Cary 300 bio

All chemicals and resins were obtained from Sigma-Aldrich unless otherwise
specified. UV/Vis spectroscopy was run on a Cary 60 (Agilent) equipped with a
single-cell temperature controller. Chromogenic substrate degradation was monitored
on a Cary 300 Bio (Agilent) with a temperature-controlled 8-cell dual-beam sample
holder. SDS-PAGE was run using Bio-Rad mini Protean TGX 4–20% gels.
Gels were imaged in a Bio-Rad Gel Doc XR+ imager. Michaelis-Menten parameters
(K_m, k_cat) were
determined by non-linear fit using the Michaelis-Menten model with or without
substrate inhibition in OriginPro 9.1. Sequence alignments were displayed using
ESPript 3.0 (Robert and Gouet 2014 (link)).

DEAB inhibition and IC₅₀ curves were assayed spectrophotometrically by monitoring the formation of NADH at 340 nm (molar extinction coefficient of 6220 MT⁻¹ cm⁻¹) on a Beckman DU-640 or Cary 300 Bio UV-vis spectrophotometer using purified recombinant ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH2, ALDH4A1, and ALDH5A1. All assays were performed at 25 °C Following a two minute incubation of enzyme with DEAB and NAD⁺, the reactions were initiated by adding substrate. With the exception of ALDH1L1, ALDH4A1 and ALDH5A1, reactions were. performed in a solution containing 100–200 nM enzyme, 200 μM NAD⁺, 1% DMSO, and 100–200 μM propionaldehyde in 50 mM sodium BES pH 7.5. For ALDH4A1, reactions contained 20 mM propionaldehyde and 1.5 mM NAD⁺. For ALDH5A1, reactions contained 2 mM propionaldehyde and 1.5 mM NAD⁺. For ALDH1L1, reactions contained 500 nM enzyme, 4 mM propionaldehyde and 500 μM NADP⁺. For enzymes that showed inhibition by DEAB, IC₅₀ values for propionaldehyde oxidation were calculated by varying the concentration of DEAB from 0 to 20 μ.M. Higher concentrations of DEAB were not used due to interference at 340 nm. However there was little to no interference at lower DEAB concentrations. Data were fit to the four parameter EC₅₀ equation using SigmaPlot (StatSys v12.3) and the values represent the average of three independent experiments (each n = 3).

UV-vis spectroscopy was used to evaluate the solubility of the ruthenium complexes. Accordingly, a 2 mL solution of each complex was prepared in full RMPI growth media (phenol red free) at the concentrations used for cellular proliferation studies (DMSO final concentration: 0.5%). After incubation for 48 h at 37°C, the solution was filtered on a celite pad, and the absorbance recorded using a Cary 300 Bio UV−vis spectrometer. The concentration of saturation of the complexes in growth media was assessed by determining the concentration at which a maximum intensity in UV absorbance (274 nm-278 nm) was observed.