Stirring rate affects thermodynamics and unfolding kinetics in isothermal titration calorimetry
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<jats:title>Abstract</jats:title> <jats:p>Isothermal titration calorimetry (ITC) directly provides thermodynamic parameters depicting the energetics of intermolecular interactions in solution. During ITC experiments, a titration syringe with a paddle is continuously rotating to promote a homogeneous mixing. Here, we clarified that the shape of the paddles (flat, corkscrew and small-pitched corkscrew) and the stirring rates influence on the thermodynamic parameters of protein–ligand interaction. Stirring with the flat paddle at lower and higher rate both yielded a lower exothermic heat due to different reasons. The complete reaction with no incompetent fractions was achieved only when the stirring was performed at 500 or 750 rpm using the small-pitched corkscrew paddle. The evaluation of the protein solution after 1,500 rpm stirring indicated that proteins in the soluble fraction decreased to 94% of the initial amount, among which 6% was at an unfolded state. In addition, a significant increase of micron aggregates was confirmed. Furthermore, a new approach for the determination of the unfolding kinetics based on the time dependence of the total reaction heat was developed. This study demonstrates that a proper stirring rate and paddle shape are essential for the reliable estimation of thermodynamic parameters in ITC experiments.</jats:p>
収録刊行物
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- The journal of biochemistry
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The journal of biochemistry 168 (1), 53-62, 2020-07
Tokyo : Japanese Biochemical Society
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詳細情報 詳細情報について
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- CRID
- 1522262180501933312
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- NII論文ID
- 40022279168
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- NII書誌ID
- AA00694073
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- ISSN
- 0021924X
- 17562651
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- NDL書誌ID
- 030502572
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- 本文言語コード
- en
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- NDL 雑誌分類
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- ZR2(科学技術--生物学--生化学)
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- データソース種別
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- NDL
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