Díaz Ayala, Ramonita

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    Methemoglobin I from lucina pectinata: A feasible alternative for a prototype biosensor for hydrogen sulfide
    (2018) Díaz Ayala, Ramonita; López Garriga, Juan; College of Arts and Sciences – Sciences; Cabrera Martínez, Carlos R.; Castro Rosario, Miguel; Vega Olivencia, Carmen A.; Department of Chemistry; Zapata Medina, Rocío
    Hydrogen sulfide (H2S) is one spiteful smelling gas that was considered for centuries extremely noxious to biological species. However, the fact that three enzymes endogenously catalyze H2S synthesis and that it has been detected in our body has changed dramatically our perceptions about H2S. The duality of this gas, its positives or negative biological effects, is responsible for the great interest of the scientific community to measure with high precision and accuracy H2S levels in biological fluids. In spite of multiple efforts as well as great number of analytical techniques, the accurate real-time H2S measurements in the nano to micromolar range will be a challenge. Thus, the search of a new, tough and precise sensor for H2S detection and analysis is one of the most important research areas in this new scientific community. Hemoglobin I from Luciana pectinata has the unique characteristic of binding and transporting H2S to symbiotic bacteria. Their high affinities toward H2S-binding suggest that this hemoprotein can be used to detect this physiologically relevant signaling molecule. Thus, recombinant hemoglobin I (r-HbI) immobilized in a conductive and biocompatible surface like multi-walled carbon nanotubes (MWCNTs) will be a suitable model to develop a unique biosensor for H2S. Analysis of each step of the MWCNTs modification is fundamental to achieve the adequate development of the H2S biosensor. Therefore, the optimal conditions to immobilize the r-Hb at the MWCNTs are the primary focus of the first part of this work. Functionalization with a biocomponent (i.e., Histidine or Lysine residue or (His)6-tagged rHbI) was first achieved by the carboxylation of MWCNTs followed by amidation with the desired species. The resulting interactions (covalent or non-covalent) were then analyzed and characterized using spectroscopy techniques. The results showed that both Histidine and Lysine amino acids were immobilized on the MWCNTs by amide bond, but the r- HbI can only interact by non-covalent interactions. Thus, the covalent immobilization of the rHbI to the MWCNTs was achieved through another route. First the section coding for the histidine-tag was removed from the cloning vector, the coding region for HbI was modified to include lysine residues at the C-terminus, followed by directional cloning into the pET28(a+) vector. In the second part of this work a poly-Lys tag was fused to Lucina pectinata’s hemoglobin I (HbI) coding sequence to immobilize the protein on a conductive and biocompatible surface for H2S detection. The (Lys)6-tagged rHbI construct was expressed in E. coli and purified by immobilization on a cation exchange matrix, followed by size exclusion chromatography. The identity, structure and function of the (Lys)6-tagged rHbI was also assessed by mass spectrometry, small and wide X-ray scattering, optical spectroscopy and kinetic analysis. The scattering, spectroscopic and kinetic results showed that the (Lys)6-tagged rHbI is structurally and functionally analogous to the native and the (His)6-tagged proteins. A high value of the association rate constant (Kon 1.4 x 105 M-1 s-1) and low value of its dissociation constant (koff 0.1 x 10-3 s-1) evidences this. This results confirmed that the (Lys)6-tagged rHbI binds H2S with the same high affinity as its homologue. The third and last part of this research focused in achieved an orientate and covalent immobilization on a conductive surface without the loss of its particular function to bind H2S at its active center as well demonstrating that (Lys)6-tagged rHbI/MWCNTs biocomposite is electrochemically active and it could be using as a long term possible bio-probe for H2S detection. Therefore, using a coupling agent, approximately 27% of the (Lys)6-tagged rHbI was immobilized via a peptide bond on to carbonyl-oxidized carbon nanotubes transducer surfaces, i.e., powder and verticallyaligned carbon nanotubes (VACNTs). The immobilization was achieved by following two steps: 1) generation of amine-reactive ester from the carboxylic acid groups of the surfaces and 2) coupling these groups with the amine groups of the Lys-tag in the (Lys)-tag rHbI moiety. We analyzed the immobilization process using different conditions and techniques to differentiate protein covalent attachment from physical adsorption. Fourier transform infrared (FTIR) microspectroscopy data showed a 14 cm-1 displacement of the protein’s amide I and amide II peaks to lower frequency after immobilization. This result indicates a covalent attachment of the protein to the surface. Differences in morphology of the carbon substrate with and without (Lys)6- tagged rHbI confirmed protein immobilization, as observed by transmission electron microscopy (TEM), supporting the FTIR findings. Electrochemical analysis also demonstrated the immobilization of the (Lys)6-tagged rHbI at the VACNTs electrode and its redox potential Ep= (-0.18 ?? 0.03)V. (Lys)6-tagged rHbI/VACNTs response toward H2S was evaluated. The results suggest the potential applicability of this electrode to detect H2S.