Characterization of the apo-form of extracellular hemoglobin of Glossoscolex paulistus (HbGp) and its stability in the presence of urea

The structural study of small heme-containing proteins, such as myoglobin, in the apo-form lacking heme has been extensively described, but the characterization and stability of the giant Glossoscolex paulistus hemoglobin (HbGp), in the absence of heme groups, has not been studied. Spectroscopic dat...

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Bibliographic Details
Published in:European biophysics journal 2020-09, Vol.49 (6), p.449-462
Main Authors: Barros, Ana E. B., Caruso, Célia Sulzbacher, Alves, Fernanda Rosa, Tabak, Marcel, Carvalho, Francisco A. O.
Format: Article
Language:English
Subjects:
Biochemistry
Biological and Medical Physics
Biomedical and Life Sciences
Biophysics
Biotechnology
Cell Biology
Electrophoresis
Fluorescent indicators
Gel electrophoresis
Glossoscolex
Heme
Hemoglobin
Life Sciences
Membrane Biology
Myoglobins
Nanotechnology
Neurobiology
Original Article
Prostheses
Prosthetic groups
Proteins
Sedimentation
Sodium lauryl sulfate
Trimers
Urea
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Summary:The structural study of small heme-containing proteins, such as myoglobin, in the apo-form lacking heme has been extensively described, but the characterization and stability of the giant Glossoscolex paulistus hemoglobin (HbGp), in the absence of heme groups, has not been studied. Spectroscopic data show efficient extraction of the heme groups from the hemoglobin, with relatively small secondary and tertiary structural changes in apo-HbGp noticed compared to oxy-HbGp. Electrophoresis shows a partial precipitation of the trimer abc (significantly lower intensity of the corresponding band in the gel), due to extraction of heme groups, and the predominance of the intense monomeric d band, as well as of two linker bands. AUC and DLS data agree with SDS-PAGE in showing that the apo-HbGp undergoes dissociation into the d and abc subunits. Subunits d and abc are characterized by sedimentation coefficients and percentage contributions of 2.0 and 3.0 S and 76 and 24%, respectively. DLS data suggest that the apo-HbGp is unstable, and two populations are present in solution: one with a diameter around 6.0 nm, identified with the dissociated species, and a second one with diameter 100–180 nm, due to aggregated protein. Finally, the presence of urea promotes the exposure of the fluorescent probes, extrinsic ANS and intrinsic protein tryptophans to the aqueous solvent due to the unfolding process. An understanding of the effect of heme extraction on the stability of hemoproteins is important for biotechnological approaches such as the introduction of non-native prosthetic groups and development of artificial enzymes with designed properties.
ISSN:0175-7571
1432-1017
DOI:10.1007/s00249-020-01449-6