Characterization of the apo-form of extracellular hemoglobin of Glossoscolex paulistus (HbGp) and its stability in the p
- PDF / 1,634,795 Bytes
- 14 Pages / 595.276 x 790.866 pts Page_size
- 59 Downloads / 131 Views
ORIGINAL ARTICLE
Characterization of the apo‑form of extracellular hemoglobin of Glossoscolex paulistus (HbGp) and its stability in the presence of urea Ana E. B. Barros1 · Célia Sulzbacher Caruso1 · Fernanda Rosa Alves1 · Marcel Tabak1 · Francisco A. O. Carvalho2 Received: 23 January 2020 / Revised: 16 May 2020 / Accepted: 8 July 2020 © European Biophysical Societies’ Association 2020
Abstract 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 nonnative prosthetic groups and development of artificial enzymes with designed properties. Keywords Heme extraction · Characterization · Urea · Fluorescent probes · Apo-HbGp
Introduction Hemoproteins are characterized by polypeptide chains combined with a non-peptide chromophore heme group (Culbertson and Olson 2010). The properties of this protein class strongly depend on the nature of the heme group (Chien et al. 2017), which can be removed from the native holoprotein through a variety of methods, resulting in an apo-protein Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00249-020-01449-6) contains supplementary material, which is available to authorized users. * Francisco A. O. Carvalho [email protected] 1
Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil
Universidade Federal do Sul e Sudeste do Pará, Marabá, PA, Brazil
2
form (Teale 1959; Ascoli et al. 1981; Woodward et al. 2007; Pires et al. 2020). The extraction of the prosthetic group leads to the easily precipitable
Data Loading...
