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Abstract
Among planktonic communities haloperoxidase enzymes may play a role in the control of intracellular and extracellular reactive oxygen species, in the generation of halogenated organic compounds and in chemical interactions between microbes. We introduce a sensitive fluorometric assay with a large dynamic range that is based on the dearylation of aminophenyl fluorescein (APF) to fluorescein by highly reactive oxygen species. Bromoperoxidase and chloroperoxidase enzymes catalyze the reaction between hydrogen peroxide and halides to generate highly reactive hypohalite intermediates able to dearylate APF. The fundamentals and standardization of the approach are illustrated using a partially purified, vanadium-dependent bromoperoxidase from the red seaweed Corallina officinalis. Laboratory cultures of two polar diatoms, Porosira glacialis and Fragilariopsis cylindrus, are used to illustrate the sensitivity and potential applications of the approach for in vitro, in vivo and in situ measurements of bromoperoxidase activity. These two diatoms differ in biovolume-specific bromoperoxidase activity by 2-orders of magnitude, from 5.4 to 0.044 fmol fluorescein μm-3 h-1, respectively. The approach is also used to investigate the partition of haloperoxidase activity between different size fractions of summer coastal planktonic communities, illustrating that generally more than 50 % of the haloperoxidase activity occurred in a > 10 μm size fraction that was dominated by diatoms. The assay has the potential to be of value in many aspects of haloperoxidase research, including developing an improved understanding of the roles of haloperoxidase enzymes in microbial planktonic communities.
Highlights
Haloperoxidase enzymes (HPO) catalyze the oxidation of halides by hydrogen peroxide (H2O2) to form a hypohalite (ClO−, BrO−, IO−) intermediate (1) that can react rapidly with organic substrates to produce halogenated compounds (2) or react with excess H2O2 to generate singlet oxygen (1O2) (Hewson and Hager, 1980; Everett et al, 1990)
The present study aimed to demonstrate the efficacy of a novel assay for BrPO activity, based on the fluorescent probe [6-(4 -amino)phenoxy-3H-xanthen-3-on9-yl]benzoic acid [aminophenyl fluorescein (APF)], designed to selectively detect highly reactive oxygen species (Setsukinai et al, 2003)
The conversion of non-fluorescent APF to fluorescein through the production of HOBr by V-BrPO of C. officinalis is shown by increases in fluorescence following the addition of H2O2 to the enzyme assay mixture at approximately 50 s after initiation of data collection (Figure 2A)
Summary
Haloperoxidase enzymes (HPO) catalyze the oxidation of halides by hydrogen peroxide (H2O2) to form a hypohalite (ClO−, BrO−, IO−) intermediate (1) that can react rapidly with organic substrates to produce halogenated compounds (2) or react with excess H2O2 to generate singlet oxygen (1O2) (Hewson and Hager, 1980; Everett et al, 1990). Haloperoxidases are generally metalloenzymes with either heme or vanadium cofactors, enzymes not requiring a metal co-factor occur in some bacteria (Littlechild, 1999). Vanadium-bromoperoxidases (V-BrPO) appear to be the most common form of haloperoxidase in the marine environment (Leblanc et al, 2015). V-BrPO activity has been demonstrated to occur in a variety of diatom species (Moore et al, 1996; Murphy et al, 2000; Hill and Manley, 2009) and a functional V-BrPO has been characterized in several strains of the globally distributed marine cyanobacterium Synechococcus sp. Information on ClPOs among planktonic microbes is limited, ClPOs have been characterized from sediment-associated bacteria (Winter et al, 2007; Bernhardt et al, 2011)
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