Modification of a Calcein-based assay for proteolytic activity [abstract]

Abstract

This investigation was aimed at developing an assay for characterizing samples gathered from tissues on which microvascular permeability studies were being conducted. In particular, we were looking for evidence of proteolytic activity. The approach we used was based on the quenching of a fluorescent dye by Cu2, and the ability of proteolytic products (amino acids and peptides) to coordinate with the Cu2, removing it from the sensor and allowing it to fluoresce. (1) After optimization of assay conditions, samples collected from two leak index experiments were tested. The results showed that there was a clear difference between the two samples with respect to proteins and small peptides. Therefore, I can conclude that this appears to be a successful assay to screen for proteolytic activity in the microvasculature. (1) Dean KES, Klein G, Renaudet O, Reymond J-L. 2003. A green fluorescent chemosensor for amino acids provides a versatile high-throughput screening (HTS) assay for proteases. Bioorganic & Medicinal Chem Letters 13:1653-1656.