D val leu lys 4 nitroanilide dihydrochloride

ELISA plate wells were coated with leptospires, standardized as described above and incubation occurred at 30 °C overnight. The plate was washed 3 times with PBS and blocked with PBS/BSA solution at 30 °C for 2 h. Next, 1 μg of Plg or PBS containing 30% (v/v) inactivated human serum (iNHS; heating at 56 °C for 20 min) was added and the plate incubated for 2 h at 30 °C. In sequence, 4 ng of uPa and the chromogenic plasmin substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (0.4 mM; Sigma) were added to each well. The plates were incubated at 37 °C and substrate degradation was determined at different times measuring absorbance at 405 nm.
For experimental control, the absorbance values of each treatment were compared with the absorbance values of the control treatments, where Plg, uPa or substrate was omitted. For statistical analysis the absorbance values of L. biflexa-LIC11711 were compared with L. biflexa-pMaOri using the Mann–Whitney U test, and a p-value below 0.05 was considered statistically significant.

The participation of rDiACT and rDiFBAL in plasmin generation was studied following the methodology previously described [17 (link)]. In brief, 2 μg of human PLG (Acris antibodies) were incubated in PBS with 3 μg of the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) in the presence of 1 μg of each recombinant protein in a final volume of 100 μl. Activation of PLG was initiated by addition of 15 ng of t-PA (Sigma), however, plasmin generation in the absence of tPA was also analyzed. After incubation of the plates (1 h at 37°C) the hydrolysis of the chromogenic substrate, which is directly proportional to the amidolytic activity of generated plasmin was revealed by measuring absorbance at 405 nm. Each sample was analyzed in triplicate.

plasminogen activation and subsequent fibrinolysis was analysed in a test volume of 100 µL by measuring the amidolytic activity of generated plasmin (González-Miguel et al., 2012 (link)). In each well, 2 µg of human plasminogen (Acris Antibodies) were incubated in PBS with 3 µg of the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) in the presence of 1 µg of FhES. Activation of plasminogen was initiated by the addition of 15 ng of tPA (Sigma). In parallel, plasmin generation was also measured in the absence of t-PA, to observe the ability of the FhES extract proteins of activating plasminogen on their own. Plates were incubated at 37° C for 1 h and the hydrolysis of the chromogenic substrate was analysed by measuring absorbance at 405 nm in an Easy Reader (Bio-Rad).