Alcohol oxidase

Silk and pollen extracts were assayed for PME activity using the modified Fluoral-P assay (Klavons and Bennett 1986; (link)Wojciechowski and Fall 1996) (link). Briefly, the reaction mixture was prepared by mixing 0.5% pectin (from citrus fruits, > 85% esterification, Sigma-Aldrich, P9561) in phosphate buffer, 200 mM sodium phosphate buffer (pH 6.6) and 0.0025 U/μl alcohol oxidase (from P. pastoris, Sigma, A2404). 20 μl of crude silk or pollen extracts were added to the reaction mixture and the reaction was incubated for 10 min at 25 °C. After incubation, 4 μg/μl (final concentration) 4-Amino-3-penten-2-one (Fluoral-P) (Sigma-Aldrich, 691,003) is added to the sample mix and incubated for another 5 min. Formaldehyde produced due to alcohol oxidase interacts with Fluoral-P to create a fluorescent product which was measured using a Tecan 96-well fluorimeter with 405 nm excitation and 510 nm emission filters. The amount of methanol released was estimated using standards with known methanol concentrations (0 to 20mM). pectinesterase (from orange peel, Sigma-Aldrich, P5400) was used as a positive control. The negative control contained distilled water in place of PME. PME activity was expressed as nmol of methanol min -1 μg protein -1 . For a given tissue, the same mass of tissue was used in each assay so PME activity data is comparable within tissues, but not across tissues.

Acetonitrile, methanol, ethanol, acetic acid and formic acid were HPLC grade from VWR. KOH Titrisol 1M was purchased from Merck. Acetone, D (+) galacturonic acid hydrated were provided by Fluka. Sodium chloride, tartaric acid, epicatechin, epigallocatechin gallate, lithium chloride, N,N-dimethylformamide, trifluoroacetic acid, myo-inositol, allose, m-hydroxydiphenyl (MHDP), alcohol oxidase from Pichia pastoris, norleucine, the 18 amino-acid standard kit and hydrochloric acid 37 % were provided by Sigma-Aldrich, sulphuric acid by Roth. Sodium hydroxide 1M was obtained from Fisher. The lithium citrate loading buffer was obtained from Biochrom.
Flavanol dimer B2, flavanol trimer C1 and Malvidin-3-O-glucoside chloride were purchased from Extrasynthese (Genay, France). Ultra-pure water was obtained from a Milli-Q Advantage A10 system (Millipore).

Fatty acid methyl ester standards, α-tocopherol standard, gallic acid, methanol, alcohol oxidase (from Pichia pastoris), purpald (4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole), phosphate buffer, safranin and sodium hydroxide were purchased from Sigma-Aldrich (Dorset, UK). All chemicals were of analytical grade.

Fluorescein isothiocyanate (FITC, 0.5 mg) and catalase (CAT; Sigma-Aldrich, catalase from bovine liver, 2000-5000 units mg -1 protein, 40 mg) were dissolved in a car-bonate-bicarbonate aqueous buffer solution (0.1 M, pH 9.2, 4 mL) and left for two hours in darkness at room temperature under gentle stirring. The FITC-tagged CAT (FCAT) was recovered by passing the reaction mixture through an Illustra NAP-25 column (GE Healthcare Life Sciences, NSW, Australia). The crude FCAT solution was concentrated through a 10 K membrane by centrifugation (4000 rpm for 20 min), followed by solvent-exchange with ultrapure water. The concentration-solvent-exchange process was repeated two times to ensure the buffer salts were completely removed from the solution. Thereafter, the concentrated FCAT aqueous solution was passed through a NAP-25 column again to ensure the complete removal of unreacted FITC. The obtained FCAT solution was stored in darkness at 4 °C.
A similar method was used to prepare fluorescein-tagged alcohol oxidase (FAOx, Sigma-Aldrich, alcohol oxidase solution from Pichia pastoris, buffered aqueous solution, 10-40 units/mg protein).