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

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Title Measuring phenol oxidase and peroxidase activities with pyrogallol, l-DOPA, and ABTS: Effect of assay conditions and soil type

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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

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Soil Biology & Biochemistry xxx (2013) 1

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Soil Biology & Biochemistry

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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

Soil Biology & Biochemistry xxx (2013) 1e9

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Soil Biology & Biochemistry

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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

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). Peroxidase ac- activity across studies (Eichlerová et al., 2012). When comparisons 206 142 tivity is measured as the rate of substrate oxidation in the presence have been made for particular soils or litter types, the substrates 207 143 of added H2O2 (Burns et al., 2013). Because this gross activity pre- tend not to produce mutually consistent results, and absolute and 208 144 sumably includes both phenol oxidase and peroxidase activities, relative oxidation rates vary widely among substrates in relation to 209 145 the phenol oxidase activity is subtracted from the gross activity to soil type and assay pH (Sinsabaugh, 2010). In our experience, oxi- 210 146 estimate the net peroxidase contribution. The three most dase assays often show no detectable activity with one substrate 211 147 commonly used substrates for phenol oxidase and peroxidase as- whereas another appears to work without interference. Given the 212 148 says in soil are pyrogallol (PYGL, 1,2,3-trihydroxybenzene), L-DOPA variation in oxidative enzyme activities among soils and substrates, 213 149 (L-3,4-dihydroxyphenylalanine), and ABTS (2,20-azino-bis(3- a negative control would be useful for determining the influence of 214 150 ethylbenzthiazoline-6-sulfonic acid); Sinsabaugh, 2010; Burns abiotic factors, such as soil mineralogy. However, negative controls 215 151 et al., 2013). Of these substrates, PYGL has the lowest redox po- for these assays have been poorly evaluated. 216 152 tential and is therefore the most easily oxidized. PYGL redox po- To assess the magnitude of these problems and provide meth- 217 153 tential decreases rapidly from 560 mV to 200 mV as pH increases odological recommendations to investigators, we compared the 218 À 154 from 1.5 to 8.5, meaning that PYGL vulnerability to oxidation in- oxidation of PYGL, L-DOPA, and ABTS across a range of assay con- 219 155 creases with increasing pH (Gao et al., 1998; Riahi et al., 2007) ditions and soil types. We aimed to determine whether relative 220 156 (Fig. 1). The redox potential of L-DOPA is less sensitive to pH, phenol oxidase and peroxidase activities can be predicted based on 221 157 declining from 525 to 460 mV over the pH range from 2.0 to 8.0 pH and the redox potential of the substrate (Fig. 1). We compared 222 158 (Serpentinia et al., 2000). ABTS has the highest redox potential substrate oxidation rates at pH values ranging from 3.0 to 10.0 in 223 159 (E0 1080 mV) (Bourbonnais et al., 1998), and because it has soils from Alaska, California, and Costa Rica and evaluated the 224 ¼ 160 no protic groups, redox potential of ABTS does not vary with pH appropriateness of autoclaved and combusted soils from these sites 225 161 (Xu, 1997). as negative controls. We chose these soils to observe whether 226 162 For any specific enzymeesubstrate combination, the optimal pH trends in substrate oxidation as a function of pH were consistent 227 163 for oxidation is a function of both the pH optimum of the enzyme across soils displaying an array of different conditions (e.g., pH, 228 164 and the response of substrate redox potential to pH. For phenol moisture content, and organic matter content). We also compared 229 165 oxidases, substrates with multiple protic groups (e.g., PYGL) should oxidation rates of PYGL, L-DOPA and ABTS in 17 soils that ranged 230 166 have maximum oxidation rates shifted toward greater pH because widely in bulk soil pH, organic matter content and other variables 231 167 the redox potential of these substrates declines with increasing pH, (Table 1). 232 168 resulting in a greater DE between the T1 Cu of the enzyme and the 233 169 substrate. On the other hand, enzymatic oxidation of non-phenolic 2. Materials and methods 234 170 substrates like ABTS should only depend on the pH optimum of the 235 171 enzyme because redox potential of these substrates does not 2.1. Enzyme assay protocol 236 172 depend on pH (Xu, 1997; Fig. 1). Similar trends should apply to 237 173 peroxidases. Enzyme assays were performed following published microplate 238 174 Although PYGL, L-DOPA and ABTS are commonly used to assay protocols (Gallo et al., 2004; Allison and Jastrow, 2006; Floch et al., 239 175 the phenol oxidase and peroxidase potentials of soils, they are 2007). Soil suspensions were prepared by homogenizing 1 g of 240

