The chromogenic substrate S-2251 (H-D-Val-Leu-Lys-pNA), a selective and sensitive substrate for plasmin activity, has made it possible to develop simple and reproducible methods for the determination of antiplasmin and plasminogen in human plasma. These methods have been optimized and studied in detail and found to be very specific for the respective factors.
Chromogenic peptide substrates for serine proteases have been designed by using two approaches: (1) by using the natural substrate as a model and (2) by structure-activity correlations obtained through screening of a large number of tripeptides. Some recent examples, substrates for kallikreins and urokinase are given.
The chromogenic peptide substrate H-D-Val-L-Leu-L-Lys-pNA (S-2251) has been chosen from a series of potent plasmin substrates. It is a highly specific and sensitive substrate towards plasmin (Km 2.5·10-4 mol/1, V 0.5·10-6 mol/min CTA-U) and its solubility in tris buffer (1·10-2 mol/1)is sufficient to obtain substrate saturation. Tris buffer pH 7.4 and I 0.15 serves as an optimal medium for the determination of plasmin. In this buffer, antiplasmins in plasma have been determined by using 0.05 CU of plasmin and 20 ul of plasma. At least two inhibitors have been found. One fast (<15 sec. incubation time) and the other(s)slow (5 min. incubation time).The substrate has also been used for plasminogen determinations after activation of 10 ul of plasma with UK (500 Ploug-U) or an excess (40 times) of SK. It was found that neither plasminogen free plasma (100% antiplasmin activity) nor an excess (>1000 times) of SBTI inhibited the substrate activity of the SK activated plasminogen. Optimal conditions for the SK-activated plasminogen was tris buffer pH 7.4, I 0.05. Km = 2.4.10-4 mol/1 for both purified plasminogen and plasma and V = 1.10-4 mol/min.CU or 3.10-6 mol/min-ml plasma.
The chromogenic substrate Bz-Ile-Glu-Gly-Arg-pNA is based on the primary structure preceding the bonds split by factor Xa in bovine prothrombin. Substitution of amino acids in this natural sequence by closely related amino acids has only given inferior substrates. Thus, the natural sequence seems very important. However, we have found that the free γ-carboxyl group of Glu is not indispensable. Substrates with the above structure but having been derivatized on the γ-carboxyl group of Glu, in the form of simple esters or amides, show improved properties. Especially Km of the new substrates compare favourably with our first substrate. Some of the amides also show increased Vmax.These improved properties have made it possible to increase sensitivity, shorten incubation times and lower substrate consumption in several methods where this type of substrate is utilized.
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