Quantitative determination of trypsinlike and chymotrypsinlike enzymes in insects

Abstract

A quantitative and highly specific assay for the determination of trypsinlike and chymotrypsinlike enzymes in insects has been developed. The assay is based on the specific binding of [1,3‐³H]diisopropylfluorophosphate to trypsinlike and chymotrypsinlike enzymes. Trypsinlike enzymes can be determined specifically in the presence of 10 mM TPCK (tosylamide‐2‐phenylethyl chloromethyl ketone; chymotrypsin inhibitor) and chymotrypsinlike enzymes can be determined in the presence of 10 mM TLCK (tosyl‐L‐lysine chloromethyl ketone HCI; trypsin inhibitor). The assay can easily detect 65 ng of either trypsinlike or chymotrypsinlike enzymes in midgut homogenates or whole extracts of many insect species. Using this assay, we have determined the amount of trypsinlike equivalents in Phlebotomus papatasi, Pediculus humanus, Stomoxys calcitrans, Musca domestica, Leishmania major promastigotes, Aedes aegypti, Culex nigripalpus, Culex quinquefasciatus, and Culicoides variipennis. No trypsinlike equivalents were found in Rhodnius prolixus or Ornithodoros moubata. The assay is useful for comparative studies and can be expanded for use as an electrophoretic fluorographic tool in the study of trypsinlike and chymotrypsinlike isozymes.