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Measuring Phenol Oxidase and Peroxidase Activities with Pyrogallol, L-DOPA, and ABTS: Effect of Assay Conditions and Soil Type

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Measuring Phenol Oxidase and Peroxidase Activities with Pyrogallol, L-DOPA, and ABTS: Effect of Assay Conditions and Soil Type UC Irvine UC Irvine Previously Published Works Title Measuring phenol oxidase and peroxidase activities with pyrogallol, l-DOPA, and ABTS: Effect of assay conditions and soil type Permalink https://escholarship.org/uc/item/3nw4m85r Authors Bach, CE Warnock, DD Van Horn, DJ et al. Publication Date 2013-12-01 DOI 10.1016/j.soilbio.2013.08.022 Peer reviewed eScholarship.org Powered by the California Digital Library University of California SBB5589_grabs ■ 10 September 2013 ■ 1/1 Soil Biology & Biochemistry xxx (2013) 1 Contents lists available at ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio 1 2 3 4 Highlights 5 6 We tested whether pH and redox potential affect three common oxidase substrates. 7 Pyrogallol and ABTS were useless under alkaline conditions for different reasons. 8 L-DOPA appears to be stable for use across a broad range of pH. 9 Autoclaved and combusted soils cannot be used as negative controls. 10 Current “oxidase” methods measure a soil property more so than enzyme activity. 11 12 0038-0717/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.soilbio.2013.08.022 Please cite this article in press as: Bach, C.E., et al., Measuring phenol oxidase and peroxidase activities with pyrogallol, L-DOPA, and ABTS: Effect of assay conditions and soil type, Soil Biology & Biochemistry (2013), http://dx.doi.org/10.1016/j.soilbio.2013.08.022 SBB5589_proof ■ 10 September 2013 ■ 1/9 Soil Biology & Biochemistry xxx (2013) 1e9 Contents lists available at ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio 1 56 2 Measuring phenol oxidase and peroxidase activities with pyrogallol, 57 3 58 4 L-DOPA, and ABTS: Effect of assay conditions and soil type 59 5 60 6 a b b c 61 Q5 Christopher E. Bachh , Daniel D. Warnockk , David J. Van Hornn , Michael N. Weintraubb , 7 Robert L. Sinsabaughh b, Steven D. Allisonn a, d, Donovan P. Germann a, * 62 8 63 9 a Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, USA 64 b 10 Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA 65 c 11 Department of Environmental Sciences, University of Toledo, Toledo, OH 43606, USA 66 d Department of Earth System Science, University of California, Irvine, CA 92697, USA 12 67 13 68 14 69 article info abstract 15 70 16 71 Article history: Microbial phenol oxidases and peroxidases mediate biogeochemical processes in soils, including mi- 17 Received 25 July 2012 crobial acquisition of carbon and nitrogen, lignin degradation, carbon mineralization and sequestration, 72 18 Received in revised form and dissolved organic carbon export. Measuring oxidative enzyme activities in soils is more problematic 73 23 August 2013 19 than assaying hydrolytic enzyme activities because of the non-specific, free radical nature of the re- 74 Accepted 28 August 2013 20 actions and complex interactions between enzymes, assay substrates, and the soil matrix. We compared 75 Available online xxx 21 three substrates commonly used to assay phenol oxidase and peroxidase in soil: pyrogallol (PYGL, 76 1,2,3-trihydroxybenzene), L-DOPA (L-3,4-dihydroxyphenylalanine), and ABTS (2,2 -azino-bis(3- 22 Keywords: 0 77 23 Laccase ethylbenzthiazoline-6-sulfonici acid). We measured substrate oxidation in three soils across a pH 78 24 Enzyme assay methods gradient from 3.0 to 10.0 to determine the pH optimum for each substrate. In addition, we compared 79 25 Extracellular enzymes activities across 17 soilsi using the three substrates. In general, activities on the substrates followed the 80 trend PYGL > L-DOPA > ABTS and were inversely related to substrate redox potential. PYGL and ABTS 26 Lignin 81 Q1 Decomposition were not suitable substrates at pH > 5, and ABTS oxidation often declined with addition of peroxide to 27 82 the assay. Absolute and relative oxidation rates varied widely among substrates in relation to soil type 28 and assay pH. We also tested whether autoclaved or combusted soils could be used as negative controls 83 29 for the influence of abiotic factors (e.g., soil mineralogy) on oxidative activity. However, neither auto- 84 30 claving nor combustion produced reliable negative controls because substrate oxidation still occurred; in 85 31 some cases, these treatments enhanced substrate oxidation rates. For broad scale studies, we recommend 86 32 that investigators use all three substrates to assess soil oxidation potentials. For focused studies, we 87 33 recommend evaluating substrates before choosing a single option, and we recommend assays at both the 88 34 soil pH and a reference pH (e.g., pH 5.0) to determine the effect of assay pH on oxidase activity. These 89 35 recommendations should contribute to greater comparability of oxidase potential activities across 90 36 studies. 