Calibration of phenol oxidase measurement in acidic wetland environments
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Phenol oxidases mediate the degradation of recalcitrant compounds, polyphenolics, in wetland soils and are considered to play a key role in the microbial carbon cycle of peatlands which predominate in boreal biomes. In order to validate a method for quantification of oxidative enzyme activity in acidic wetland environments, the relationship between pH and substrate oxidation was studied using the standard enzyme tyrosinase and in soils collected from six freshwater wetlands including three marshes in north Florida and peatlands of northern Minnesota. Phenol oxidase (PO) activity was quantified with two commonly used assay substrates, ABTS (2,2'-azino-bis(3-ethylobenzthiazoline-6-sulfonic acid) and L-DOPA (L-3,4-dihydroxyphenylalanine), across a pH range of 4 to 7 which matched the in situ pH range of the studied wetlands. The PO assay is sensitive and activity could be detected with either substrate across a pH range of 4 to 7. However, with the standard enzyme tyrosinase, it was shown that a large change or threshold in oxidation rates occurred at pH 5. At pH < 5, L-DOPA oxidation rates were greatly diminished and ABTS oxidation was at a maximum. Above pH 5, ABTS oxidation occurred at much slower rates and L-DOPA oxidation was at a maximum. The pH response of PO activity in wetland soils corroborated observations made with tyrosinase. Thus, ABTS is recommended to be an effective substrate for the quantification of PO activity at an in situ pH of < 5, while L-DOPA is recommended at an in situ pH of > 5. In soils collected from a northern Minnesota peatland, assays conducted at an in situ pH of 4 showed one to two orders of magnitude higher rates of PO activity in solid phase peat in comparison to porewater, indicating that the majority of PO activity is associated with the peat. At three Minnesota peatland sites, PO activity was shown to attenuate with depth in agreement with the activities of other enzymes and with rates of peat decomposition.