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241 Table 1 306 242 Potential phenol oxidase and gross peroxidase activities for 17 soils measured with three substrates: PYGL (pyrogallol, 1,2,3-trihydroxybenzene), L-DOPA (L-3,4- 307 dihydroxyphenylalanine), and ABTS (2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). 243 0 308 244 Sample origin Coordinates SOM (%) Bulk pH Phenol oxidase Peroxidase Phenol oxidase Peroxidase 309 245 Assay pH 5.0 Assay pH 5.0 Assay pH 8.0 Assay pH 8.0 310 246 311 PYGL DOPA ABTS PYGL DOPA ABTS DOPA DOPA 247 312 a 248 Boreal Forest, AK 63.91N 145.73W 16.80 5.0 N.D. N.D. 0.44 5.48 1.66 0.83 N.D. 5.0 313 Hardwood Forest, OH 41.66 N 83.78 W 7.40 6.6 3.21 N.D. 0.38 15.4 12.3 0.36 7.3 49.6 249 314 Hardwood Forest, OH 41.66N 83.78W 7.40 6.6 N.D. N.D. N.D. 31.8 3.79 N.D. 9.7 32.5 250 Hardwood Forest, OH 41.57N 83.86W 4.60 6.1 6.43 0.38 0.64 19.7 8.10 1.57 17.6 30.9 315 251 Tall Grass Prairie, OH 41.65N 83.77W 10.40 7.2 N.D. N.D. 1.93 N.D. N.D. 0.54 57.9 99.4 316 252 Maple Litter Microcosm 41.68N 83.71W 6.20 4.5 1.72 N.D. N.D. 7.50 0.65 N.D. 10.7 19.8 317 253 Semiarid Grassland, NM 34.40N 106.68W 1.80 8.2 10.8 5.75 0.39 24.2 2.17 0.22 140.0 267.0 318 Creosote Shrubland, NM 34.35 N 106.69 W 2.40 7.5 5.11 2.72 N.D. 4.54 0.25 N.D. 69.5 29.6 254 319 Juniper Savannah, NM 34.43N 105.86W 3.70 7.2 N.D. N.D. 0.38 5.59 0.05 N.D. 60.2 441.0 255 Piñon Juniper Woodland, NM 34.36N 106.27W 6.00 6.0 13.3 7.08 0.48 4.94 N.D. N.D. 1.7 141.0 320 256 Ponderosa Pine Forest, NM 35.86N 106.60W 11.60 5.5 3.30 1.75 0.03 8.04 8.56 0.41 63.6 481.0 321 Subalpine Spruce Forest, NM 35.89N 106.53W 4.80 5.2 3.25 1.73 0.30 2.87 0.59 N.D. 32.0 497.0 257 a 322 Semiarid Grassland, CA 33.73N 117.70W 3.10 6.8 N.D. N.D. 0.50 5.53 1.79 N.D. 11.3 19.7 258 a 323 Trop. Rainforest, Costa Rica 8.71N 83.61W 15.10 5.8 1.83 1.42 3.63 13.3 5.75 2.37 N.D. N.D. 259 Antarctic Dry Valley, Antarctica 77.61S 163.00E 0.10 10.0 2.57 0.09 0.01 3.11 2.70 0.21 52.3 160.0 324 260 Antarctic Dry Valley, Antarctica 77.61S 163.00E 0.11 8.7 0.33 0.17 0.06 1.55 4.54 0.11 3.2 63.1 325 261 Antarctic Dry Valley, Antarctica 77.61S 163.00E 0.02 7.9 0.03 N.D. 0.05 7.32 N.D. 0.32 N.D. N.D. 326 262 Mean 4.32 2.34 0.66 10.05 3.78 0.69 38.36 155.77 327 Standard error 1.17 0.82 0.26 2.15 0.95 0.23 10.69 47.78 263 328 AK Alaska, USA. OH Ohio, USA. NM New Mexico, USA. CA California, USA. 264 ¼ ¼ 1 ¼ ¼ 329 Activity units are mmol hÀ g dry soil. Because sample size was low (n 1) in most locations, no error values are reported for individual sites. At pH 5, two-way ANOVA on ¼ 265 log 1 transformed data showed a significant effect of substrate (Phenol oxidase F 11.32, P < 0.001; Peroxidase F 32.27, P < 0.001) on enzyme activities, whereas site 330 þ 2,17 ¼ 2,21 ¼ 266 was significant for peroxidase only (Phenol oxidase: F 1.86, P 0.109; Peroxidase: F 2.48, P 0.026). N.D. Not detectable. 331 15,27 ¼ ¼ 16,21 ¼ ¼ ¼ 267 a Denotes sample origin used in the pH gradient study. 332 268 333 269 334 270 fresh soil in 125 ml of buffer using a hand held Bamix Homogenizer Peninsula, Costa Rica (8.71N, 83.61W). Three replicate samples 335 (BioSpec Products, Bartlesville OK, USA). Enzyme activities were were taken with a soil corer (2.5 cm diameter 5 cm depth), at 271 Â 336 272 measured by combining 200 mL of soil suspension with 50 mL of least 10 m apart, from each sampling location, individually sealed in 337 273 substrate solution. Controls included in each microplate were: plastic bags, and homogenized by hand before freezing. Detailed 338 substrate buffer, soil suspension buffer, and buffer-only blank. descriptions of these soils and their handling can be found in 274 þ þ 339 275 For peroxidase assays, all wells received 10 mL of 0.3% hydrogen German et al. (2012), and bulk soil properties are shown in Table 1. 