Spectrophotometric method to quantify and discriminate urokinase and tissue-type plasminogen activators.
Plasminogen activator and urokinase are often used as biological markers of cell activation. However, the methods currently used are cumbersome, make no discrimination between tissue-type plasminogen activator and urokinase, and do not allow expression of the results of the overall reaction in International Units. The one-step method described in this paper lacks these drawbacks. Moreover, we propose use of H-D-Val-Phe-Lys-4-nitroanilide as substrate which has a lower Km than the standard H-D-Val-Leu-Lys-4-nitroanilide which is commercially available. Low concentrations of sodium dodecyl sulfate in the reaction mixture dramatically and preferentially accelerate the reaction catalyzed by tissue-type plasminogen activators. Identical results are obtained under kinetic or fixed-time assay conditions using either a photometer or 96-well plate reader. The corresponding formulae are provided.
Schnyder
J
J
Sandoz Research Institute Berne Ltd., Switzerland.
Marti
R
R
Cooper
P H
PH
Payne
T G
TG
D000818:Animals; D002478:Cells, Cultured; D005337:Fibrin / Q000494:pharmacology; D005340:Fibrinogen / Q000494:pharmacology; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013696:Temperature; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification*; D014568:Urokinase-Type Plasminogen Activator / Q000302:isolation & purification*
D012967:Sodium Dodecyl Sulfate; D005337:Fibrin; D005340:Fibrinogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator