BIBLIOS

Document type
Journal articles

Document subtype
Full paper

Title
Characterization of plasma labile heme in hemolytic conditions

Participants in the publication
Zélia Gouveia (Author)
Ana R. Carlos (Author)
Xiaojing Yuan (Author)
Frederico Aires-da-Silva (Author)
Roland Stocker (Author)
Ghassan J. Maghzal (Author)
Sónia S. Leal (Author)
Cláudio M. Gomes (Author)
Dep. Química e Bioquímica
BioISI
Smilja Todorovic (Author)
Olga Iranzo (Author)
Susana Ramos (Author)
Ana C. Santos (Author)
Iqbal Hamza (Author)
João Gonçalves (Author)
Miguel P. Soares (Author)

Summary
Extracellular hemoglobin, 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 when generated during hemolytic conditions labile heme 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, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme-binding capacity, 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.


Institution
FACULDADE DE CIÊNCIAS DA UNIVERSIDADE DE LISBOA

Where published
The FEBS Journal

Publication Identifiers
ISSN - 1742-464X

Publisher
Wiley



Document Identifiers
DOI - https://doi.org/10.1111/febs.14192
URL - http://dx.doi.org/10.1111/febs.14192

Rankings
SCIMAGO Q1 (2018) - 2.202 - Biochemistry
SCIMAGO Q1 (2017) - 2.058 - Biochemistry
SCOPUS Q1 (2017) - 2.058 - Biochemistry
SCOPUS Q1 (2017) - 2.058 - Molecular Biology
Web Of Science Q1 (2018) - 4.739 - BIOCHEMISTRY & MOLECULAR BIOLOGY - SCIE