REACTIVATION OF C1-INHIBITOR POLYMERS BY DENATURATION AND GEL-FILTRATION CHROMATOGRAPHY

C1-inhibitor is a proteinase inhibitor in the serpin family. It is an important inhibitor of complement C1, plasma kallikrein, and factor XI Ia, and as such is involved in regulating inflammatory pathways. Studi es on the plasma-derived protein are hampered by the relative ease wit h which the protein converts to an inactive state on storage, under mi ld denaturing conditions, or by incubating in some unfavorable buffers . This inactivation is caused by formation of soluble polymers which c an be visualized on native electrophoresis. In order to facilitate stu dies on both the plasma-derived protein and recombinant variants plann ed for the future, it was necessary to devise a method for the rapid r eactivation of the polymers in high yield. It was found that nonionic detergents did not dissociate the polymers, but they were readily diss ociated in 0.1% SDS. Treatment with 0.1% SDS followed by rapid removal of the SDS and refolding on an FPLC Superose 6 column allowed for rec overy of about 15% of the protein in the active monomeric form. Eighty -five percent eluted as a range of higher order polymers. Using 8 M ur ea as the denaturant a 25% yield of active monomer was recovered. Howe ver, with 6 M guanidine hydrochloride as the denaturant, the yield of active monomer was almost 50%. The remaining material was not present as a range of polymeric species but was probably a dimer. Therefore th is method is a useful technique to facilitate studies on C1-inhibitor. Moreover, the ability to produce monomer, dimer, and polymer forms of C1-inhibitor is useful for studies investigating the conformational c hanges which have occurred in the different forms. (C) 1997 Academic P ress.