Conformational analysis of hemopexin by Fourier-transform infrared and circular dichroism spectroscopy.
Hemopexin is a serum glycoprotein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptor-mediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% beta-structure, 15% alpha-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme-hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the heme-protein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis.
Division of Moelcular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 64110.
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D002942:Circular Dichroism; D017666:Deuterium Oxide; D006418:Heme / Q000737:chemistry*; D006466:Hemopexin / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared
D006418:Heme; D006466:Hemopexin; D017666:Deuterium Oxide