The Multifunctional Catalytic Hemoglobin from Amphitrite ornata: Protocols on Isolation, Taxonomic Identification, Protein Extraction, Purification, and Characterization

The multifunctional catalytic hemoglobin from the terebellid polychaete <i>Amphitrite ornata</i>, also named dehaloperoxidase (<i>Ao</i>DHP), utilizes the typical oxygen transport function in addition to four observed activities involved in substrate oxidation. The multifunctional ability of <i>Ao</i>DHP is presently a rare observation, and there exists a limitation for how novel dehaloperoxidases can be identified from macrobenthic infauna. In order to discover more infaunal DHP-bearing candidates, we have devised a facilitated method for an accurate taxonomic identification that places visual and molecular taxonomic approaches in parallel. Traditional visual taxonomic species identification by the non-specialist, at least for <i>A. ornata</i> or even for other marine worms, is a very difficult and time-consuming task since a large diversity is present and the method is restricted to adult worm specimens. The work herein aimed to describe a method that simplifies the taxonomic identification of <i>A. ornata</i> in particular through the assessment of its mitochondrial cytochrome c oxidase subunit I gene by employing the DNA barcoding technique. Furthermore, whole-worm specimens of <i>A. ornata</i> were used to extract and purify <i>Ao</i>DHP followed by an H₂O₂-dependent peroxidase activity assay evaluation against substrate 2,4,6-trichlorophenol. <i>Ao</i>DHP isoenzyme A was also overexpressed as the recombinant protein in <i>Escherichia coli</i>, and its peroxidase activity parameters were compared to <i>Ao</i>DHP from the natural source. The activity assay assessment indicated a tight correlation for all Michaelis-Menten parameters evaluated. We conclude that the method described herein exhibits a streamlined approach to identify the polychaete <i>A. ornata</i>, which can be adopted by the non-specialist, and the full procedure is predicted to facilitate the discovery of novel dehaloperoxidases from other marine invertebrates.