AUTHOR=Karnaukhova Elena , Rutardottir Sigurbjörg , Rajabi Mohsen , Wester Rosenlöf Lena , Alayash Abdu I. , Åkerström Bo 

TITLE=Characterization of heme binding to recombinant α1-microglobulin

JOURNAL=Frontiers in Physiology

VOLUME=5

YEAR=2014

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2014.00465

DOI=10.3389/fphys.2014.00465

ISSN=1664-042X

ABSTRACT=<p><bold>Background</bold>: Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme<xref ref-type="fn" rid="fn0001"><sup>1</sup></xref> and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M <italic>in vitro</italic> using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions.</p><p><bold>Methods</bold>: To examine heme binding properties of A1M we utilized UV/Vis absorption spectroscopy, visible circular dichroism (CD), catalase-like activity, migration shift electrophoresis, and surface plasmon resonance (SPR), which was specifically developed for the assessment of His-tagged A1M.</p><p><bold>Results</bold>: The results of this study confirm that A1M is a heme binding protein that can accommodate heme at more than one binding site and/or in coordination with different amino acid residues depending upon heme concentration and ligand-to-protein molar ratio. UV/Vis titration of A1M with heme revealed an unusually large bathochromic shift, up to 38 nm, observed for heme binding to a primary binding site. UV/Vis spectroscopy, visible CD and catalase-like activity suggested that heme is accommodated inside His-tagged (tgA1M) and tagless A1M (ntA1M) in a rather similar fashion although the His-tag is very likely involved into coordination with iron of the heme molecule. SPR data indicated kinetic rate constants and equilibrium binding constants with K<sub>D</sub> values in a μM range.</p><p><bold>Conclusions</bold>: This study provided experimental evidence of the A1M heme binding properties by aid of different techniques and suggested an analytical methodology for a rapid evaluation of ligand-binding properties of recombinant A1M versions, also suitable for other His-tagged proteins.</p>