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Macromolecular chromogenic substrates for measuring proteinase activity
G L Hortin1, I Warshawsky, M Laude-Sharp
1Clinical Pathology Department, National Institutes of Health, Bldg. 10, Room 2C-407, 10 Center Dr., Bethesda, MD 20892-1508, USA. ghortin@cc.nih.gov
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 alpha(2)-macroglobulin-proteinase complexes was monitored spectrophotometrically.
Results:
Macrosubstrates had hydrodynamic radii of approximately 20 A, 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 alpha(2)-macroglobulin had approximately 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 alpha(2)-macroglobulin-proteinase complexes. Thus, macrosubstrates may provide more accurate functional assays of proteinases such as coagulation factors.