Gouveia, Zélia and Carlos, Ana Rita and Yuan, Xiaojing and Aires-da-Silva, Frederico and Stocker, Roland and J Maghzal, Ghassan and Leal, Sónia S and Gomes, Cláudio M and Todorovic, Smilja and Iranzo, Olga and Ramos, Susana and Santos, Ana Catarina and Hamza, Iqbal and Gonçalves, João and Soares, Miguel P (2017) Characterization of Plasma Labile Heme in Hemolytic Conditions. The FEBS Journal, 284 (19). pp.3278-3301. ISSN 1742-4658 (OA)
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Abstract
Extracellular hemoglobin (Hb), a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches we demonstrate that labile heme generated during hemolytic conditions is bound to plasma molecules with an affinity higher than 10(-7) M and that 2-8% (~2-5 μM) of the total amount of heme detected in plasma can be internalized by bystander cells, i.e. bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme binding capacity (HBC1/2 ), that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10(-7) M. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme. This article is protected by copyright. All rights reserved.
| Item Type: | Article |
|---|---|
| Additional Information: | This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. |
| Subjects: | R Medicine > R Medicine (General) |
| Depositing User: | Repository Administrator |
| Date Deposited: | 08 Aug 2017 21:44 |
| Last Modified: | 22 Oct 2017 23:25 |
| URI: | https://eprints.victorchang.edu.au/id/eprint/614 |
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