Quantifying Long-Time\nHydrogen–Deuterium Exchange\nof Bovine Serum Albumin with Hydrogen–Deuterium Exchange Small-Angle\nNeutron Scattering

Hydrogen–deuterium exchange (HDX) measured by\nsmall-angle\nneutron scattering (HDX-SANS) is used to measure HDX in bovine serum\nalbumin (BSA) under different temperatures and formulation conditions.\nHDX-SANS measurements are performed at 40, 50, and 60 °C in D<sub>2</sub>O after storing proteins at 4 °C for 1 week to pre-exchange\nthe readily accessible hydrogens. This enables us to probe the long-time\nHDX of protons at the core of the BSA proteins, which is more challenging\nfor solvent molecules to access. The HDX kinetics are observed to\nfollow an Arrhenius behavior with an apparent activation energy of\n81.4 ± 1 kJ/mol, which is composed of the energy for protein\nconformational fluctuations and that for exchanging an amide hydrogen.\nAdding a tonicity agent of 150 mM NaCl has only a very slight effect\non the HDX kinetics. Interestingly, we also observed that the formulation\nwith faster HDX kinetics has a lower onset temperature of denaturation.\nThis observation is qualitatively consistent with a previous study\nof HDX-SANS on a monoclonal antibody (mAb), despite the large difference\nof the secondary structure between BSA, dominated by alpha helices,\nand mAb, which is predominantly composed of β-sheets.