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Biotinylated trypsin and its application as a sensitive, versatile probe for the detection and characterisation of an ovine chondrocyte serine proteinase inhibitor using Western blotting
Author(s) -
Rodgers Kenneth John,
Melrose James,
Ghosh Peter
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170135
Subject(s) - biochemistry , chondrocyte , microbiology and biotechnology , biotinylation , chemistry , fast protein liquid chromatography , blot , trypsin , gel electrophoresis , trypsin inhibitor , isoelectric focusing , polyacrylamide gel electrophoresis , serine proteinase inhibitors , sodium dodecyl sulfate , chromatography , affinity chromatography , biology , serine protease , enzyme , protease , gene , in vitro
Biotinylated trypsin (bT) was used as a probe on Western blots of 10–20% polyacrylamide gradient Tris‐Tricine gels for the detection of serine proteinase inhibitors (SPIs) isolated from extracts of ovine articular cartilage and from chondrocyte conditioned culture medium. The major cartilage SPI, a 58 kDa glycoprotein, was purified by sequential Sephacryl S–300 gel permeation, concanavalin A affinity, anion exchange and Superose 12 fast protein liquid chromatography (FPLC). A low molecular weight SPI of ∼ 6 kDa was also detectable in cartilage extracts after prolonged storage at 4°C and following affinity chromatography on immobilised chymotrypsin, suggesting that the 6 kDa inhibitor may have arisen from proteolytic modification of the 58 kDa SPI. Analysis of chondrocyte conditioned culture medium using the bT detection system revealed that the major SPI present was the 6 kDa species although a small amount of the 58 kDa SPI was also detectable. The large molecular weight ‘native’ cartilage SPI was distinct from ovine α1‐proteinase inhibitor (α 1 ‐PI) when examined by native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). The 6 kDa cartilage SPI was indistinguishable from basic pancreatic trypsin inhibitor (BPTI, aprotinin) following sodium dodecyl sulfate (SDS)‐PAGE under reduced and nonreduced conditions. Amino terminal sequencing of the 6 kDa cartilage SPI revealed a strong (90%) homology with BPTL.