Insertion of Heme b into the Structure of the Cys34‐Carbamidomethylated Human Lipocalin α1‐Microglobulin: Formation of a [(Heme)2(α1‐Microglobulin)]3 Complex

Abstract

α₁-Microglobulin (α₁m) 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 α₁m–heme interactions are lacking. Here, we report for the first time the preparation and analysis of a stable α₁m–heme complex upon carbamidomethylation of the reactive Cys34 by using recombinantly expressed human α₁m. Analytical size-exclusion chromatography coupled with a diode-array absorbance spectrophotometry demonstrates that at first an α₁m–heme monomer is formed. Subsequently, a second heme triggers oligomerization that leads to trimerization. The resulting (α₁m[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.