Figure 1
Left panel: schematic of the hypothesised reaction on circular β2GPI, highlighting the intact disulfide bond in the fifth domain (DV). The protein undergoes selective, enzymatic driven reduction by TRX-1 (recycled by TCEP) and liberates the two thiols, which are subsequently labelled by MBP to avoid re-oxidation. All reaction steps were performed under argon atmosphere at 25 °C in HBS buffer (pH 7.4) with mild shaking. 50 µM TRX-1 was reduced by 50 µM TCEP in a total volume of 100 µL HBS buffer for 1 h. 900 µL HBS buffer and 0.2 µM β2GPI were added and incubated for 1 h. 15 µM MPB were added for 15 min to label free thiols. Finally, 200 µM reduced GSH was used to quench unbound MPB (15 min). Right panel: C-terminal, fifth domain (DV) of β2GPI with the disulfide Cys288/Cys326 highlighted in red.
