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 XIIa, and as such is involved in regulating inflammatory pathways. Studies on the plasma-derived protein are hampered by the relative ease with which the protein converts to an inactive state on storage, under mild denaturing conditions, or by incubating in some unfavorable buffers. This inactivation is caused by formation of soluble polymers which can be visualized on native electrophoresis. In order to facilitate studies on both the plasma-derived protein and recombinant variants planned for the future, it was necessary to devise a method for the rapid reactivation of the polymers in high yield. It was found that nonionic detergents did not dissociate the polymers, but they were readily dissociated 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 recovery 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 urea as the denaturant a 25% yield of active monomer was recovered. However, 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 this 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 changes which have occurred in the different forms.
Gauthier
M
M
Department of Oral Medicine and Diagnostic Sciences, College of Dentistry, University of Illinois at Chicago 60612, USA.
Patston
P A
PA
D002850:Chromatography, Gel; D003174:Complement C1 Inactivator Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D003902:Detergents; D019281:Dimerization; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D066298:In Vitro Techniques; D011108:Polymers / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D014508:Urea
D003174:Complement C1 Inactivator Proteins; D003902:Detergents; D006146:Guanidines; D011108:Polymers; D014508:Urea; D019791:Guanidine