Activity assays for characterizing the thrombolytic protein fibrolase.
Characterization of the thrombolytic agent fibrolase was accomplished employing specific proteolytic and thrombolytic assays. This paper describes a method to measure enzyme proteolytic activity using the oxidized beta-chain of insulin as a substrate. Advantages of this method include a short incubation time for substrate cleavage followed by an isocratic HPLC method with a retention time of approx. 5 min. Proteolytic activity can be rapidly and easily quantitated with this procedure. An azocasein assay was also used to quantitate proteolytic activity. This method was optimized with respect to substrate concentration and incubation time allowing for the rapid quantitation of fibrolase activity. A thrombolytic assay is described which employs fibrin plate clearance and has the advantage of rapid and accurate quantitation compared with previously described methods. It also allows for the standardization of fibrolase in plasmin-equivalent units.
Patton
L M
LM
Department of Pharmacology, Marion Merrell Dow Inc., Kansas City, MO 64134-0627.
Pretzer
D
D
Schulteis
B S
BS
Saggart
B S
BS
Tennant
K D
KD
Ahmed
N K
NK
D000818:Animals; D002364:Caseins; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis; D007328:Insulin; D007700:Kinetics; D008666:Metalloendopeptidases / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments; D012910:Snake Venoms; D012911:Snakes; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
D002364:Caseins; D007328:Insulin; D010446:Peptide Fragments; D012910:Snake Venoms; C026018:azocasein; D005340:Fibrinogen; D008666:Metalloendopeptidases; C058789:fibrolase