Novaspec plus spectrophotometer

Assays were performed in either 1 cm/0.2 cm path cUVettes (UVette; Eppendorf, Milan, Italy) in a final volume of 1 mL, or 96-microwell plates in a final volume of 0.2 mL. In the former case, OD₃₄₀ was determined with a Novaspec plus spectrophotometer (Amersham Biosciences, Milan, Italy) equipped with a Peltier device set to 37°C and a UVette adaptor. In the latter case, the plate was equilibrated at 37°C prior to enzyme addition, and absorbance was measured using a Ledetect plate reader (Labexim, Lengau, Austria) equipped with a LED plugin at 340 nm. Each sample was carried out in triplicate (technical replications). Each determination was repeated with at least three different enzyme preparations (biological replications), obtaining almost identical patterns. Presented data refer to a single enzyme preparation, and are means ± SE over technical replicates. Linear and non-linear regressions of data were computed using Prism 6 for Windows, version 6.03 (GraphPad Software, San Diego, CA, United States).

MDH (0.5 or 2 μM) was denatured in buffer C for 30 min at 47 °C or for 60 min at 41 °C, respectively. Protein disaggregation and refolding were started by diluting aggregated proteins or sHsp/protein complexes and chaperones 1:1 (2 μM Ssa1, 1 μM Sis1, 0.1 μM Sse1, 1 μM Hsp104, 1 μM GroEL, 1 μM GroES) in buffer C containing 0.1 mg ml⁻¹ BSA at 30 °C. The reactivation was monitored as published previously16 (link) using an Amersham Biosciences Novaspec Plus spectrophotometer. The same assay was used to follow the inactivation of MDH (0.5 μM) in the presence or absence of sHsps on incubation at 47 °C or 41 °C, respectively.