340 276 peroxide, including controls. Assays were incubated for 3 or 4 h Assays were conducted using PYGL, L-DOPA, and ABTS at pH values 341 277 (depending on the experiment; see below) at the recorded tem- from 3.0 to 10.0, at 0.5 pH unit intervals, with and without the 342 278 perature of the site where the sample was collected. Preliminary addition of 10 mL of 0.3% hydrogen peroxide. We used 50 mM so- 343 fi 279 assays revealed that incubation times of 3e4 h were suf cient to dium acetate buffer for pH values ranging from 3.0 to 5.0 and 344 280 produce linear accumulation of reaction products over time. Both adjusted the pH with glacial acetic acid. We used 25 mM maleic 345 281 shorter and longer incubation times were investigated, and results acid buffer for pH values ranging from 5.5 to 7.5, and adjusted the 346 282 from longer incubations tended to be less reliable, as the accumu- pH with 3 M hydrochloric acid. We used 50 mM sodium bicar- 347 283 lation of reaction product over time often became non-linear in bonate buffer for pH values ranging from 8.0 to 10.0, and adjusted 348 284 longer incubations. Buffers were 50 mM sodium acetate, 25 mM the pH with 3 M hydrochloric acid or 10 M sodium hydroxide so- 349 285 maleic acid disodium salt hydrate, or 50 mM sodium bicarbonate lution. Assays were incubated for 3 h (a duration that was 350 fi 286 (depending on assay pH; see below). Substrates were purchased con rmed to produce a linear increase in absorbance over time) in 351 287 from SigmaeAldrich (St. Louis, Missouri, USA) and solutions were the absence of light at temperatures appropriate for each site: 10 C 352 288 freshly prepared for each trial (5 mM PYGL, 25 mM L-DOPA, or for Alaska, 22 C for California, and 28 C for Costa Rica (German 353 289 2 mM ABTS in deionized water), following the published protocols et al., 2011, 2012). Absorbance was read at 3 h, and potential 354 fi 290 cited above. Micromolar extinction coef cients were determined phenol oxidase and peroxidase activities were calculated as 355 291 for the oxidation products of each substrate, as described in the described previously (German et al., 2011). 356 292 protocols, and calculated on the basis of mmole of product with a 357 293 path length of 10 mm (German et al., 2011). The extinction co- 2.3. Potential negative controls for phenol oxidase and peroxidase 358 fi 294 ef cients used in our calculations were 4.2 for PYGL, 7.9 for L-DOPA assays 359 295 and 73 for ABTS, which are similar to values reported in the liter- 360 296 ature (Allison and Vitousek, 2004; DeForest, 2009; Saiya-Cork et al., Approximately 10 g of soil from Alaska, California, and Costa 361 297 2002; Shi et al., 2006; Yao et al., 2009). Activity values were 362 1 1 Rica were either autoclaved for 30 min at 121 C or combusted at calculated as mmol hÀ gÀ soil. Soil replicates chosen from each 298 550 C for 3 h to destroy oxidative enzymes. Combustion destroys 363 location were stored after the initial collection at 80 C and sub- 299 À enzymes by oxidizing and removing all organic compounds, 364 sequently stored at 20 C until analysis. 300 À including enzymes, from soil. Autoclaving is often used to perma- 365 301 nently denature proteins using heat and pressure (Sinsabaugh, 366 302 2.2. Activity as a function of pH 2010), although it is possible that mineral-stabilized enzymes 367 303 could withstand autoclaving (Stursova and Sinsabaugh, 2008). 368 304 Soils were collected from Delta Junction, Alaska (63.91N, These ‘negative control’ soils were assayed at pH 5.0 using 50 mM 369 305 145.73W), Irvine, California (33.73N, 117.70W), and Nicoya sodium acetate buffer under the same temperature conditions as 370

