Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.7/782
Título: Characterization of Plasma Labile Heme in Hemolytic Conditions
Autor: Gouveia, Zélia
Carlos, Ana Rita
Yuan, Xiaojing
Aires-da-Silva, Frederico
Stocker, Roland
J. Maghzal, Ghassan
Leal, Sónia S.
Gomes, Cláudio M.
Todorovic, Smilja
Iranzo, Olga
Ramos, Susana
Santos, Ana Catarina
Hamza, Iqbal
Gonçalves, João
Soares, Miguel P.
Palavras-chave: hemolysis
sickle cell disease
antibody engineering
single‐domain antibody
Data: Ago-2017
Editora: Wiley
Citação: Gouveia, Z., Carlos, A. R., Yuan, X., Aires-da-Silva, F., Stocker, R., J. Maghzal, G., Leal, S. S., Gomes, C. M., Todorovic, S., Iranzo, O., Ramos, S., Santos, A. C., Hamza, I., Gonçalves, J. and Soares, M. P. (), Characterization of Plasma Labile Heme in Hemolytic Conditions. FEBS J. Accepted Author Manuscript. doi:10.1111/febs.14192
Resumo: 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.
Descrição: The deposited article is the accepted manuscript (post-print version) posted online 7 August 2017 and provided by The Febs Journal. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. The deposited article version contains attached the supplementary materials within the pdf. This publication hasn't any creative commons license associated, although it is in open access.
Peer review: yes
URI: http://hdl.handle.net/10400.7/782
DOI: 10.1111/febs.14192
Versão do Editor: http://onlinelibrary.wiley.com/doi/10.1111/febs.14192/abstract
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