Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, k_cat, and the rate constant of productive binding, k_on, of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, revealing that inactive monomer units of ALP are deficient in disulfide bond formation and zinc ion coordination, and also offering the way to prepare homogenous and active populations of ALP for quantitative digital bioassays of ALP.

中文翻译：

---

阐明和控制低和高活性碱性磷酸酶分子的定量数字生物测定

碱性磷酸酶（ALP）是一种同型二聚酶，已广泛用于各种生物测定中，作为疾病标记和酶探针。从数字ELISA的快速增长和单分子ALP异构体的新兴多重分析中可以看出，数字生物测定的最新进展彻底改变了基于ALP的诊断测定。但是，在ALP分子中发现的固有异质性妨碍了基于ALP的定量数字生物测定。这项研究旨在定量分析大肠杆菌中ALP的单分子活性并揭示了ALP催化活性的静态异质性，该活性具有两个不同的种群：半活性部分和全活性部分。通过连续缓冲液交换进行数字化分析，发现了ALP的单分子Michaelis-Menten动力学；半活性分子的催化转换速率k _cat和生产结合速率常数k _on减半_。全活性分子。这些发现表明，半活性ALP分子是由非活性和活性单体单元组成的异源二聚体，而完全活性的ALP分子包含两个活性单元。还观察到了其他来源的ALP的静态异质性：小牛肠或虾，显示了如何将发现的结果推广到整个物种。带有二硫键增强剂和尖峰锌离子的ALP的无细胞表达导致均一的ALP群体具有完整活性，这表明ALP的非活性单体单元缺乏二硫键的形成和锌离子配位，并提供了制备均一的方法和ALP活跃人群进行ALP定量数字生物测定。