4 C.E. Bach et al. / Soil Biology & Biochemistry xxx (2013) 1e9

371 the fresh soils using PYGL, L-DOPA, and ABTS as substrates. Absor- California soils (Table 2). Peroxidase activities were measurable 436 372 bance was read at 30 min intervals for 3 h, and the absorbance was with ABTS in Alaskan soils at pH values under 6.0, but were near 437 373 regressed against time for each substrate and soil collection loca- zero in Costa Rican and Californian soils (Fig. 2). In Alaskan soil, 438 374 tion. Linear relationships between absorbance and time were taken there was no significant relationship between pH and peroxidase 439 375 as oxidative activity of a soil against a particular substrate. measured with ABTS. 440 376 441 377 2.4. Oxidase activity in diverse soils 3.2. Potential negative controls for phenol oxidase and peroxidase 442 378 assays 443 379 We collected soils from different ecosystems to compare the 444 380 behavior of each substrate in assays with different soil properties. The oxidative activity of autoclaved and combusted soils varied 445 381 One soil sample (n 1) was collected by corer or auger (to a depth with soil and substrate. For Alaskan soils, autoclaved samples 446 ¼ 382 of 10 cm) from 17 locations, representing a diverse range of vege- readily oxidized PYGL and L-DOPA with and without peroxide, 447 383 tation and soil types (Table 1). There was no plot-scale replication showing steady increases in absorbance over time, with R2 values 448 384 within sites because samples were collected as part of a broad 0.75 (Table 3). Moreover, the slopes of these lines were greater 449 385 survey for a different study. Following collection, all soil samples than those of the native soil, showing that greater oxidation was 450 386 were frozen at 20 C until analyzed. For all soils, assays were detected in the potential negative controls than in the soils con- 451 À 387 conducted at pH 5 using 50 mM sodium acetate buffer and at pH 8 taining active oxidases and peroxidases. ABTS was not oxidized by 452 388 using 50 mM sodium bicarbonate buffer. For each substrate, assays autoclaved Alaskan soils in the absence of peroxide, but peroxide 453 389 were conducted with and without addition of hydrogen peroxide. addition provided a linear increase in absorbance over time against 454 390 Assays were read in a spectrophotometer at 4 h; detailed kinetic this substrate, again, with the autoclaved soils showing greater 455 391 analyses were not conducted for this part of the study. Readings activity than the native soil. Autoclaved Californian and Costa Rican 456 392 were adjusted for absorbance due to substrate and homogenate soils showed no oxidative activity against any substrate in the 457 393 controls, as described in the assay protocol section. absence of peroxide, but showed activity against PYGL and L-DOPA 458 394 in the presence of peroxide, with R2 values 0.65 (Table 3). The 459 395 2.5. Statistical analysis autoclaved Costa Rican soils had lower slopes than the native soils 460 396 in the presence of peroxide. ABTS was not oxidized by autoclaved 461 397 To measure the effects of pH on substrate oxidation in soils from Californian or Costa Rican soils. 462 398 Alaska, California, and Costa Rica, we performed linear regression Combusted Alaskan soil showed oxidative activity only against 463 399 analyses to obtain slope, R2 values, and associated P-values for the PYGL without peroxide, but oxidized all three substrates in the 464 400 dependence of oxidation on pH. We used two-way ANOVA to presence of peroxide, with R2 values 0.85 (Table 3). As with 465 401 examine the effects of soil collection site and assay substrate on autoclaved soils, the oxidation shown by the combusted soils in the 466 402 measured phenol oxidase and peroxidase activities in diverse soils. presence of peroxide exceeded the oxidation observed in the native 467 403 To satisfy normality assumptions, data were log 1 transformed soils. In contrast, combusted Californian soils showed no oxidative 468 þ 404 before running the ANOVA. P-values below 0.05 were considered activity against PYGL, and variable oxidation against ABTS, all in the 469 405 significant. absence of peroxide. L-DOPA was oxidized by combusted Cali- 470 406 fornian soils, with or without peroxide, with R2 values 0.66, and 471 407 3. Results all three substrates were oxidized in the presence of peroxide 472 408 (Table 3). Oxidation rates in combusted Californian soil exceeded 473 409 3.1. Activity levels across a pH gradient oxidation rates in the native soils with added peroxide. With the 474 410 exception of ABTS in the presence of peroxide, combusted Costa 475 411 Phenol oxidasedPYGL oxidation was most readily detected at Rican soils readily oxidized all substrates, with R2 values 0.90 476 412 pH values ranging from 6 to 7 (Alaska and California), but was also (Table 3). In contrast to the other soils, the combusted Costa Rican 477 413 detected under more acidic conditions in Costa Rican soils (Fig. 2 soil had lower oxidative activity than the native Costa Rican soils 478 414 AeC). At pH > 7.5, PYGL rapidly auto-oxidized, making activity with added peroxide. 