Sodium dodecyl sulfate-induced dissociation of complexes between human tissue plasminogen activator and its specific inhibitor.

The stability of complexes between serine proteinases and their inhibitors after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis has been claimed to indicate covalent bond formation. In this work we have investigated the effects of SDS on the stability of complexes between single-chain or two-chain tissue plasminogen activator (t-PA) and its inhibitor (PAI-1). Complexes formed by incubation of t-PA with PAI-1 for 15 min at 22 degrees C were further incubated with various amounts of SDS before being subjected to SDS-polyacrylamide gel electrophoresis. The molecular species in the gels were identified both by zymography or by autoradiography after immunoblotting with antibodies directed against either t-PA or PAI-1. It was demonstrated that the interaction of SDS with t-PA.PAI-1 complexes before electrophoresis resulted in a transition from the complexed state to the free forms of t-PA and PAI-1 in a time- and dose-dependent manner. The first-order dissociation rate constant in the presence of 35 mM SDS at 22 degrees C had a koff value of 1.4 x 10(-2) min-1, which corresponds to a half-life of 49.5 min. The t-PA released from the complexes was fibrinolytically active, whereas the released PAI-1 inhibited activator-dependent fibrinolysis. In a similar fashion, the well characterized nonacylated pair alpha 1-proteinase inhibitor-elastase was dissociated by SDS treatment, confirming the validity of our experimental approach to demonstrate the reversibility of t-PA.PAI-1 complexes. These results demonstrate that SDS-polyacrylamide gel electrophoresis traps the molecular species in the state in which the proteins existed prior to the analysis, and they suggest that under the conditions used, the interaction of t-PA with PAI-1 results in the formation of nonacylated reversible complexes. This phenomenon may be relevant to the pathophysiology of fibrinolysis and to the general mechanism of serine proteinase-inhibitor complex formation.