Ultrasensitive fluorogenic substrates for serine proteases.

S Butenas; M E DiLorenzo; K G Mann

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PubMed · 9364984

Ultrasensitive fluorogenic substrates for serine proteases.

S Butenas·M E DiLorenzo·K G Mann

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Abstract

Selective, sensitive assays for the quantitation of serine proteases involved in coagulation and fibrinolysis have been developed employing fluorogenic substrates containing a 6-amino-1-naphthalenesulfonamide leaving group (PNS-substrates). Over one hundred substrates were evaluated for hydrolysis by the serine proteases of blood coagulation and fibrinolysis, and substrate structure-efficiency correlations were examined. PNS-substrates which contain Lys in the P1 position are specific for Lys-plasmin and are either not hydrolyzed or hydrolyzed at a relatively low rate by factor Xa, thrombin, or urokinase-type plasminogen activator (uPA). These substrates allow quantitation of Lys-plasmin at concentrations as low as 1 pM. Eighteen of over 90 substrates tested for factor XIa are hydrolyzed by this enzyme at a relatively high rate reaching a k(cat), value of 170 s(-1) and allowing quantitation of factor XIa at 10 fM. Eighteen of almost 90 PNS-substrates tested display high specificity for thrombin, some exceeding that for factor Xa by >10,000-fold and >100-fold for activated protein C (APC). Seven of these substrates have a k(cat) over 100 s(-1) and three of them have a K(M) below 1 microM. They allow the quantitation of thrombin at concentrations as low as 20 fM. For APC, uPA and the factor VIIa/tissue factor complex, quantitation is feasible at 1 pM concentration. For factor Xa and factor VIIa the limits are 0.4 pM and 40 pM respectively. The PNS-substrates presented in this study may be employed for the development of direct and sensitive serine protease assays.

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Anilino Naphthalenesulfonates Chromogenic Compounds Factor XIa Fibrinolysin Fluorescent Dyes Humans Kinetics Peptide Fragments Recombinant Proteins Sensitivity and Specificity Serine Endopeptidases Structure-Activity Relationship Substrate Specificity Thrombin Urokinase-Type Plasminogen Activator Comparative Study Journal Article Research Support, U.S. Gov't, P.H.S.

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S Butenas, M E DiLorenzo, K G Mann. 1997. Ultrasensitive fluorogenic substrates for serine proteases.. https://pubmed.ncbi.nlm.nih.gov/9364984/

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