Role of lysine residues in recombinant protein-PLG interaction and activation to enzymatically active plasmin.

<p><b>A:</b> Binding of the immobilized proteins LipL32, Lp29, Lp49, LipL40, MPL36, rLIC10494, rLIC12730 and rLIC12238 (10 µg/ml) to PLG (10 µg/ml) was carried out in the presence (PLG+ACA) or absence (PLG) of the lysine analogue 6-aminocaproic acid. BSA was used as a negative control for nonspecific binding. The bound PLG was detected and quantified by specific antibodies. Bars represent the mean absorbance at 492 nm ± the standard deviation of four replicates for each sample and are representative of two independent experiments. Statistically significant differences are shown by * (<i>P</i><0.001) and ** (<i>P</i><0.0001). <b>B:</b> Cleavage of specific plasmin substrate by PLG bound to recombinant proteins was assayed by modified ELISA as immobilized proteins received the following treatment: PLG+uPA+specific plasmin substrate (PLG+uPA+S), or controls lacking one of the three components (PLG+uPA; PLG+S; uPA+S). BSA was employed as a negative control. Bars represent the mean absorbance values at 405 nm, as a measure of relative substrate cleavage, ± the standard deviation of four replicates for each experimental group and are representative of two independent experiments. Statistically significant differences are shown by * (<i>P</i><0.00001), ** (<i>P</i><0.005) and *** (<i>P</i><0.05).