Work

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

Year: 2024

Type: article

Abstract: The multifunctional catalytic hemoglobin from the terebellid polychaete Amphitrite ornata , also named dehaloperoxidase ( Ao DHP), utilizes the typical oxygen transport function in addition to four observed activities involved in substrate oxidation. The multifunctional ability of Ao 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 A. ornata , 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 particularly simplifies the taxonomic identification of A. ornata through the assessment of its mitochondrial cytochrome c oxidase subunit I gene by employing the DNA barcoding technique. Furthermore, whole worm specimens of A. ornata were used to extract and purify Ao DHP followed by an H 2 O 2 -dependent peroxidase activity assay evaluation against substrate 2,4,6-trichlorophenol. Ao DHP isoenzyme A was also overexpressed as the recombinant protein in Escherichia coli , and its peroxidase activity parameters were compared to Ao 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 A. ornata , 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.

Source: Methods and Protocols

DOI: 10.3390/mps7060100