Biotinylated aprotinin: a versatile probe for the detection of serine proteinases on western blots.
The present study was undertaken to provide a highly sensitive detection system for the identification and characterisation of serine proteinases separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Biotinylated aprotinin of high specific activity (88-92% active) was prepared (i) by reaction of aprotinin directly with N-hydroxy sulfosuccinimidyl-6-(biotinamido) hexanoate, and (ii) by reaction of aprotinin-trypsin complex with N-hydroxy succinimidobiotin. Both biotinylated aprotinin samples were suitable as probes for the detection of the serine proteinases, neutrophil elastase and cathepsin G, pancreatic trypsin and chymotrypsin and plasmin on nitrocellulose blots. Specific irreversible chloromethyl ketone proteinase inhibitors used in combination with this detection system enabled respective proteinases to be selectively inactivated and thus positively identified. The biotinylated aprotinin detection system was highly sensitive and could detect as little as 0.2 ng (8.5 fmol) of active proteinase (trypsin). In summary, a method has been developed for the sensitive detection of serine proteinases separated by SDS-PAGE. The method is more sensitive and convenient to perform than conventional zymography and significantly, when used in conjunction with specific serine proteinase inhibitors or specific antibodies can yield appreciable information on the identity of the respective serine proteinases being examined. Furthermore the molecular mass of the serine proteinase may be reliably obtained by this method. This method should find application in identifying the role that serine proteinases play in the etiopathogenesis of connective tissue disorders.
Melrose
J
J
Raymond Purves Bone and Joint Research Laboratories, Royal North Shore Hospital, St Leonards, NSW, Australia.
Ghosh
P
P
Patel
M
M
D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000737:chemistry*; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry*; D015153:Blotting, Western / Q000379:methods*; D056649:Cathepsin G; D002403:Cathepsins / Q000032:analysis; D002417:Cattle; D002918:Chymotrypsin / Q000032:analysis; D005341:Fibrinolysin / Q000032:analysis; D006801:Humans; D019272:Leukocyte Elastase; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000032:analysis; D012697:Serine Endopeptidases / Q000032:analysis*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry; D014357:Trypsin
D015842:Serine Proteinase Inhibitors; D013388:Succinimides; C061443:sulfosuccinimidyl 6-(biotinamido)hexanoate; D001710:Biotin; D007611:Aprotinin; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D014357:Trypsin; D005341:Fibrinolysin