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Measuring Enzymatic Stability by Isothermal Titration Calorimetry
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校准ITC仪器:弱基中和问题
Žiga Medoš1, Marija Bešter-Rogač1, Epameinondas Leontidis2
1Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000, Ljubljana, Slovenia.
Analytical biochemistry
|July 8, 2024
概括
精确的异热定位热量计 (ITC) 校准需要考虑定位器体积损失,特别是在精确的测量中. 一个可变体积的协议有助于纠正从注射器尖端的扩散性HCl损失,提高热因子的准确性.
科学领域:
- 分析化学 分析化学
- 生物物理化学 生物物理化学
背景情况:
- 同热度定位热度计 (ITC) 仪器提供高精度,但需要化学校准才能准确.
- 盐酸 (HCl) 与弱基TRIS的反应是确定热因子的标准方法.
研究的目的:
- 用现代ITC仪器调查校准问题,特别关注HCl/TRIS反应.
- 识别和量化影响ITC测量的精度的错误来源.
主要方法:
- 使用VP-ITC和两个纳米ITC进行校准实验.
- 采用了可变标称体积协议来检测和纠正标称损失.
- 进行了数学建模,以分析从注射器尖端散发性丢失的HCl.
- 从复制数据中估计的数据方差函数,以改进最小正方形的拟合.
主要成果:
- 观察到显著的标量体积不足 (Δv ≈ 0.3 μL),归因于从注射器尖端的HCl扩散.
- 证明了可变体积协议在弥补这一缺口方面的有效性.
- 发现碳酸盐污染和弱基解可以引入实质性错误.
- 在不同的仪器中量化低信号精度 (σ ≈ 1 μJ) 和标称体积不确定性.
结论:
- 标称器的扩散损失是影响ITC准确性的关键因素,需要使用可变体积协议进行精确的校准.
- 标准校准协议可能不足,因为未解决的错误来源,如扩散,污染和水解.
- 准确确定热因子需要仔细考虑这些系统错误和仪器特定的不确定性.
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