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Structural characterization of the extracellular hemoglobin of Amynthas gracilis (HbAg) by different biophysical techniques

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Author(s):
Jonathan Brito Souza de Oliveira
Total Authors: 1
Document type: Master's Dissertation
Press: Araraquara. 2018-08-08.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Química. Araraquara
Defense date:
Advisor: Patrícia Soares Santiago
Abstract

The extracellular hemoglobin molecules present high oligomeric stability, resistance to oxidation, cooperativity and affinity to bind oxygen, besides the potential use in biotechnological applications a s blood substitute and biosensors of environmental contam ination. Considering that, this dissertation aimed to characterize the structure and stability of the giant extracellular hemoglobin of the annelid Amynthas gracilis (HbAg) by different biophysical t echniques, such as optical absorption, light scattering intensity (LSI), dynamic light scattering (DLS), SDS - PAGE electrophoresis, analytical ultracentrifugation (AUC), Matrix Assisted Laser Desorption Ionization - Time of Fly - Mass Spectrometry (MALDI - TOF - MS ) and atomic force microscopy . The HbAg had an optical absorption spectrum with a maximum of 415 nm (Soret band) , and 540 and 575 nm (Q bands) at pH 7.0 . The alk alinization of the medium shifted the absorption maxima to 405 nm and a single broad Q band at 54 0 nm due to the oxidation of the heme group. The protein profile presented by HbAg by SDS - PAGE electropho resis was similar but not identical to the extracellular hemoglobin of Glossoscolex paulistus (HbGp). HbAg presented three bands with molecular mass (MM) ranging 25 - 37 KDa associated with the L chains, whereas HbGp presented only two bands in this MM range. MALDI - TOF - MS data show ed that HbAg presents four isoforms for the monomer d ( d 1 - d 4 ) with MM ranging 16.2 - 16.8 kDa, three linkers ( L 1 , L 2 and L 3 ), 2 5.8 - 26.8 kDa, two isoforms for the abc trimer and a single isoform for the abcd tetramer (67.7 kDa). By AUC , the MM of whole HbAg was determined in 3.9 MDa, a higher value than that observed for HbGp (3.6 MDa). The highest MM value observed was associated with self - aggregation of HbAg as a function of storage time. The isoelectric point value (pI) estimated by Zeta Potential wa s 6.0 ± 0.3. Native HbAg had a h ydrodynamic d iameter (D h ) of 28 nm determined by DLS. However, at pH less than 6.5 the protein sho we d tendency to form aggregates; at pH above 8.0 the hemoglobin undergoes dissociation concomitant wit h aggregation. Atomic Force M icroscopy (AFM) of HbAg films at pH 7.0 revealed a height between 15 - 20 nm that was associated with two overlapping hexagonal l ayers. Initial studies of peroxidase activity (POD) indicate d higher stability of HbAg against high concentrations of hydrogen peroxide (H 2 O 2 ) relative to HbGp. Finally, the results presented in this dissertation show a great advance in the characterizatio n of HbAg, a giant extracellular hemoglobin not previously reported in the literature. In addition, it has enabled the creation of new study strands that in the future may result in the creation of products of great biotechnological application, such as bi osensors destined to the medical and environmental areas, as well as products of oxygen supply to tissues under the condition of hypoxia. (AU)