title: Characterization of heme binding to recombinant α1-microglobulin.
abstract: BACKGROUND
  Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme 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 in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions.
  
  
  METHODS
  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.
  
  
  RESULTS
  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 KD values in a μM range.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Biochemistry and Vascular Biology, Division of Hematology Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration Silver Spring, MD, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Karnaukhova
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences in Lund, Lund University Lund, Sweden.
authors.forename: Sigurbjörg
authors.initials: S
authors.lastname: Rutardottir
authors.affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, Food and Drug Administration Silver Spring, MD, USA.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Rajabi
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences in Lund, Lund University Lund, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Wester Rosenlöf
authors.affiliation: Laboratory of Biochemistry and Vascular Biology, Division of Hematology Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration Silver Spring, MD, USA.
authors.forename: Abdu I
authors.initials: AI
authors.lastname: Alayash
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences in Lund, Lund University Lund, Sweden.
authors.forename: Bo
authors.initials: B
authors.lastname: Åkerström
keywords: alpha-1-microglobulin; circular dichroism; heme binding; surface plasmon resonance