L-DOPA assay

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Method synopsis

Spectrophotometric assay measuring phenol oxidation capacity using L-3,4-dihydroxyphenylalanine substrate with absorbance detection at 460 nm. Widely used to quantify oxidative enzyme activity including laccase, polyphenol oxidase, and peroxidase in soil samples.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Reactive iron, not fungal community, drives organic carbon oxidation potential in floodplain soils (2023), Soil Biology and Biochemistry. Implementations in other papers (listed below) may differ.

1. L-DOPA oxidative potential assays
   Soil slurries prepared by homogenizing 0.5-1.0 g field-moist soil with 200 μL sodium acetate buffer (pH 5.5) using blender at high speed for 1 min. In 96-well plates, 50 μL soil slurry added to 200 μL soil homogenate plus 50 μL L-DOPA substrate (250 μM final concentration) and standard control (200 μL buffer plus 50 μL L-DOPA). Plates centrifuged 3 min at 3000 rpm, supernatant pipetted to new plates, absorbance read at 460 nm.
   Quantities: 0.5-1.0 g soil per slurry, 250 μM L-DOPA final concentration, 8 replicate wells per treatment in 96-well formatDuration: 2 h incubation in dark at room temperatureConditions: Room temperature, dark conditions, pH 5.5 acetate buffer
   Equipment: Hamilton blender, 96-well plates, microplate reader (BioTek Synergy HTX), centrifuge
2. Metal addition experiments
   Mn(III) added to 125 mL soil slurries as 1.25 mL from 40 mg Mn L⁻¹ stock of Mn(III) acetate dihydrate in sodium acetate buffer. Fe(II) added as 1.25 mL from 200 mg Fe L⁻¹ stock composed of FeCl₂ in 2% HCl. Both metals chosen to match high end concentrations found in soil slurries by ICP-OES analysis.
   Quantities: 40 mg Mn L⁻¹ and 200 mg Fe L⁻¹ addition concentrations, 1.25 mL additions to 125 mL slurriesDuration: Same 2 h incubation as standard assaysConditions: Concentrations chosen to match native soil levels, acidic conditions for Fe(II) stock
   Equipment: stock solutions, micropipettes, same 96-well plate setup

Typical Equipment

• Hamilton blender
• 96-well plates
• microplate reader (BioTek Synergy HTX)
• centrifuge

Output Measurements

• phenol oxidative potential (μmol g⁻¹ h⁻¹)

Papers Using This Protocol (1)

Species Studied With This Protocol