Nbd cl

Substrates NBD-Cl, CDNB, NBC, DCNB, ECA, 4-NPA, and Cum-OOH (Merck, Germany), which are typical substrates for detecting detoxification and GPOX activities of tau class GSTs, were used to investigate the enzyme activities of wild-type and mutants. The activity towards NBD-Cl was determined according to the method described by Ricci et al. [43 (link)], the activity towards CDNB, NBC, DCNB, and ECA was determined according to the methods described by Habig et al. [44 (link)], and the activity towards Cum-OOH was determined according to the method described by Edwards and Dixon [45 (link)]. Taking NBD-Cl and GSH as substrates (Merck, Germany), the kinetic parameters of mutant enzymes were monitored according to the description of Yang et al. [21 (link)]. The K_m and V_max values for GSH were obtained by fixing the NBD-Cl concentration at 1.0 mM and detecting the enzyme activity with a GSH concentration in the range of 0.2 to 1.0 mM. The K_m and V_max values for NBD-Cl were obtained by fixing the GSH concentration at 1.0 mM and detecting the enzyme activity with NBD-Cl concentrations ranging from 0.2 to 1.0 mM. Non-linear regression analysis of Graphpad Prism 10.0 (Graphpad Software Inc., San Diego, CA, USA) was applied to the data to obtain the kinetic parameters of mutants. The experimental data were represented as the average of three independent experiments.

Protein (10 μm) was exposed to oxidants (90 μm) for 30 min. NBD‐Cl (Sigma‐Aldrich, 100 μm) was added to the samples and was incubated for 30 min, at 37 °C, after the samples were subjected to spin column (Amicon Ultra 10 kDa, Millipore, Ireland). Electronic absorption spectra were carried out in a photodiode Multispec 1501 spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD), using quartz cuvettes with 1.0‐ and 0.1‐cm optical path and 0.5‐nm slit 66.

To quantify the free N termini level, soluble proteins were extracted from leaves in 50 mM sodium citrate buffer containing 1 mM EDTA (pH 7.0) and desalted using PD SpinTrap G-25 columns (GE Healthcare) to remove free amino acids. Crude protein extract (2.5 μM) was incubated with 0.5 mM NBD-Cl (Sigma-Aldrich) in 50 mM sodium citrate buffer containing 1 mM EDTA (pH 7.0) for 14 h at room temperature. The fluorescence intensity was quantified with a FLUOstar Omega plate reader (BMG Labtech; excitation: 470±10; emission: 520 nm). For the quantification of free N termini in response to ABA (Sigma-Aldrich), leaf discs were floated (30 mg) in ½ × Hoagland medium supplemented with 50 μM ABA for 2, 4, 6 and 8 h before analysis.