Phosphate buffer

Animals were euthanized and excised inner ears were fixed overnight in 2.5 % gluteraldehyde in 0.1 M phosphate buffer (Sigma-Aldrich). Fixed ears were decalcified for 48 hours in 4.3 % EDTA in 0.1 M phosphate buffer (Sigma-Aldrich). Fine dissection was performed to reveal the organ of Corti, before osmium tetroxide (Agar Scientific)–thiocarbohydrazide (Fluka) processing (adapted from Hunter-Duvar [18 (link)]) was carried out. The inner ears were then dehydrated through increasing strength ethanol solutions (Fisher Scientific) and critical point dried using an Emitech K850 (EM Technologies Ltd). The specimens were then mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150R S sputter coater (Quorum Technologies). Prepared cochlea were visualised with a JEOL LSM-6010 (Jeol Ltd) scanning electron microscope. Hair cell stereocilia bundle counts were performed by counting the number of adjacent inner hair cells (IHCs) and outer hair cells (OHCs) to ten pillar cells. For the analysis the cochlea was divided into three separate regions (turns), apical (<180° from apex), mid (180–450° from apex), and basal (450–630° from apex). Four ears (one ear per mouse) were analysed for each genotype at each region.

Room-temperature burst kinetics were obtained under burst-phase conditions using an SX20 stopped flow (Applied Photophysics) by monitoring substrate depletion by absorbance at 260 nm. Enzyme and substrate were mixed 1:1 at 25 °C and in 0.1 M phosphate buffer (Sigma-Aldrich, pH 7.2) supplemented with 50 mM NaHCO₃ (Sigma-Aldrich). Burst kinetics were assayed at final enzyme concentrations of 10 μM and final substrate concentrations varying between 50 and 400 μM (equation (1)). $\frac{P}{E_0}=v_{\mathrm{steady}}\times t-\left(v_{\mathrm{steady}}-v_{\mathrm{burst}}\right)\times \left(1-e^{-k\times t}\right)/k$
Catalytic parameters (k_cat, K_M and k_cat/K_M) were determined under burst-phase and steady-state conditions using CAZ (Δξ = −9,000 M⁻¹ cm⁻¹) at 260 nm by measuring the initial enzymatic reaction rate in an Epoch plate-reader (Biotek). Burst phase rates were determined at 4 °C, and steady-state parameters were determined at 25 °C. Reactions rates were obtained in at least duplicates at a final enzyme concentration of 1 μM (final assay volume of 100 μl). Ultraviolet-transparent 96-well plates (Corning) were used. Assays were performed in 0.1 M phosphate buffer (Sigma-Aldrich, pH 7.2), supplemented with 50 mM NaHCO₃ (Sigma-Aldrich).