P49 (Lys)-tagged sulfide-reactive hemoglobin I from Lucina pectinata designed for covalent immobilization

Abstract

Background Recently, the importance of hydrogen sulfide (H 2 S) in human health has been a focus of many studies. Nevertheless, there is an increased need for robust and flexible H 2 S sensors. Immobilized proteins have provided remarkable advantage for the efficient assembly of biomaterials. Hemoglobin I (Phe68, Phe29, Gln64, Phe43) from Lucina pectinata is a high affinity H 2 S binding hemoprotein, which can be used to detect its natural ligand. Recombinant hemoglobin I (rHbI) has been shown to have similar kinetic properties to those of native HbI, except for a histidine tag added for affinity purification. A potential biosensor for H 2 S detection could be designed using rHbI immobilized in a conductive and biocompatible surface, like multi-walled carbon nanotubes (MWCNTs). Methods In this study we analyzed and optimized conditions for rHbI immobilization at the MWCNTs. Functionalization with a biocomponent (i.e., Histidine or Lysine residue or rHbI) was achieved by the carboxylation of MWCNTs followed by amidation with the desired species. 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. The resulting (Lys) 6 -tagged rHbI will be expressed in E. coli and purified by ion exchange chromatography. Results X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR) analyses revealed successful modification of the walls by an amide bond between the amine group of the amino-acids (Histidine or Lysine) and the carboxylic groups of the oxidized MWCNTs (MWCNTs-COOH), in both cases. However, this is not the case with rHbI. Our results suggest that even though rHbI has a 6-His tag, the primary amino groups of each residue that could have reacted with the MWCNTs-COOH groups were already forming peptide bonds to rHbI. The secondary amine in the imidazole group is not capable of forming an amide bond. Considering that the lateral chain of Lysine offers another amino group capable of forming an amide bond, it may be preferable to employ a Lysine tag instead of using histidine tags. Conclusions The results of histidine or lysine modified MWCNTs used as reference gave the optimum conditions for the immobilization of the H 2 S reactive hemoprotein in the MWCNTs. The successful fusion of rHbI with a Lys tag should provide the conditions required for covalent immobilization of this sulfide-reactive hemoglobin.