Assessing chaperone function of Hsp70s

The chaperone function of PfHsp70-x_F, PfHsp70-x_T and hHsp70 was investigated by monitoring their ability to suppress heat-induced aggregation of a model substrate, malate dehydrogenase (MDH) from the porcine heart (Sigma-Aldrich, USA) as previously described (Shonhai et al. 2008 (link); Zininga et al. 2016 (link)). Briefly, the capability of each protein to suppress thermally induced aggregation of MDH was monitored spectrophotometrically. MDH (0.6 μM) was added to the preheated buffer (50 mM Tris, pH 7.4, 100 mM NaCl) at 51 °C. The temperature was maintained at 51 °C for 80 min, and the absorbance was monitored at 340 nm in 5-min intervals using a SpectraMax M3 spectrometer (Molecular Devices, USA). BSA was used as a non-chaperone control in this assay. The data were analysed using GraphPad Prism 9.0 software (GraphPad Software, CA, USA).