Collagenolytic enzymes assayed by spectrophotometry with suspensions of reconstituted collagen fibrils.
Collagenolytic enzymes were quantitated by a method based on spectrophotometry of suspended reconstituted collagen fibrils. To obtain optically stable suspensions it was necessary to perform a short sonication of the aggregated fibrils at 10 degrees C. When fibrils were cleaved with mammalian fibroblast collagenase at 35 degrees C the triple helical collagen fragments (TCA and TCB) would uncoil spontaneously and the decreasing turbidity was used as an estimate of enzyme activity. The method is a specific collagenase assay since a possible cleavage in the non-helical parts of the collagen molecule with contaminating proteinases is without effect on the turbidity of the suspension and the collagen substrate is not converted to gelatin at 35 degrees C. After 1 h of incubation 0.2 U (equivalent to 0.2 micrograms) of fibroblast collagenase could be detected. In purification procedures with microbial collagenases many fractions were tested by overnight incubations in disposable cuvettes. Sealing of cuvettes with square silicone stoppers allowed rotation of enzyme-substrate mixtures directly in the cuvettes. Only standard laboratory equipment is required for this assay, which is not dependent on radiolabeling or preparation of specific immunologic reagents.
Bleeg
H S
HS
Dept. Oral Biology, Royal Dental College, Arhus C, Denmark.
D000818:Animals; D002417:Cattle; D003094:Collagen / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D003012:Microbial Collagenase / Q000032:analysis* / Q000302:isolation & purification; D009391:Nephelometry and Turbidimetry / Q000379:methods; D011247:Pregnancy; D011489:Protein Denaturation; D051381:Rats; D013010:Sonication; D013053:Spectrophotometry / Q000379:methods*; D013535:Suspensions / Q000737:chemistry; D013710:Tendons / Q000737:chemistry
D013535:Suspensions; D003094:Collagen; D003012:Microbial Collagenase