Title

Kinetics Of Hemoprotein Reduction And Interprotein Heme Transfer

Document Type

Article

Publication Date

1985

Published In

Biochemistry

Abstract

The transfer of hemin from one protein to another is an event biologically important for the conservation of heme iron. Hemin entering the circulation (or added to serum) is mainly bound by albumin and then transferred to hemopexin [Morgan, W. T., Liem, H. H., Sutor, R. P., & Muller-Eberhard, U. (1976) Biochim. Biophys. Acta 444, 435-445], and we are now investigating which mechanisms may be operative in enhancing this process. The presence of imidazole has been demonstrated to accelerate hemin transfer from albumin to hemopexin [Pasternack, R. F., Gibbs, E. J., Hoeflin, E., Kosar, W. P., Kubera, G., Skowronek, C. A., Wong, N. M., & Muller-Eberhard, U. (1983) Biochemistry 22, 1753-1758]. The present work is an examination of the effect of the reduction of albumin-bound hemin on the rate of its transfer to hemopexin. Hemin (Hmᴵᴵᴵ; ferriprotoporphyrin IX) was reduced to Hmᴵᴵ (ferroprotoporphyrin IX) by the addition of sodium dithionite under argon. The reduction kinetics of Hmᴵᴵᴵ to Hmᴵᴵ were studied separately in the two complexes: with human serum albumin (HSA), which binds up to 20 mol of heme/mol (the first mole with K ≃ 10⁸), and with hemopexin (HHx), which binds heme equimolarly (K ≃ 10¹³). The rate of reduction of Hmᴵᴵᴵ to Hmᴵᴵ on HSA was first order over several half-lives and linearly dependent on [S₂O₄²−]½. At [HSA]₀/[Hmᴵᴵᴵ] ~ 3, the k_obsd was (5 X 10−³)+ 0.75[S₂O₄²−]½, and with [HSA]/[Hmᴵᴵᴵ] ~ 25, the k_obsd was (2 X 10−³) + 0.25[S₂O₄²-]½. The reduction of Hmᴵᴵᴵ to Hmᴵᴵ on human hemopexin (HHx) is much more rapid with k_obsd = (2.5 X 10−³) [S₂Od²−]½. The transfer of Hmᴵᴵ from HSA to HHx was studied by adding dithionite to Hmᴵᴵᴵ·HSA and mixing this with HHx. The transfer was biphasic, consisting of two first-order processes, k_f and k_s, independent of [Hmᴵᴵᴵ·HSA]₀, [HSA]₀, and [HHx]₀, but with a slight dependence on pH and ionic strength. The transfer of Hmᴵᴵᴵ from HSA involves two steps that may be due to HSA existing as two noninterconverting conformers [Moehring, G. A., Chu, A. H., Kurlansik, L., & Williams, T. J. (1983) Biochemistry 22,3381-3386]. Since the overall rate of the "redox" transfer pathway of Hmᴵᴵ from HSA to HHx is as efficient as the pathway catalyzed by the presence of 50 mM imidazole, a catalyst much more effective than this nitrogen base would have to be present in vivo to enhance the transfer of heme from HSA to HHx.

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