479 415 calculations impossible under alkaline conditions with this sub- See Supplemental Tables S1, S2, and S3 in the online version of 480 416 strate. Overall no significant trends in PYGL oxidation with respect this article for the complete dataset on the autoclaved and com- 481 417 to pH were found at any of the sites. In Alaskan soil with a native pH busted soils. 482 418 of 5.0, L-DOPA oxidation was limited to pH values ranging from 3.5 483 419 to 7.0, whereas oxidation was detected under more alkaline pH 3.3. Oxidase activities in diverse soils 484 420 values (5.0e9.0) for soils from California and Costa Rica (Fig. 2 Ae 485 421 C). Similar to PYGL, no significant linear trends in phenol oxidase Consistent with our pH gradient analysis, rapid auto- 486 422 activity with pH were found for L-DOPA. ABTS oxidation rates oxidation precluded assays with PYGL at pH 8 in any soil. 487 423 decreased significantly (P < 0.05) with pH in Alaskan and California Moreover, at pH 8, ABTS was not oxidized in any soil with or 488 424 soils, and ABTS was not oxidized above pH values of 7.5 in any soil without peroxide. Thus, L-DOPA was the only substrate suitable 489 425 (Fig. 2 AeC; Table 2). for assays at both pH 5 and 8. For soils with measurable phenol 490 426 PeroxidasedWhen using PYGL as a substrate, peroxidase ac- oxidase activity at both pH values, oxidation rates at pH 8 were 491 427 tivity was detectable between pH values of 3.0 and 7.5, even when 93.90 65.77 (mean SEM) times greater than rates at pH 5. 492 Æ Æ 428 phenol oxidase activity was undetectable in a particular soil (Fig. 2). With the exception of two soils that showed no activity at either 493 429 With PYGL there was a significant negative relationship between pH, soils with no detectable activity at pH 5 showed measurable 494 430 net peroxidase activity and assay pH for soils collected from Alaska activity at pH 8 (Table 1). With peroxide addition, L-DOPA 495 431 and Costa Rica (Table 2; Fig. 2DeF). In California soils, the rela- oxidation at pH 8 was 776.42 673.21 times greater than activity 496 Æ 432 tionship between activity and pH switched from negative to posi- at pH 5 for soils with measurable activity at both pH values. 497 433 tive at pH 5.5 (P 0.01; Table 2; Fig. 2E). For L-DOPA, there were Furthermore, soils with no detectable peroxidase activity at pH 5 498 ¼ 434 positive relationships between peroxidase activity and pH above displayed activity at pH 8, except one soil (from the McMurdo 499 435 pH 6.0 (Fig. 2 DeF). This trend was significant for Alaska and Dry Valleys, Antarctica) that had no activity at either pH. At pH 5, 500

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.2013.08.022 526 525 524 523 522 521 520 519 518 517 516 515 514 513 512 511 510 509 508 507 506 505 504 503 502 501 565 564 563 562 561 560 559 558 557 556 555 554 553 552 551 550 549 548 547 546 545 544 543 542 541 540 539 538 537 536 535 534 533 532 531 530 529 528 527 laect hsatcei rs s ah .. ta. esrn hnloiaeadprxds ciiiswt pyrogallol, with activities peroxidase and oxidase phenol Measuring al., et C.E., Bach, as: press in article this cite Please fasycniin n oltp,Si ilg iceity(03,http://dx.doi.org/10.1016/j.soilbio.2013.08.022 (2013), Biochemistry & Biology Soil type, soil and conditions assay of ..Bc ta./Si ilg iceityxx(03 1 (2013) xxx Biochemistry & Biology Soil / al. et Bach C.E. e 9 SBB5589_proof ■ 0Spebr2013 September 10 L

DP,adAT:Effect ABTS: and -DOPA, Fig. 2. Phenol oxidase (Panels AeC) and peroxidase (Panels DeF) activities as a function of pH in soils collected from Alaska, California and Costa Rica. Activities were determined using pyrogallol (PYGL), L-3,4-dihydroxyphenylalanine (L-DOPA) and 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrates. Values are mean SEM (n 3 cores for each enzyme and pH). The inset shows high phenol oxidase activities measured using DOPA in California 0 Æ ¼ soils at pH 8e9. ■ 5/9 5 601 600 599 598 597 596 595 594 593 592 591 590 589 588 587 586 585 584 583 582 581 580 579 578 577 576 575 574 573 572 571 570 569 568 567 566 630 629 628 627 626 625 624 623 622 621 620 619 618 617 616 615 614 613 612 611 610 609 608 607 606 605 604 603 602 SBB5589_proof ■ 10 September 2013 ■ 6/9

