School of Chemical and Biomolecular Engineering, Georgia Institute of Technology.
Johns Hopkins University School of Medicine.
J Vis Exp. 2024 Sep 27(211). doi: 10.3791/67216.
The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature increases. For a therapeutic enzyme, other measures of stability may also be critical, particularly its ability to retain function in human serum at 37 °C, which we refer to as serum stability. Here, we describe an in vitro assay to assess the serum stability of the wildtype Homo sapiens adenosine deaminase I (HsADA1) enzyme using an absorbance-based microplate procedure. Specifically, this manuscript describes the preparation of buffers and reagents, a method arranging for the coincubation of HsADA1 in serum, a method to analyze the test samples using a microplate reader, and an accompanying analysis to determine the fraction of activity that an HsADA1 enzyme retains in serum as a function of time. We further discuss considerations to adapt this protocol to other enzymes, using an example of a Homo sapiens kynureninase enzyme, to help aid the protocol's adaptation to other enzymes where serum stability is of interest.
酶稳定性的概念通常是指酶的热稳定性——随着温度升高，酶保留结构和活性的能力。对于治疗性酶，其他稳定性的衡量标准也可能至关重要，特别是其在 37°C 人血清中保留功能的能力，我们称之为血清稳定性。在这里，我们描述了一种使用基于吸光度的微孔板程序评估野生型人类腺苷脱氨酶 I（HsADA1）酶血清稳定性的体外测定方法。具体来说，本文描述了缓冲液和试剂的制备、在血清中同时孵育 HsADA1 的方法、使用微孔板读数器分析测试样品的方法以及伴随的分析，以确定 HsADA1 酶在血清中随时间保留的活性分数。我们进一步讨论了适应本方案以用于其他酶的注意事项，以人类犬尿氨酸酶为例，以帮助将该方案适应对血清稳定性感兴趣的其他酶。
