FIG. 5.

Measurement of active plasmin bound to B. burgdorferi as determined by release of radioactivity from radiolabeled R22 ECM. Standard curves generated for unbound plasmin degradation of ECM labeled with [³⁵S]methionine-cysteine (A), [³H]fucose (B), and [³H]proline (C) were used to calculate the amount of plasmin bound per organism. Tissue culture plate wells containing labeled ECM were incubated for 6 h with 2.5 × 10⁷, 5 × 10⁷, and 10⁸ plasmin-coated spirochetes and graded concentrations of unbound plasmin per well. Released (supernatant) and unreleased (2 N NaOH digest of undegraded ECM minus supernatant) radioactivity was counted for each of three replicate wells. Percent release of the total radioactive counts present in each well was calculated as described in Materials and Methods. Linear curve fits were calculated by the least squares method, using the DeltaGraph 4.0 software package. The experiment was performed twice with similar results.