Effect of the chromogenic substrate on the colorimetric determination of plasmin and plasminogen in milk

From animals in different lactation phases, 56 samples of bovine milk and 40 of ovine milk were collected and assessed for plasmin (PL) and plasminogen (PG) activities with the same method, by using both D-Val-Leu-Lys 4-nitroanilide (VAL) and N-(p-Tosyl)-Gly-Pro-Lys 4-nitroanilide (TOSYL). The mean values of PL (VAL vs TOSYL) were 6.58 vs 4.31 and 15.49 vs 27.25 U/ml, respectively, in bovine and ovine milk; those of PG were 26.10 vs 23.49 (bovine) and 16.07 vs 26.13 U/ml (ovine). The linearity between results was evaluated with a linear regression approach, assuming TOSYL-PL and -PG values as dependent and VAL-PL and -PG as independent variables, respectively. Intercept and slope for PL were 0.059 and 0.646 in bovine, 8.155 and 1.237 in ovine milk; those of PG were 15.037 and 0.324 (bovine), 14.197 and 0.744 (ovine). The different mean values and intercepts of regression curves were attributed to a divergent response of bovine and ovine PL to the chromogenic substrate. Moreover, it was speculated that VAL and TOSYL might differently affect the conversion of PG to PL, that takes place during the assay, due to complex interactions with the plasmin-plasminogen modulatory system.