pH- and metal ion-linked stability of the hemopexin-heme complex.
Thermal denaturation of the human hemopexin-heme complex was investigated under a variety of solution conditions to identify factors that influence heme release. The midpoint temperature for the transition between the folded and folded states, T(m), of the hemopexin-ferriheme complex exhibits a significant dependence on pH. When the pH is reduced from 7 to 5 (50 mM BisTris buffer and 50 mM NaCl), T(m) decreases by approximately 23 degrees C despite the relatively higher chloride concentration that tends to stabilize the protein. The thermal stability of the hemopexin-ferroheme complex was examined at pH 7.4 to yield a T(m) that is 3.2 degrees C lower than that of the hemopexin-ferriheme complex under identical conditions. The effect of transition metal ions, which hemopexin has recently been shown to bind [Mauk, M. R., Rosell, F. I., Lelj-Garolla, B., Moore, G. R., and Mauk, A. G. (2005) Biochemistry 44, XXXX-XXXX], was also considered. Cu(2+) and Zn(2+) had the greatest effect, reducing T(m) for the transition by 4.8 and 6.5 degrees C, respectively, relative to the value for the protein in the absence of metal ions [T(m) = 64.9 degrees C [10 mM sodium phosphate buffer (pH 7.4)]]. These metal ions also interfered significantly with the recovery of the native state from the unfolded protein when the protein on returning to 20 degrees C. The current results demonstrate how the conditions within the endosomes of hepatocytes (pH approximately 5.0, [Cl(-)] approximately 60 mM) and the potential presence of transition metal ions or heme iron reduction contribute to the membrane receptor-mediated process of heme release from hemopexin.
Rosell
Federico I
FI
Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Mauk
Marcia R
MR
Mauk
A Grant
AG
D002021:Buffers; D002413:Cations, Divalent; D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006466:Hemopexin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019216:Metals, Heavy / Q000737:chemistry* / Q000378:metabolism; D009566:Nitrates / Q000737:chemistry; D010084:Oxidation-Reduction; D011199:Potentiometry; D011489:Protein Denaturation; D012965:Sodium Chloride / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D002021:Buffers; D002413:Cations, Divalent; D019216:Metals, Heavy; D009566:Nitrates; D006418:Heme; D012965:Sodium Chloride; C031618:sodium nitrate; D006466:Hemopexin