Ion-Specific Protein/Water Interface Determines the Hofmeister Effect on the Kinetic Stability of Glucose Oxidase Erik Sedlák Dagmar Sedláková Jozef Marek Jozef Hančár Katarína Garajová Gabriel Žoldák 10.1021/acs.jpcb.9b05195.s001 https://acs.figshare.com/articles/journal_contribution/Ion-Specific_Protein_Water_Interface_Determines_the_Hofmeister_Effect_on_the_Kinetic_Stability_of_Glucose_Oxidase/9805352 Homodimeric glucose oxidase (GOX) from Aspergillus niger is a prominent enzyme used for a number of applications in biotechnology and clinical diagnostics. For robust and long-term functional applications of GOX, the stability of the protein is of utmost importance. In vitro, GOX is irreversibly inactivated over time by a mechanism that is poorly understood, and hence, it presents a significant drawback for the development of strategies to stabilize the enzyme. We show that the nonequilibrium stability of GOX is fully described by a one-step conformational unfolding kinetics. To explore the strategies for improving GOX nonequilibrium stability, the effect of salts of the Hofmeister series is examined using microcalorimetry. We obtain activation energies <i>E</i><sub>a</sub> and inactivation temperatures <i>T</i><sub>k</sub> (at which the irreversible step is 1.0 min<sup>–1</sup>) as a function of the salt types and concentrations. Based on the analysis by the extended Langmuir model, we find that at high salt concentrations (>1 M) the Hofmeister effect on inactivation temperature is determined by the universal ion-specific effect on the protein/water interface, which apparently does not depend significantly on a particular amino-acid sequence and 3D protein structure. Our findings identify protein/water interfacial tension as a critical physicochemical attribute of excipients that is crucial for increasing enzyme kinetic stability. 2019-09-11 14:44:49 inactivation temperatures T k enzyme Kinetic Stability ion-specific effect salt types Hofmeister series amino-acid sequence inactivation temperature Glucose Oxidase Homodimeric glucose oxidase strategy GOX nonequilibrium stability Hofmeister effect Aspergillus niger 3 D protein structure concentration activation energies E application nonequilibrium stability Hofmeister Effect Langmuir model