Biotin-labeled potato chymotrypsin inhibitor-1: a useful probe for the detection and quantitation of chymotrypsin-like serine proteinases on western blots and its application in the detection of a serine proteinase synthesised by articular chondrocytes.
Potato chymotrypsin inhibitor-1 (pCTI-1) was biotinylated by reaction with sulfosuccinimidyl-6-(biotinamido)hexanoate. This derivative was used as a probe on Western blots for the detection and quantitation of chymotrypsin and the detection of a chymotrypsin-like serine proteinase synthesized by ovine chondrocytes in alginate bead culture. Densitometric analysis demonstrated that there was a linear relationship between the amount of chymotrypsin electrophoresed, over the range 0.1 to 10 ng, and the intensity of the band detected on Western blots using biotinylated pCTI-1 as probe, indicating that the technique could be used for the quantification of active proteinases. The biotinylated pCTI-1 detection technique was convenient to use, reproducible, and more sensitive than zymography.
Rodgers
K J
KJ
Raymond Purves Bone and Joint Research Laboratories, Royal North Shore Hospital, St. Leonards, New South Wales, Australia.
Melrose
J
J
Ghosh
P
P
D000818:Animals; D001710:Biotin / Q000737:chemistry*; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000201:enzymology*; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002863:Chromogenic Compounds / Q000378:metabolism; D002918:Chymotrypsin / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism; D012697:Serine Endopeptidases / Q000032:analysis* / Q000378:metabolism; D012756:Sheep
D002863:Chromogenic Compounds; D009842:Oligopeptides; D010940:Plant Proteins; D011480:Protease Inhibitors; C023578:proteinase inhibitor I (plants); D001710:Biotin; C043307:succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide; D012697:Serine Endopeptidases; D002918:Chymotrypsin