Formaldehyde dehydrogenase

A fluorescence-based enzyme coupled assay was used to detect the formaldehyde product of demethylation of H3K4me3 peptide under multiple turnover conditions (excess of substrate peptide over KDM5C). Various concentrations of H3K4me3 (1–21) substrate peptide (GenScript) were added with 1 mM alpha-ketoglutarate to initiate demethylation by ~1 μM KDM5C in 50 mM HEPES pH 7.5, 50 mM KCl, 50 μM ammonium iron(II) sulfate, 2 mM ascorbic acid, 2 mM NAD+, and 0.05 U formaldehyde dehydrogenase (Sigma-Aldrich) at room temperature. Upon initiation, fluorescence (350 nm excitation, 460 nm emission) was measured in 20 sec intervals over 30 min using a Molecular Devices SpectraMax M5e plate reader. NADH standards were used to convert fluorescence to the rate of product concentration formed. Initial rates of the first 3 min of demethylation were plotted as a function of substrate concentration and fit to the tight-binding quadratic velocity equation $\mathrm{Y}={\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}{}^{\mathrm{*}}\left(\left(\mathrm{X}+{\mathrm{E}}_{\mathrm{T}}+{\mathrm{K}}_{\mathrm{m}}\right)-\mathrm{s}\mathrm{q}\mathrm{r}\mathrm{t}\left({\left(\mathrm{X}+{\mathrm{E}}_{\mathrm{T}}+{\mathrm{K}}_{\mathrm{m}}\right)}^{\wedge }2-4^{*}{\mathrm{E}}_{\mathrm{T}}{}^{*}\mathrm{X}\right)\right)/\left(2^{*}{\mathrm{E}}_{\mathrm{T}}\right)$ using GraphPad Prism to determine Michaelis-Menten kinetic parameters of demethylation.

Formaldehyde dehydrogenase (EC 1.2.1.46) collected from Pseudomonas putida (specific activity 1.6 U/mg) and β-nicotinamide adenine nucleotide (NAD⁺) obtained from Sigma (Spruce Street, St. Louis, USA). Methylene blue (MB) was purchased from Sigma (Spruce Street, St. Louis, USA). Formaldehyde (37%) and acetic acid were purchased from Merck company. Gold nanoparticles (AuNPs) and ionic liquid [EMIM][OTF] were purchased from Sigma (Spruce Street, St. Louis, USA). All chemicals used in the experiments were analytical reagent grade. Deionized water was obtained from a Millipore Milli-Q purification system. The Malabar Red Snapper (Lutjanus malabaricus) and Longtail Tuna (Thunnus Tonggol) samples were collected from the three different local wet markets in Kota Kinabalu, Sabah, Malaysia.