Macromolecular chromogenic substrates for measuring proteinase activity.
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.
Hortin
G L
GL
Clinical Pathology Department, National Institutes of Health, Bldg. 10, Room 2C-407, 10 Center Dr., Bethesda, MD 20892-1508, USA. ghortin@cc.nih.gov
Warshawsky
I
I
Laude-Sharp
M
M
D002850:Chromatography, Gel; D002863:Chromogenic Compounds / Q000138:chemical synthesis / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D010450:Endopeptidases / Q000032:analysis* / Q000737:chemistry; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D012680:Sensitivity and Specificity; D000511:alpha-Macroglobulins / Q000737:chemistry
D002863:Chromogenic Compounds; D004151:Dipeptides; D009842:Oligopeptides; D000511:alpha-Macroglobulins; D011092:Polyethylene Glycols; C021129:S 2238; C043307:succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide; C041319:isoleucyl-prolyl-arginine-4-nitroanilide; D010450:Endopeptidases