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Quantifying Long-Time Hydrogen–Deuterium Exchange of Bovine Serum Albumin with Hydrogen–Deuterium Exchange Small-Angle Neutron Scattering

Published

Author(s)

Roisin Donnelly, Norman J. Wagner, Yun Liu

Abstract

Hydrogen−deuterium exchange (HDX) measured by small-angle neutron scattering (HDX-SANS) is used to measure HDX in bovine serum albumin (BSA) under different temperatures and formulation conditions. HDX-SANS measurements are performed at 40, 50, and 60 °C in D2O after storing proteins at 4°C for 1 week to pre-exchange the readily accessible hydrogens. This enables us to probe the long-time HDX of protons at the core of the BSA proteins, which is more challenging for solvent molecules to access. The HDX kinetics are observed to follow an Arrhenius behavior with an apparent activation energy of 81.4 ± 1 kJ/mol, which is composed of the energy for protein conformational fluctuations and that for exchanging an amide hydrogen. Adding a tonicity agent of 150 mM NaCl has only a very slight effect on the HDX kinetics. Interestingly, we also observed that the formulation with faster HDX kinetics has a lower onset temperature of denaturation. This observation is qualitatively consistent with a previous study of HDX-SANS on a monoclonal antibody (mAb), despite the large difference of the secondary structure between BSA, dominated by alpha helices, and mAb, which is predominantly composed of β-sheet
Citation
Journal of Physical Chemistry B
Volume
129
Issue
1

Keywords

SANS, HDX, HDX-SANS, protein, dynamics, stability, BSA

Citation

Donnelly, R. , Wagner, N. and Liu, Y. (2025), Quantifying Long-Time Hydrogen–Deuterium Exchange of Bovine Serum Albumin with Hydrogen–Deuterium Exchange Small-Angle Neutron Scattering, Journal of Physical Chemistry B, [online], https://doi.org/10.1021/acs.jpcb.4c03967, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=960404 (Accessed July 8, 2025)

Issues

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Created January 9, 2025, Updated July 7, 2025
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