Purification of hemopexin and its domain fragments by affinity chromatography and high-performance liquid chromatography.

A method is described for the preparation of apohemopexin from Cohn Fraction IV-4 of human serum by one-step affinity chromatography on a heme-agarose column and separation of its tryptic domain fragments by high-performance liquid chromatography (HPLC). Limited tryptic digestion cleaved human apohemopexin after Arg-216 into half molecules and the N-terminal half was degraded very rapidly, whereas heme-saturated hemopexin was cleaved after Lys-101. These results suggest that hemopexin is composed of two domains that are connected by an exposed histidine-rich hinge-like region in apohemopexin which becomes inaccessible to trypsin in heme-saturated hemopexin. Also described is the preparation of apohemopexin from whole rabbit serum in two steps, heme-affinity chromatography and ion-exchange HPLC, and separation of its tryptic domain fragments by HPLC. Limited tryptic digestion also cleaves rabbit apohemopexin into half-molecules but the N-terminal half is more stable than the C-terminal half in this case. This lends support to the idea of functional differences between domains.