Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes.

TELLECHEA, M. E.; GARCIA-PARDO, J.; COTABARREN, J.; LUFRANO, D.; BAKAS, L.; AVILÉS, F. X.; OBREGON, W. D.; LORENZO, J.; TANCO, S.

Bio-protocol

Bio-protocol LLC

Año: 2016 vol. 6

Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze thehydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range ofphysiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas etal., 2007). In this context, the study and discovery of new MCP inhibitors from plants constitute avaluable approach for the development of new therapeutic strategies. Herein we describe a simple andaccessible microplate method for the study of the specific and dose-response carboxypeptidase Ainhibitory activities present in Andean potato tubers. Our protocol combines an extraction methodoptimized for small protein inhibitors in plant tissues, with the measurement of enzyme kinetics using amicroplate reader. These instruments are capable of reading small sample volumes, for many samplesin a very short time-frame, therefore reducing the time and costs of high-throughput screeningexperiments. Although this protocol describes the study of Andean potatoes, our approach is alsoapplicable to the analysis other plant samples.