CD and inductively coupled plasma mass spectrometry (ICP-MS) analyses using Chelex treatment

To clarify the Cd-binding of rcgPDI and detect binding of rcgPDI with Cd or other metals during protein preparation, treatment with Chelex chelating metal ion exchange resin (Bio-Rad) was performed. Approximately 5 mg of resin was used for every 100 µL of cgPDI. The resin was added to the cgPDI and stirred gently for 1 h. The cgPDI was filtered from the resin to obtain the metal-free cgPDI (cgPDI-Chelex) group.

The CD spectrum was obtained using a J-820 spectropolarimeter (Jasco). rcgPDI was used as the protein sample. The samples were divided into five groups. In the pure rcgPDI, rcgPDI was dissolved in ultrapure water. In the second group, rcgPDI + Cd, rcgPDI was dissolved in ultrapure water with the addition of Cd at a final concentration of 1 mM. In the third group, rcgPDI treated with Chelex reagent, rcgPDI was dissolved in ultrapure water treated with the Chelex treatment as described above. In the fourth group, rcgPDI treated with Chelex reagent + Cd, rcgPDI was dissolved in ultrapure water treated with the Chelex treatment as explained above, followed by addition of Cd to a final concentration of 1 mM. In the fifth group, rcgPDI + Cd treated with Chelex reagent, rcgPDI was dissolved in ultrapure water with the addition of Cd at a final concentration of 1 mM, and then treated with the Chelex treatment as described above. The rcgPDI concentration was adjusted to 0.5 mg/mL. Measurements were performed at wavelengths ranging from 200 to 250 nm. The cuvette path length was 0.1 cm, scan speed was 50 nm/min, response time was 1 s, bandwidth was 1 nm, and data pitch was 0.1 nm. All samples were scanned three times and the average was taken. Secondary structure analysis was done by the BsStSel protein secondary structure analysis program³⁷.

The metal concentrations were determined by ICP-MS using the 7900/MassHunter system (Agilent). The measurement conditions are summarized in Table ^S2. The organic matter in each sample was decomposed using nitric acid in an open wet ashing method. The sample was placed in a glass test tube, and 1 mL of concentrated nitric acid was added. The mixture was heated at 90 °C for 1 h and then at 120 °C until the nitric acid was completely evaporated. Concentrated nitric acid (1 mL) was added again and the two heating steps were repeated. Finally, 1 mL of 30% hydrogen peroxide solution (for precision analysis) was added, heated at 90 °C for 1 h, and completely evaporated at 120 °C. Heating was performed using a model DTU-2CN aluminum block constant-temperature bath (TIETECH Co., Ltd.). The decomposition product was dissolved in 5 mL of 0.08 M nitric acid and used as the measurement sample. One hundred microliters of 1000 mg/L standard solution of each metal was mixed and scaled up to 100 mL with 0.1 M nitric acid to prepare a 1 ppm standard solution.