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

α(1)-Microglobulin (α(1)m) 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(1)m-heme interactions are lacking. Here, we report for the first time the preparation and analysis of a stable α(1)m-heme complex upon carbamidomethylation of the reactive Cys34 by using recombinantly expressed human α(1)m. Analytical size-exclusion chromatography coupled with a diode-array absorbance spectrophotometry demonstrates that at first an α(1)m-heme monomer is formed. Subsequently, a second heme triggers oligomerization that leads to trimerization. The resulting (α(1)m[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).