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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
Author(s) -
Siebel Judith F.,
Kosinsky Robyn L.,
Åkerström Bo,
Knipp Markus
Publication year - 2012
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100808
Subject(s) - heme , chemistry , hemeprotein , electron paramagnetic resonance , resonance raman spectroscopy , beta 2 microglobulin , raman spectroscopy , crystallography , biochemistry , nuclear magnetic resonance , biology , physics , optics , immunology , enzyme
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 .