Asymmetrical flow field-flow fractionation to probe the dynamic association equilibria of β-D-galactosidase
نویسندگان
چکیده
Protein dynamics play a significant role in many aspects of enzyme activity. Monitoring structural changes and aggregation biotechnological enzymes under native conditions is important to safeguard their properties function. In this work, the potential asymmetrical flow field-flow fractionation (AF4) study dynamic association equilibria β-D-galactosidase (β-D-Gal) was evaluated. Three commercial products β-D-Gal were investigated using carrier liquids containing sodium chloride or ammonium acetate, effect adding magnesium (II) liquid assessed. Preservation protein integrity during AF4 analysis essential influence several parameters, such as focusing step (including use frit-inlet), cross flow, injected amount, studied. Size-exclusion chromatography (SEC) light scattering (DLS) used corroborate in-solution oligomerization observed with AF4. contrast SEC, provided sufficiently mild separation monitor conformations without disturbing equilibria. showed that acetate concentrations above 40 mM led further dimers (“tetramerization”) β-D-Gal. Magnesium ions, which are needed activate β-D-Gal, appeared induce dimer association, raising justifiable questions about divalent metal ions on whether tetramers most active form
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ژورنال
عنوان ژورنال: Journal of Chromatography A
سال: 2021
ISSN: ['1873-3778', '0021-9673']
DOI: https://doi.org/10.1016/j.chroma.2020.461719