8 anilino 1 naphtalenesulfonic acid ans

Fluorescence measurements with the wild-type and variant MCAD enzymes (0.5 μg/μl) diluted in 20 mM HEPES buffer, pH 7.0 and 200 mM NaCl were performed on a Cary Eclipse fluorescence spectrophotometer equipped with a temperature-controlled Peltier multicell holder (Varian). Intrinsic FAD fluorescence and binding of the hydrophobic dye 8-anilino-1-naphtalenesulfonic acid (ANS, Sigma-Aldrich) to surface exposed hydrophobic groups was co-monitored (multiwavelength program) during heat-induced denaturation experiments in the temperature range from 25 °C to 65 °C at a heating rate of 1 °C/min. Changes in ANS fluorescence emission were detected at 450 nm (excitation at 395 nm, 5.0/10.0 nm slit widths), whereas intrinsic FAD emission was monitored simultaneously at 530 nm (excitation at 450 nm, 5.0/10.0 nm slit widths). We ensured that no fluorescence resonance energy transfer occurred at the wavelengths used. Transition midpoints (T_m1/2) were determined graphically with T_m1/2 being the temperature at which half denaturation occurred. Significances between wild-type and variant proteins were calculated by one-way ANOVA followed by a Dunnett’s post test (GraphPad Prism 5.0).

Dithiotreitol (DTT), N-ethylmaleimide (NEM), beta-mercaptoethanol (β-ME), reduced nicotinamide adenine dinucleotide phosphate (NADPH), isopropyl-β-d-thiogalactopyranoside (IPTG), kanamycin sulphate, ampicillin, 2-iodoacetamide, diethylenetriaminepentaacetic acid (DTPA), 8-anilino-1-naphtalene sulfonic acid (ANS) and imidazol were from Sigma-Aldrich (Darmstadt, Germany). H₂O₂ was obtained from Mallinckrodt Chemicals (St. Louis, MO, USA). 15(S)-HpETE (≥98% pure), 15(S)-HpEPE (≥98% pure), 9α,11α-epidioxy-15S-hydroperoxy-prosta-5Z,13E-dien-1-oic acid (PGG₂, ≥95% pure), arachidonic acid (AA, ≥98% pure) and eicosapentaenoic acid (EPA, ≥98% pure) (Supplementary Figure S1) were obtained from Cayman Chemical (Ann Arbor, MI, USA) as ethanolic solutions (or in acetone solution in the case of PGG₂) and stored under argon at −80 °C. All other reagents were obtained from standard commercial sources and used as received. Experiments were performed in 50 mM phosphate buffer plus 0.1 mM DTPA, pH 7.8 and 25 °C, except otherwise indicated.