FIGURE 2.

Plasminogen contains unpaired cysteine thiols. A, purified plasma plasminogen was incubated without or with 20 mm iodoacetamide (IAM) for 24 h prior to incubation with the biotin-linked maleimide (MPB) for a further 24 h. Unreacted MPB was quenched with glutathione, and the plasminogen was resolved on SDS-PAGE and either stained with colloidal Coomassie or transferred to PVDF membrane and blotted with streptavidin-peroxidase to detect the MPB label. B, purified plasma plasminogen was incubated without or with 10 mm pegylated maleimide for 1 h, resolved on SDS-PAGE, and stained with colloidal Coomassie. C, K1–3 or K5-serine protease plasminogen fragments were generated by limited proteolysis with porcine elastase. The fragments (5 μg) were resolved on SDS-PAGE and stained with colloidal Coomassie. D, K1–3 or K5-serine protease fragments were incubated with MPB for 4 h, the unreacted MPB was quenched with glutathione, and the fragments (1 μg) were resolved on SDS-PAGE and blotted with streptavidin-peroxidase to detect the MPB label. E, K5-serine protease was incubated with MPB for 1, 4, or 24 h, the unreacted MPB was quenched with glutathione, and the fragment was resolved on SDS-PAGE and blotted with streptavidin-peroxidase. The positions of molecular mass markers are shown at left.