6 C.E. Bach et al. / Soil Biology & Biochemistry xxx (2013) 1e9

631 all substrates provided useable data (Table 1). Using two-way Table 2 696 2 632 ANOVA, we detected significant effects of substrate Slope, R and associated P-values for regressions of potential activity against pH for 697 phenol oxidase and net peroxidase; activity was measured with PYGL, L-DOPA and 633 (F 11.32, P < 0.001), but not site (F 1.86, P 0.109) on 698 2,17 ¼ 15,17 ¼ ¼ ABTS in soils from Alaska, California, and Costa Rica. Only significant trends in 634 phenol oxidase activities. Mean phenol oxidase activities varied substrate oxidation with respect to pH are reported along with the pH range over 699 635 inversely with substrate redox potential with a PYGL:L-DOP- which the trend occurred. 700 636 701 A:ABTS activity ratio of 7:4:1 (see Supplemental Table S4 in the Location Substrate pH R2 P-value n Trend 637 online version of this article for activity ratios for each site). 702 638 However, four of the 17 soils showed activity toward ABTS, but Phenolo oxidas 703 Delta Junction, AK ABTS 3.5e6.5 0.73 <0.01 7 Decreasing 639 not toward L-DOPA or PYGL, which have lower redox potentials. Irvine, CA ABTS 3.5e7.5 0.94 <0.01 9 Decreasing 704 640 Gross peroxidase activities differed by substrate (F 32.27, Net peroxidase 705 2,21 ¼ 641 P < 0.001) and site (F 2.48, P 0.026), and were also Delta Junction, AK PYGL 3.0e7.5 0.43 0.04 10 Decreasing 706 16,21 ¼ ¼ 642 inversely related to substrate redox potential; mean peroxidase Delta Junction, AK DOPA 6.0e8.5 0.73 0.03 6 Increasing 707 Irvine, CA PYGL 3.0e5.0 0.92 0.01 5 Decreasing 643 L 708 activity ratios (PYGL: -DOPA:ABTS) were 15:5:1. One soil showed Irvine, CA PYGL 5.5e7.5 0.94 0.01 5 Increasing 644 activity toward ABTS, but not toward L-DOPA or PYGL. Irvine, CA DOPA 6.0e10.0 0.75 <0.01 8 Increasing 709 645 Ratios of activity with versus without added peroxide ranged Nicoya Peninsula, CR PYGL 3.0e5.5 0.96 <0.01 6 Decreasing 710 646 widely by substrate. Excluding soils with no oxidase activity, ratios Nicoya Peninsula, CR DOPA 3.0e5.5 0.76 0.02 5 Decreasing 711 647 of oxidation rates with peroxide to those without peroxide were 712 648 generally lowest for ABTS (median 1.9) followed by PYGL (median 713 fi 649 3.1) and L-DOPA (median value 4.5 at pH 5 and 3.2 at pH 8). At pH 5, Despite dif culties in measuring oxidase activities consistently 714 650 four of five soils with no measurable phenol oxidase activity on across substrates and soil types, our data generally support the 715 651 PYGL showed activity with added peroxide. For L-DOPA, six of eight predictions illustrated in Fig. 1. Average oxidase activities across soil 716 652 soils with no measurable phenol oxidase activity showed peroxi- types at pH 5.0 were highest for PYGL, which has the lowest redox 717 653 dase activity; one soil (Piñon Juniper Woodland, NM) showed potential, and lowest for ABTS, which has the highest redox po- 718 654 phenol oxidase activity, but no detectable activity with added tential (Table 1; Table S4). In our pH study (Fig. 2), oxidative re- 719 655 peroxide. For ABTS, three soils showed no activity with or without sponses were consistent with the interplay between the pH optima 720 656 peroxide, whereas four soils that showed phenol oxidase activity of the enzymes and pH effects on substrate redox potential. 721 657 had no detectable activity with peroxide addition. At pH 8, one of 722 658 three soils with no phenol oxidase activity on L-DOPA showed ac- 4.1. Assay pH 723 659 tivity with added peroxide. 724 660 Phenol oxidase activities observed using PYGL and L-DOPA were The patterns of ABTS oxidation rates as a function of assay pH 725 661 highly correlated across the data in Table 1 (r 0.90, P 0.001), were similar to those reported by Xu (1997), Floch et al. (2007), and 726 ¼ ¼ 662 whereas activities on ABTS were not correlated with those for PYGL Eichlerová et al. (2012). Xu (1997) suggested that oxidation of non- 727 663 (r 0.07, P 0.838) or L-DOPA (r 0.05, P 0.908). No patterns phenolic substrates such as ABTS declines as pH increases due to 728 ¼ ¼ ¼ ¼ 664 were apparent for gross peroxidase activity: activities measured inhibition of the T1 reaction center of laccase. Eichlerová et al. 729 665 with PYGL and L-DOPA were not significantly correlated (r 0.37, (2012) attributed this inhibition to hydroxyl ions. The decrease in 730 ¼ 666 P 0.179), and other substrate combinations also showed poor ABTS oxidation with increasing pH in the presence of hydrogen 731 ¼ 667 correlations (PYGL vs. ABTS: r 0.31, P 0.423; L-DOPA vs. ABTS peroxide may also be explained by the low pH optimum of per- 732 ¼ ¼ 668 r 0.12, P 0.786). oxidases. For instance, the pH optimum for lignin peroxidase is 3.0 733 ¼ ¼ 669 (Marquez et al., 1988), so one would expect lower rates of ABTS Q3 734 670 4. Discussion oxidation in the presence of peroxide at pH values greater than 3.0. 735 671 Moreover, the oxidation of ABTS can be decreased by fulvic acids, 736 672 Oxidation rates for commonly used phenol oxidase and humic acids, and low molecular mass compounds extracted from 737 673 peroxidase substrates depended on assay pH and soil type. Both soils (Eichlerová et al., 2012), which may partly explain difficulties 738 674 PYGL and ABTS were unsuitable for assays at alkaline pH because in using ABTS with bulk soil homogenates. 739 675 of auto-oxidation with PYGL, and prohibitively high redox po- For PYGL, there was no significant relationship between oxida- 740 676 tentials with ABTS. L-DOPA appears to be the only substrate that is tion and assay pH in the absence of hydrogen peroxide, mostly 741 677 suitable for use across a broad range of pH values. Eichlerová et al. because activities were often undetectable. Similar to PYGL, no 742 678 (2012) also observed that L-DOPA was oxidized across a broader significant trends were found for L-DOPA, but activity substantially 743 679 range of pH values than ABTS. However, L-DOPA was not oxidized increased at high pH in Californian and Costa Rican soils. This result 744 680 in all our samples (even if other substrates were), and its oxida- was surprising given that phenol oxidase activities are generally 745 681 tion was sometimes negatively affected by peroxide addition. We inhibited under alkaline pH. However, pH optima for certain laccase 746 682 even observed variation in our ability to detect the same enzyme isozymes have been reported up to pH 7.5 (Bollag and Leonowicz, 747 683 under the same conditionsdfor example, native AK and CA soils 1984). This observed increase in L-DOPA oxidation might reflect 748 684 showed phenol oxidase activity on PYGL at pH 5.0 in our negative favorable pH conditions for particular laccase isozymes in the soil. It 749 685 control study (Table 3) but not in our pH study (Fig. 2AeB). It is is difficult to conclude whether this increase in oxidative activity 750 686 possible that our detection limits were better in the negative with L-DOPA is a function of the enzyme or the substrate because 751 687 control study because absorbances were consistently measured we could not evaluate phenol oxidase activity under high pH with 752 688 over multiple time points. In order to improve detection limits, the other substrates due to auto-oxidation of PYGL and the high 753 689 shorter assay incubation times of 5 min or 1 h have been used for redox potential of ABTS. 754 690 ABTS and PYGL (Allison and Jastrow, 2006; Floch et al., 2007). In In the presence of hydrogen peroxide, PYGL oxidation decreased 755 691 our preliminary assays, reaction rates for ABTS and PYGL from pH 3.0 to 5.5, as predicted based on the pH optimum (w3.0) of 756 692 remained linear over 3e4 h, but longer incubation times pro- the enzyme (Figs. 1 and 2). This pattern was also observed with L- 757 693 duced inconsistent results, possibly because reaction product DOPA in Costa Rican soil. At higher pH, auto-oxidation made 758 694 accumulation rates are more likely to become non-linear with enzymatic activity determination impossible with PYGL, whereas 759 695 longer incubations. peroxidase activities on L-DOPA increased at pH values above 6.0 in 760

