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Clinical Chemistry 47: 215-222, 2001;
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(Clinical Chemistry. 2001;47:215-222.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Macromolecular Chromogenic Substrates for Measuring Proteinase Activity

Glen L. Hortin1,a, Ilka Warshawsky1 and Maryline Laude-Sharp2

1 Clinical Pathology Department, National Institutes of Health, Bldg. 10, Room 2C-407, 10 Center Dr., Bethesda, MD 20892-1508.

2 Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110.
a Author for correspondence. Fax 301-402-1885; e-mail ghortin{at}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 ~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 {alpha}2-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 {alpha}2-macroglobulin-proteinase complexes. Thus, macrosubstrates may provide more accurate functional assays of proteinases such as coagulation factors.






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Copyright © 2001 by the American Association for Clinical Chemistry.