2.13. Bilayer Interferometry (BLI)

For the BLI assay on Octet^Red 96 (ForteBio, Fremont, CA, USA), proteins were reduced with 20 mM DTT for 30 min at room temperature. DTT was removed using a Hitrap^® desalting column (Cytiva, Marlborough, MA, USA) equilibrated in 25 mM Tris/HCl, pH 7.4, 25 mM NaCl. To block all free Cys, Prdxs, MBP-ASK1-TBD, and MBP were treated with 20 mM iodoacetamide (IAM) at room temperature for 30 min, and excess IAM was removed on Bio-spin columns (Bio-Rad Laboratories, Richmond, VA, USA). Next, the number of free thiols was determined using Ellman’s reagent (5,5-dithio-bis-(2-nitrobenzoic acid)) (DTNB assay) to confirm that all potential thiol groups were blocked [47].

The protein MBP was used as a reference (negative control) to eliminate the binding possibility of Prdx1 WT to the MBP part of the MBP-ASK1 fusion protein, and Prdx2 WT was used to determine the selectivity of the Prdx1:MBP-ASK1-TBD interaction. All proteins were prepared in 25 mM Tris/HCl, pH 7.4, 25 mM NaCl, 1% bovine serum albumin (BSA), 0.05% Tween 20, and 0.01 mM maltose. His-tagged MBP-ASK1-TBD and His-tagged MBP were loaded onto the Ni²⁺-NTA sensors (ForteBio, Fremont, CA, USA) at a concentration of 0.45 µM and 0.15 µM, respectively. The concentration of analytes (Prdx1 WT, Prdx1 C52A, Prdx1 C173A, and Prdx2 WT) was fixed at 1 µM. The assay was done in the presence of 10 µM DTT for the reducing condition and 10 µM H₂O₂ for the oxidizing condition. Data were obtained with the Data Acquisition 9.0 (ForteBio, Fremont, CA, USA) software of the instrument. To calculate association (k_on) and dissociation rate (k_off) constants, different concentrations of the analyte (Prdx1s) were utilized, and the data were analyzed by the Data Analysis 9.0 software (ForteBio, Fremont, CA, USA). In this software, the association curves were fitted through the equation:

The dissociation curves were fitted through the following equations:

where y is the BLI signal (in nm), indicating the level of binding as nm shift, while y₀ represents the nm shift at the beginning of the dissociation phase, t is the time (s), and t₀ is the time at the beginning of the dissociation phase. [Analyte] is the given concentration of Prdx1 or its variants, and R_max is the fitted maximum binding of the analyte to a given immobilized ligand on the biosensor surface.