Insertion of Heme b into the Structure of the Cys34-Carbamidomethylated Human Lipocalin α _1-Microglobulin: Formation of a [(Heme) _2(α _1-Microglobulin)] _3 Complex

摘要

α _1-Microglobulin (α _1m) is a 26 kDa plasma and tissue protein belonging to the lipocalin protein family. Previous investigations indicate that the protein interacts with heme and suggest that it has a function in heme metabolism. However, detailed characterizations of the α _1m-heme interactions are lacking. Here, we report for the first time the preparation and analysis of a stable α _1m-heme complex upon carbamidomethylation of the reactive Cys34 by using recombinantly expressed human α _1m. Analytical size-exclusion chromatography coupled with a diode-array absorbance spectrophotometry demonstrates that at first an α _1m-heme monomer is formed. Subsequently, a second heme triggers oligomerization that leads to trimerization. The resulting (α _1m[heme] _2) _3 complex was characterized by resonance Raman and EPR spectroscopy, which support the presence of two ferrihemes, thus indicating an unusual spin-state admixed ground state with S= ~3/ _2, ~5/ _2. Heme induces trimer: A stable α _1-microglobulin-heme complex results after carbamidomethylation of the reactive Cys34. Size-exclusion chromatography and absorbance spectroscopy reveal the formation of a heme-dependent trimer. The resulting ferriheme complex was characterized by resonance Raman and EPR spectroscopy, which indicate an unusual spin-state admixed ground state with S= ~3/ _2, ~5/ _2.