Macromolecular chromogenic substrates for measuring proteinase activity

Abstract

Background: Proteinase activities are often measured using chromogenic substrates that are much smaller than physiological substrates. Methods: The hydrodynamic size of macromolecular substrates (macrosubstrates) prepared by linking small chromogenic substrates to polyethylene glycol was determined by gel filtration. Efficiency of macrosubstrate cleavage by proteinases and α2-macroglobulin-proteinase Complexes was monitored spectrophotometrically. Results: Macrosubstrates had hydrodynamic radii of ∼20 Å, similar to proteins with a molecular weight of 18 000. Different macrosubstrates served as efficient substrates for chymotrypsin, trypsin, and thrombin. Linking small substrates to a polymer variably affected substrate efficiency, with the impact on activity ranging from a 60-fold decrease to a 30-fold increase. Proteinases complexed with α2-macroglobulin had ∼10-fold lower activity vs macrosubstrates than small substrates. Conclusions: Macrosubstrates are efficient substrates that allow decreased measurement of sterically hindered proteinase molecules such as α2-macroglobulin-proteinase complexes. Thus, macrosubstrates may provide more accurate functional assays of proteinases such as Coagulation factors. © 2001 American Association for Clinical Chemistry.