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761 Table 3 826 2 762 Equations and R values for signiﬁcant (P < 0.05) regressions of net absorbance units against time for phenol oxidase and net peroxidase in native, autoclaved, and combusted 827 soils from Alaska, California, and Costa Rica. Assays were conducted at pH 5.0 and read at 30 min intervals over a period of 3 h. Time interval represents the entire period over 763 828 which positive net absorbance was observed. N.D. Not detectable. N.S. Not signiﬁcant. 764 ¼ ¼ 829 2 765 Location Substrate Equation R P-value Time interval (h) n 830 766 Native soil: phenol oxidase 831 Delta Junction, AK PYGL 0.018x 0.020 0.90 <0.01 0.0e3.0 7 767 þ 832 768 Delta Junction, AK DOPA N.D. N.D. 833 Delta Junction, AK ABTS N.D. N.D. 769 Irvine, CA PYGL 0.006x 0.001 0.87 0.02 1.0e3.0 5 834 þ 770 Irvine, CA DOPA N.D. N.D. 835 771 Irvine, CA ABTS 0.123x 0.168 0.93 <0.01 0.0e3.0 7 836 þ 772 Nicoya Peninsula, CR PYGL N.D. N.D. 837 Nicoya Peninsula, CR DOPA N.D. N.D. 773 838 Nicoya Peninsula, CR ABTS N.S. 0.0e3.0 7 774 Autoclaved soil: phenol oxidase 839 775 Delta Junction, AK PYGL 0.035x 0.0381 0.95 <0.01 0.0e3.0 7 840 þ Delta Junction, AK DOPA 0.016x 00.014 0.94 <0.01 0.0e3.0 7 776 þ 841 777 Delta Junction, AK ABTS N.D. N.D. 842 Irvine, CA PYGL N.D. N.D. 778 Irvine, CA DOPA N.D. N.D. 843 779 Irvine, CA ABTS N.D. N.D. 844 780 Nicoya Peninsula, CR PYGL N.D. N.D. 845 781 Nicoya Peninsula, CR DOPA N.D. N.D. 846 Nicoya Peninsula, CR ABTS N.D. N.D. 782 847 Combusted soil: phenol oxidase 783 Delta Junction, AK PYGL 0.032x 0.0283 0.97 <0.01 1.0e3.0 5 848 À 784 Delta Junction, AK DOPA N.D. N.D. 849 785 Delta Junction, AK ABTS N.D. N.D. 850 786 Irvine, CA PYGL N.D. N.D. 851 Irvine, CA DOPA 0.006x 0.011 0.66 0.05 0.5e3.0 6 þ 787 Irvine, CA ABTS N.S. 0.0e3.0 7 852 788 Nicoya Peninsula, CR PYGL 0.084x 0.012 0.94 <0.01 0.5e3.0 6 853 À 789 Nicoya Peninsula, CR DOPA 0.033x 0.001 0.90 <0.01 0.0e3.0 7 854 À Nicoya Peninsula, CR ABTS 0.131x 00.10 0.93 <0.01 0.0e3.0 7 790 þ 855 Native soil: net peroxidase 791 856 Delta Junction, AK PYGL 0.045x 0.097 0.77 <0.01 0.0e3.0 7 þ 792 Delta Junction, AK DOPA 0.043x 00.093 0.7600 0.02 0.5e3.0 6 857 þ 793 Delta Junction, AK ABTS 0.065x 00.023 0.96 <0.01 0.0e3.0 6 858 þ Irvine, CA PYGL 0.012x 0.0260 0.89 <0.010 0.5e3.0 6 794 þ 859 Irvine, CA DOPA 0.018x 0.0070 0.94 <0.010 0.5e3.0 6 795 þ 860 Irvine, CA ABTS N.D. N.D. 796 Nicoya Peninsula, CR PYGL 0.134x 0.269 0.90 <0.01 0.5-3.0 6 861 þ 797 Nicoya Peninsula, CR DOPA 0.153x 0.1640 0.77 <0.010 0.0e3.0 7 862 þ 798 Nicoya Peninsula, CR ABTS 0.009x 0.033 0.84 0.0300 0.5e3.0 5 863 À þ 799 Autoclaved soil: net peroxidase 864 Delta Junction, AK PYGL 0.089x 0.172 0.75 <0.01 0.0e3.0 7 800 þ 865 Delta Junction, AK DOPA 0.075x 0.1950 0.95 <0.010 0.5e3.0 5 þ 801 Delta Junction, AK ABTS 0.166x 0.4230 0.76 0.0100 0.0e.0 7 866 þ 802 Irvine, CA PYGL 0.022x 0.0060 0.95 <0.01 1.0e3.0 5 867 À 803 Irvine, CA DOPA N.D. N.D. 868 804 Irvine, CA ABTS N.D. N.D. 869 Nicoya Peninsula, CR PYGL 0.068x 0.044 0.84 0.01 0.5e3.0 6 þ 805 Nicoya Peninsula, CR DOPA 0.053x 0.0740 0.65 0.03 0.0e3.0 7 870 þ 806 Nicoya Peninsula, CR ABTS N.D. N.D. 871 807 Combusted soil: net peroxidase 872 Delta Junction, AK PYGL 0.138x 0.073 0.96 <0.01 0.5e3.0 6 808 þ 873 Delta Junction, AK DOPA 0.070x 00.041 0.8500 0.01 0.5e3.0 6 809 þ 874 Delta Junction, AK ABTS 0.176x 00.130 0.87 <0.01 0.0e3.0 7 þ 810 Irvine, CA PYGL 0.117x 00.014 0.97 <00.01 0.5e3.0 6 875 þ 811 Irvine, CA DOPA 0.074x 00.026 0.99 <00.01 0.5e3.0 6 876 þ Irvine, CA ABTS 0.134x 00.025 0.93 <00.01 0.5e3.0 6 812 þ 877 Nicoya Peninsula, CR PYGL 0.055x 00.011 0.97 <00.01 1.0e3.0 5 813 þ 878 Nicoya Peninsula, CR DOPA 0.032x 00.014 0.93 <00.01 0.5e3.0 6 þ 814 Nicoya Peninsula, CR ABTS N.D. N.D. 879 815 880 816 881 817 882 818 Alaskan and Californian soils (Table 2). This pattern suggests that substrate reaches low enough redox potentials to auto-oxidize, 883 819 even as pH values increasingly exceed the enzyme optimum, L- thereby precluding activity measurements at high pH values in 884 820 DOPA oxidizes more readily due to declines in substrate redox most soils. 885 821 potential. Under neutral and alkaline conditions, the catechol group 886 822 present in L-DOPA may increase the formation of hydroxyl radicals 4.2. Negative controls 887 823 in the presence of hydrogen peroxide and iron and potentially 888 824 contribute to enhanced substrate oxidation (Iwahashi et al., 1989). In general, autoclaving and combusting soils did not prevent 889 825 The same pattern probably occurs with PYGL (i.e. Fig. 2E), but this oxidative reactions, meaning that these treatments cannot be used 890

