NanoDrop fluorometry adopted for microassays of proteasomal enzyme activities

Abstract

NanoDrop spectrophotometry and NanoDrop fluorospectrometry are used almost exclusively to determine the concentrations of nucleic acids and proteins. We propose that NanoDrop fluorospectrometry can also be applied for measuring enzyme activities using fluorogenic substrates such as the proteolytic activities of the 26S proteasome. Because the NanoDrop ND-3300 device requires only 2 μl of sample, the amount of sample extract, substrate, and cofactors used for an enzyme assay can be significantly reduced. In this report, we present exemplary microassays for proteasomal activities (chymotrypsin-, trypsin-, and PGPH [peptidyl-glutamyl peptide hydrolase]-like sites) in extracts of isolated hemocytes from a marine crab, Cancer pagurus (Crustaceae).