Uv star

The activities of the purified transaminase variants were studied using the conversion of (R)-1-phenylethylamine resulting in the formation of acetophenone [51 (link)], which was quantified photometrically at 245 nm over time using the Infinite^® 200 PRO (TECAN) plate reader in UV-transparent microtiter plates (UV-Star, Greiner Bio-One GmbH, Berlin, Germany). The assay was performed with 2.5 mM (R)-1-phenylethylamine as amine donor and 2.5 mM pyruvate as amine acceptor in 1.25–2.5% DMSO, 50 mM Davies buffer pH 6.5–9.5 at 30 °C. One unit (U) activity was defined as the formation of 1 µmol acetophenone per minute. All measurements were performed in triplicates.

Cardiac samples were homogenised in HEPES. Homogenates were centrifuged at 15,000 rpm for 5 min in a Centrikon H-401 (Germany) centrifuge at 4°C. Following centrifugation, the supernatant was collected and frozen at −30°C until analysed. All enzymatic assays were carried out at 25 ± 0.5°C using a Power Wavex microplate scanning spectrophotometer (Bio-Tek Instruments, USA) in duplicate in 96-well microplates (UV Star, Greiner Bio-One, Germany). The enzymatic reactions began with the addition of the tissue extract. The specific assay was measured.

To assess the spectral compatibility with DUV fluorescence microscope, the absorption and the excitation spectra of the fluorescent probes Hoechst 33258 (0.08 mg/ml, H341, Dojindo Laboratories, Kumamoto, Japan), propidium iodide (0.1 mg/ml, P1304MP, Invitrogen, USA) and Alexa Fluor 594-conjugated antibody (1:50, ab150076, Donkey anti-rabbit IgG H&L, Abcam plc., Cambridge, UK) were obtained using spectrophotometer microplate reader (Varioskan LUX, ThermoFisher Scientific, Waltham, MA, USA). The absorption spectrum (240–800 nm range) and emission spectrum (400–800 nm range) with excitation at 280 nm were measured using UV light transparent 96-well plate (UV-Star, Greiner Bio-One, Longwood, FL, USA).

The direct inhibitory effect of BLEx and its fractions on XOD activity was evaluated as the conversion of xanthine to Uric acid under XOD (from bovine milk, Sigma). Uric acid, xanthine, and allopurinol (Fujifilm Wako) were dissolved in 0.1 M phosphate buffer (PB; pH 8.0). Next, 50 μL PB, 50 μL samples (appropriate concentrations of BLEx, fractions, or 1 mM allopurinol), and 100 μL of 1 mM xanthine were added to a 96-well plate (UV-star, Greiner Bio-One, Kremsmünster, Austria). Finally, XOD was added to a final concentration at 0.1 mU and reacted for 3 min at 37 °C. The production of Uric acid was measured as the absorbance at 293 nm using SpectraMax (Molecular Devices, San Jose, CA, USA).

The pKa values of the residue H53 of ATA-Afu or its E49Q variant were predicted by the program PropKa 3.3 (developed by the Jensen group) [52 (link)]. PropKa 3.3 predicts the pKa values of ionizable groups in proteins based on their 3D structure. The 3D structure of ATA-Afu was downloaded from the PDB database (PDB code: 4CHI) and was used as a structure template to predict the 3D homology models of its E49Q variant, using the program YASARA based on its homology model generation algorithm [45 ]. The pdb file served as input for PropKa 3.3 and the predicted pKa values of all the ionizable residues were calculated automatically.
The pKa value of the internal aldimine group of the cofactor of ATA-Afu-E49Q was measured using a literature method [42 (link)]. The absorption spectra of ATA-Afu-E49Q at pH 6.5–9.5 were determined using the Infinite^® 200 PRO (TECAN) plate reader in UV-transparent microtiter plates (UV-Star, Greiner Bio-One GmbH). These pH-dependent spectral changes have been known to reflect the ionization state of the imine nitrogen of the internal aldimine bond formed between K179 and PLP, and the protonated form of the internal aldimine of ATA-Afu-E49Q is thought to have an absorption band around 410 nm, which was titratable and resulted in a pKa value of 9.0 (Figure S3, Supplementary Materials).