Husted Anna L, Sutton Victoria R, Presnar Lauren A, Blackburn R Kevin, Staton Joseph L, Borgianini Stephen A, D'Antonio Edward L
Department of Natural Sciences, University of South Carolina Beaufort, 1 University Boulevard, Bluffton, SC 29909, USA.
Department of Molecular and Structural Biochemistry, North Carolina State University, 120 W Broughton Drive, Raleigh, NC 27607, USA.
Methods Protoc. 2024 Dec 11;7(6):100. doi: 10.3390/mps7060100.
The multifunctional catalytic hemoglobin from the terebellid polychaete , also named dehaloperoxidase (DHP), utilizes the typical oxygen transport function in addition to four observed activities involved in substrate oxidation. The multifunctional ability of 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 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 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 were used to extract and purify DHP followed by an HO-dependent peroxidase activity assay evaluation against substrate 2,4,6-trichlorophenol. DHP isoenzyme A was also overexpressed as the recombinant protein in , and its peroxidase activity parameters were compared to 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 , 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.
来自蛰龙介科多毛纲动物的多功能催化血红蛋白，也被称为脱卤过氧化物酶（DHP），除了具有参与底物氧化的四种已知活性外，还具备典型的氧气运输功能。DHP的多功能性目前较为罕见，而且从大型底栖动物中鉴定新型脱卤过氧化物酶存在一定局限性。为了发现更多携带DHP的底栖动物候选物种，我们设计了一种简便方法，将视觉分类和分子分类方法并行使用，以实现准确的分类鉴定。非专业人员通过传统视觉分类法对物种进行鉴定，至少对于[具体物种名称缺失]甚至其他海洋蠕虫来说，是一项非常困难且耗时的任务，因为存在大量的物种多样性，并且该方法仅限于成年蠕虫标本。本文的工作旨在描述一种方法，特别是通过利用DNA条形码技术评估其线粒体细胞色素c氧化酶亚基I基因，来简化[具体物种名称缺失]的分类鉴定。此外，使用[具体物种名称缺失]的全虫标本提取并纯化DHP，随后针对底物2,4,6 - 三氯苯酚进行依赖血红素加氧酶的过氧化物酶活性测定评估。DHP同工酶A也作为重组蛋白在[具体表达系统缺失]中过表达，并将其过氧化物酶活性参数与天然来源的DHP进行比较。活性测定评估表明，所有评估的米氏参数之间存在紧密相关性。我们得出结论，本文所述方法展示了一种简化的方法来鉴定多毛纲动物[具体物种名称缺失]，非专业人员可以采用，并且整个过程预计将有助于从其他海洋无脊椎动物中发现新型脱卤过氧化物酶。
