Figure 5

Interactions of rEno and its mutants with plasminogen. A Far-Western blotting analysis. WT rEno, single mutants K451L and K452L, and double mutant K451L-K452L were transferred to a PVDF membrane and incubated with plasminogen and anti-plasminogen antibody. BSA served as a negative control. Protein bands were visualized using ECL substrate. B Activity of bound plasminogen. Plasminogen placed in the microtiter plates coated with WT rEno, each of the three mutants, or BSA. After washing, bound plasminogen was treated with tPA, followed by substrate, and the OD value was measured at 405 nm. Results are expressed as means ± SD of three experiments with triplicate samples (**p < 0.01). C SPR analysis. WT rEno and the three mutants were separately injected over immobilized plasminogen. Sensorgrams show the binding of immobilized plasminogen to WT rEno and the three mutants. The arrow indicates the end of the injection period, at which point dissociation of WT rEno and the three mutants from plasminogen can be observed. The different proteins are represented by different colored lines. RU, resonance units.