FIGURE 1.

A, HMGB1 and its three cysteine residues. Cys-23 and Cys-45 form a disulfide bond under oxidative conditions. B, experimental scheme for the in situ protein NMR approach to investigate the behavior of HMGB1 in actual extracellular fluids. C, ¹H-¹⁵N SOFAST-HMQC spectra recorded with eight scans at 37 °C for all-thiol HMGB1 (red) and disulfide HMGB1 (blue) immediately after dissolving in human serum. D, ¹H-¹⁵N SOFAST-HMQC spectra of all-thiol HMGB1 (red) and disulfide HMGB1 (blue) immediately after dissolving in 20 mm HEPES-NaOH (pH 7.4), 120 mm NaCl, and 5% D₂O. 135 μm ¹⁵N-labeled HMGB1 was used for both the serum and buffer samples. Line shapes of NMR signals from HMGB1 in serum were substantially broader than those in buffer presumably due to the molecular crowding environment of the serum. E, SDS-PAGE of the serum samples used for the NMR experiments. 3 μl of serum before and after dissolving 135 μm ¹⁵N-labeled HMGB1 was loaded. The arrow indicates the position of the HMGB1 band. The strongest bands are albumin. F, changes in the ¹H-¹⁵N SOFAST-HMQC spectra recorded at 37 °C for all-thiol HMGB1 in human serum. Due to oxidation, signals from all-thiol HMGB1 became weaker, whereas those from disulfide HMGB1 became stronger.