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 inhibi tors after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophor esis has been claimed to indicate covalent bond formation. In this wor k we have investigated the effects of SDS on the stability of complexe s between single-chain or two-chain tissue plasminogen activator (t-PA ) and its inhibitor (PAI-1). Complexes formed by incubation of t-PA wi th PAI-1 for 15 min at 22-degrees-C were further incubated with variou s amounts of SDS before being subjected to SDS-polyacrylamide gel elec trophoresis. 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 i nteraction of SDS with t-PA.PAI-1 complexes before electrophoresis res ulted 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 dis sociation rate constant in the presence of 35 mm SDS at 22-degrees-C h ad a k(off) value of 1.4 x 10(-2) min-1, which corresponds to a half-l ife of 49.5 min. The t-PA released from the complexes was fibrinolytic ally active, whereas the released PAI-1 inhibited activator-dependent fibrinolysis. In a similar fashion, the well characterized non-acylate d pair alpha1-proteinase inhibitor-elastase was dissociated by SDS tre atment, confirming the validity of our experimental approach to demons trate the reversibility of t-PA.PAI-1 complexes. These results demonst rate that SDS-polyacrylamide gel electrophoresis traps the molecular s pecies in the state in which the proteins existed prior to the analysi s, and they suggest that under the conditions used, the interaction of t-PA with PAI-1 results in the formation of nonacylated reversible co mplexes. This phenomenon may be relevant to the pathophysiology of fib rinolysis and to the general mechanism of serine proteinase-inhibitor complex formation.