PURIFICATION OF HUMAN PLASMINOGEN-ACTIVATOR INHIBITOR (PAI-1) FROM ESCHERICHIA-COLI AND SEPARATION OF ITS ACTIVE AND LATENT FORMS BY HYDROPHOBIC INTERACTION CHROMATOGRAPHY

Human recombinant PAI-1, expressed in Escherichia coli, was purified a nd separated into its active and latent components by chromatography o n heparin- and phenyl-substituted agarose under conditions which favou r the stability of the active inhibitor, Two columns, with a combined volume of less than 40ml, were used to purify and separate up to 40mg of PAI-I in one day. Purified fractions of PAI-1 were analysed by SDS- PAGE, fluorescence spectroscopy and thermostability measurements, A me thod for concentrating the inhibitor and conditions for storage of con centrated PAI-1 were established, Since PAI-1 spontaneously refolds it s reactive-centre loop in a way similar to what is believed to occur i n the proteinase-serpin complexes, studies with this inhibitor may pla y an important role in elucidating the mechanism of serpin action. The method we are presenting yields highly purified fractions of active a nd latent PAI-1 with relative ease and facilitates detailed investigat ions of its reaction mechanism.