91 Ó 2013 Published by Elsevier Ltd. 37 92 38 93 39 94 40 95 41 1. Introduction cellular processes and defense, as well as carbon (C) and nitrogen 96 42 (N) acquisition (Sinsabaugh, 2010). Once in the soil environment, 97 43 Extracellular enzymes are the proximate agents of organic these enzymes mediate the biogeochemical processes of lignin 98 44 matter decomposition, and thus the activities of hydrolytic en- degradation, carbon mineralization and sequestration, and dis- 99 45 zymes (e.g. cellulases, phosphatases, chitinases, proteases) are solved organic C export. 100 46 widely measured in ecosystem studies (German et al., 2011). In Phenol oxidases oxidize phenolic compounds using oxygen as 101 47 contrast, phenol oxidase and peroxidase activities have been an electron acceptor. These enzymes include fungal laccases and 102 48 measured in a smaller number of soil enzyme studies. Fungi and prokaryotic laccase-like enzymes that typically have multiple 103 49 bacteria express oxidases for a variety of functions, including copper (Cu) atoms at the reaction center (Baldrian, 2006; Hoegger 104 50 et al., 2006). The ability of a laccase to oxidize a particular substrate 105 fi 51 is de ned by the parameter kcat, or the number of substrate mol- 106 52 * Corresponding author. Tel.: 1 9498245368; fax: 1 9498242181. ecules oxidized per enzyme active site per unit time. This param- 107 þ þ 0 53 E-mail address: [email protected] (D.P. German). eter is related to the difference in redox potential (DE ) between the 108 54 109 e 55 0038-0717/$ see front matter Ó 2013 Published by Elsevier Ltd. 110 http://dx.doi.org/10.1016/j.soilbio.2013.08.022 Please cite this article in press as: Bach, C.E., et al., Measuring phenol oxidase and peroxidase activities with pyrogallol, L-DOPA, and ABTS: Effect of assay conditions and soil type, Soil Biology & Biochemistry (2013), http://dx.doi.org/10.1016/j.soilbio.2013.08.022 SBB5589_proof ■ 10 September 2013 ■ 2/9 2 C.E. Bach et al. / Soil Biology & Biochemistry xxx (2013) 1e9 111 first Cu in the reaction chain (designated T1) and the substrate (Xu, 176 112 1997). Electrons are transferred from the T1 Cu to T2 and T3 reac- 177 113 tion centers and ultimately used to reduce molecular oxygen to 178 114 water (Baldrian, 2006). Thus, in laccases, redox potential is a 179 115 measure of the potential for a reaction center to acquire electrons. 180 116 The redox potential of laccases ranges widely (450e800 mV), as do 181 117 pH optima (4.0e7.5) and substrate preferences, because the poly- 182 118 peptide configuration that surrounds the redox center is highly 183 119 variable (Baldrian, 2006). As pH increases beyond the optimum, 184 120 hydroxyl ions inhibit the transfer of electrons from the T1 Cu to the 185 121 T2/T3 reaction centers, which progressively reduces enzyme ac- 186 122 tivity (Xu, 1997; Eichlerová et al., 2012). The redox potentials of 187 123 laccases and similar enzymes are too low to directly oxidize the 188 124 non-phenolic linkages of lignin. However, laccases catalyze the 189 125 production of a variety of organic radicals, called redox mediators, 190 126 that are able to break non-phenolic linkages and thereby depoly- 191 127 Q2 merize lignin (Bourbonnais et al., 1998; Leonowicz et al., 2001; 192 128 Camarero et al., 2005). 193 129 Peroxidases such as manganese peroxidase and lignin peroxi- 194 130 dase use H2O2 as an electron acceptor. These enzymes have Fe- 195 131 containing heme prosthetic groups with redox potentials up to 196 Fig. 1. Expected trends for oxidative enzyme activity based on pH optima and redox 132 1490 mV, giving them the capacity to break aryl and alkyl bonds 197 potentials of phenol oxidases and peroxidases and the redox-pH relationships of the 133 198 within lignin either directly or through redox intermediates such as substrates 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), L-3,4- 3 134 Mn þ (Kersten et al., 1990; Higuchi, 1990; Rabinovich et al., 2004). dihydroxyphenylalanine (L-DOPA) and pyrogallol (PYGL) (Xu, 1997; Gao et al., 1998; 199 135 The pH optimum corresponding to the maximum redox potential of Riahi et al., 2007). Enzyme redox potential is shown as a solid line and substrates 200 136 Phanerochaete lignin peroxidase is about 3.0 (Oyadomari et al., are shown as dashed lines. The non-phenolic linkages of lignin are oxidized at redox 201 2 potentials >1000 mV. Some phenol oxidases can generate soluble redox mediators 137 2003). Manganese peroxidase, which oxidizes Mn þ, has a pH op- whose redox potentials are comparable to those of peroxidases. 202 138 timum of 4.5 (Mauk et al., 1998). 203 139 In soils, potential phenol oxidase activity is typically measured 204 140 as the rate of oxidation of a model substrate added to soil sus- rarely used concurrently, which limits comparisons of oxidative 205 141 pensions (German et al., 2011; Burns et al., 2013).
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