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891 as negative controls (Carter et al., 2007; Stursova and Sinsabaugh, involving natural redox mediators and minerals, as was observed 956 892 2008; Sinsabaugh, 2010). Autoclaved soils displayed no phenol with the autoclaved and combusted soils. Unfortunately, it is 957 893 oxidase activity in Californian and Costa Rican soils, but showed difficult to identify indicators of these effects a priori. Within our 958 894 elevated activity in Alaskan soil. In the presence of peroxide, sub- data, there is no discernible pattern to these inconsistencies in 959 895 strate oxidation was observed in nearly all autoclaved and com- relation to soil pH, SOM concentration, or site characteristics. 960 896 busted soils, except with ABTS (Table 3). Given that the majority of One way to test for interference in oxidase assays is to add 961 897 autoclaved and combusted soils oxidized PYGL, L-DOPA, and ABTS, known amounts of commercially available oxidases (e.g. tyrosi- 962 898 oxidative activity should probably be considered a whole soil nase) and/or peroxidases (e.g., horseradish peroxidase) to soil 963 899 property rather than a measure of specific enzyme concentrations. slurries and examine the oxidation rates of the different substrates 964 900 This interpretation is also relevant to our pH study, in which min- (Allison, 2006). If activity does not increase linearly with the con- 965 901 eral catalysis of substrate oxidation may have occurred at alkaline centration of added enzyme (under saturating substrate concen- 966 902 pH values due to reduced redox potentials of PYGL and L-DOPA. trations), then side reactions within the soil environment may 967 903 Substrate oxidation in sterilized treatments could be explained prevent the accurate measurement of potential oxidase and 968 904 by minerals present in the soil such as Fe(II), which actively par- peroxidase activity in bulk soil samples. If this is the case, oxidases 969 905 ticipates in redox cycling and can generate reactive oxygen species could be isolated from soil prior to assays, but then there may be 970 906 through the Fenton reaction (Chacon et al., 2006; Hall and Silver, challenges in extrapolating the assay results to the soil environ- 971 907 2013). Manganese oxides are also produced biotically and abioti- ment (Eichlerová et al., 2012). 972 908 cally in soils, and may contribute to soil organic matter oxidation 973 909 (Spiro et al., 2009). Recently, Hall and Silver (2013) used L-DOPA to 4.4. Conclusions 974 910 assess phenol oxidative activity of Fe(II) in tropical soils and re- 975 911 ported a linear increase in L-DOPA oxidation with increasing soil We found that three substrates commonly used in oxidase as- 976 912 Fe(II) concentrations. The authors also used autoclaved soil to says each revealed distinct trends with respect to pH and soil type. 977 913 destroy enzymatic activity and found little change in rates of L- Thus measuring soil oxidase activity remains a challenge. Our re- 978 914 DOPA oxidation. Thus abiotic reactions in soil probably contribute sults lead to several recommendations for overcoming this chal- 979 915 to substrate oxidation, especially with L-DOPA and PYGL. lenge and improving oxidase techniques. For research questions 980 916 Oxidation rates and linearity over time were generally lower in that require standardized comparisons across multiple soil types, 981 917 native soils relative to combusted soils, potentially due to the we recommend conducting assays with multiple substrates at a 982 918 presence of antioxidant compounds in native soil organic material reference pH (e.g. 5.0) to obtain a more complete profile of oxida- 983 919 (Rimmer and Smith, 2009). Our high-temperature treatments likely tion potentials and identify potential sources of interference. 984 920 caused changes in soil physical and chemical properties that Furthermore, by standardizing assay conditions and verifying re- 985 921 increased the availability of reactive mineral species to participate action linearity, inconsistencies due to methodological differences 986 922 in redox reactions (Trevors, 1996). Consistent with this idea, the can be minimized, allowing for a sharper focus on the oxidative 987 923 clay-sized fraction of soils, which is low in organic matter and rich variation across soil types due to enzymatic or abiotic factors. For 988 924 in minerals, can show higher oxidative activities than organic-rich studies that focus on a single system, researchers should evaluate 989 925 soil fractions (Allison and Jastrow, 2006). multiple substrates at an assay pH that approximates the soil pH 990 926 before proceeding (Burns et al., 2013). Our data on autoclaved and 991 927 4.3. Soil survey combusted soils suggest that assays with common oxidase sub- 992 928 strates detect oxidative potential of the entire soil matrix rather 993 929 For most of the soils analyzed, oxidative enzyme activities were than just potential enzyme activity (Sinsabaugh, 2010). Minerals 994 930 consistent with the redox characteristics of the substrates. probably contribute to this oxidative function, particularly at 995 931 Furthermore, activity measurements based on PYGL and L-DOPA neutral to alkaline pH. Therefore measured oxidation rates should 996 932 were more similar to one another than to measurements based on not necessarily be interpreted as a metric of enzyme abundance in 997 933 ABTS (Table 1; Fig. 1). However, phenol oxidase activities in five of the soil. Overall, our findings and recommendations should help 998 934 the 17 soils were inconsistent with our hypothesized dependence generate more reliable assays of soil oxidase activities that improve 999 935 on redox potential because ABTS was oxidized at a higher rate than our understanding of soil carbon cycling in ecosystems. 1000 936 PYGL or L-DOPA (Table 1). This pattern could occur if PYGL and L- 1001 937 DOPA are more subject to chemical interference than ABTS. We also Acknowledgments 1002 938 observed several instances in the soil survey and the negative 1003 939 control experiment where peroxide addition unexpectedly reduced We thank Cristina Takacs-Vesbach, Zachary Rinkes, and Cory 1004 940 oxidative activity (Tables 1, S1eS3). Such a reduction could occur if Cleveland for help in collecting soil and/or enzyme data. This study 1005 941 peroxide addition increases the reactivity of chemical compounds was funded by the Office of Science, U.S. Department of Energy (to 1006 942 that interfere with the assay. Alternatively, inconsistencies in our SDA), University of California, Irvine, Undergraduate Research Op- 1007 943 soil survey dataset could have occurred because we did not opti- portunity Program (to CEB), NSF grant DEB-0918718 (to MNW), the 1008 944 mize incubation times and check for reaction linearity in every soil Sevilleta LTER Program, and a University of California President’s 1009 945 type, as we did with our Alaskan, Californian, and Costa Rican soils Postdoctoral Fellowship (to DPG). 1010 946 (German et al., 2011). 1011 947 The relatively high frequency of inconsistent results for phenol Appendix A. Supplementary data 1012 948 oxidase and peroxidase assays is a cause for concern and probably 1013 949 contributes to the high coefficients of variation typically reported Supplementary data related to this article can be found at http:// 1014 950 for these activities (Sinsabaugh et al., 2008). There are many po- dx.doi.org/10.1016/j.soilbio.2013.08.022. 1015 951 tential sources of interference including substrate sorption, 1016 952 dimerization, and condensation of reaction intermediates with Uncited references Q4 1017 953 other organic molecules, as well as the existence of catalytic cycles 1018 954 involving minerals (Spiro et al., 2009; Sinsabaugh, 2010; Eichlerová Anderson-Teixeira et al., 2011; Bottner et al., 1999; Chen et al., 1019 955 et al., 2012). There is also the potential for synergistic interactions 2000; Ekberg et al., 2007; Furuno et al., 2002; Koranda et al., 1020

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