and hemoglobin. Substrates containing longer peptide sequences and more sensitive leaving groups are employed in assay of stem bromelain. The substrate, Z-Arg-Arg-NHMec, which is scarcely fluorescent, is hydrolyzed to liberate 7-amino-4-methylcoumarin, and this is quantified fluorimetrically once the enzyme is inactivated with chloroacetate. The fluorescence of the free aminomethylcoumarin is determined by excitation at 360 nm and emission at 460 nm. All enzymes purified by active-site-directed affinity chromatography are reversibly blocked by mercuric chloride or a disulfide. As no further activation is performed and disulfide is routinely added to column fractions, the enzymes remain in the reversibly blocked state following freeze-drying.