Chromatographically distinguishable heme insertion isoforms of human hemopexin.
Two spectroscopically distinct, non-interconverting forms of human hemopexin have been isolated by immobilized metal ion affinity chromatography and characterized spectroscopically. Form alpha (characterized by a bisignate Soret CD spectrum) and form beta (Soret CD characterized by a positive Cotton effect) exhibit different spectroscopic responses to addition of Zn2+ or Cu2+, yet both forms exhibit the same metal ion-induced decrease in Tm for the thermally induced release of the heme prosthetic group. Far UV-CD spectra indicate that the two isoforms possess essentially identical secondary structures, but their differential retention during metal ion affinity chromatography indicates slight differences in exposure of His residues on the protein surface. We propose that these observations result from the binding of heme in form beta with an orientation that differs from the crystallographically observed binding orientation for rabbit hemopexin by rotation of the heme prosthetic group by 180 degrees about the alpha-gamma meso-carbon axis and from interaction of metal ions at two separate binding sites.
Department of Biochemistry and Molecular Biology and the Centre for Blood Research, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC V6T 1Z3 Canada.
Marcia R
MR
Mauk
Federico I
FI
Rosell
A Grant
AG
Mauk
D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004583:Electrons; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D006466:Hemopexin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D019216:Metals, Heavy / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry
D019216:Metals, Heavy; D020033:Protein Isoforms; D006418:Heme; D006466:Hemopexin