C. Eugene Bennett Department of Chemistry, West Virginia University , Morgantown, West Virginia 26506, United States.
Anal Chem. 2017 Jan 3;89(1):929-936. doi: 10.1021/acs.analchem.6b04074. Epub 2016 Dec 21.
Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis-Menten constants (K) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A K value of 3.3 ± 0.8 mM (V, 2100 ± 200 μM/min) was obtained for 3'-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a K of 2 ± 1 mM (V, 400 ± 100 μM/min) was obtained for the 6'-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a K value of 3 ± 2 mM (V, 900 ± 300 μM/min) for 3'-sialyllactose. With a knowledge of V, the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3' and 6' sialic acid linkages.
磷脂纳米凝胶增强了外糖苷酶神经氨酸酶的稳定性和性能，并用于在毛细管内创建酶的固定区域。使用纳米凝胶，不需要通过共价键固定酶，因为它受到物理限制。这使得能够快速定量不同底物的米氏常数 (K)，并最终提供一种量化唾液酸连接（即 2-3 与 2-6）的方法。酶的固定区域价格低廉，易于在毛细管内定位，以支持使用神经氨酸酶进行电泳介导的微分析，从而分析唾液酸连接。为了避免在静态孵育过程中扩散的限制，通过在毛细管内电泳混合使底物通过固定的静止区域进行正向和反向通过的次数变化，可重现地实现孵育期。用来自产气荚膜梭菌的神经氨酸酶对用 2-氨基苯甲酸标记的 3'-唾液乳糖进行分析，得到 K 值为 3.3 ± 0.8 mM（V，2100 ± 200 μM/min），该酶可裂解具有α2-3,6,8,9 连接的唾液酸单体，这与文献中报道的需要台式分析的数值相似。酶裂解 2-3 连接的速度快于 2-6，对于 6'-唾液乳糖底物，得到 K 值为 2 ± 1 mM（V，400 ± 100 μM/min）。对选择性裂解 2-3 唾液酸连接的替代神经氨酸酶，得到 3'-唾液乳糖的 K 值为 3 ± 2 mM（V，900 ± 300 μM/min）。根据 V 的知识，该方法应用于 2-3 和 2-6 唾液乳糖的混合物以及 2-3 和 2-6 唾液酸化三触角聚糖。纳米凝胶电泳是一种廉价、快速且简单的替代当前技术，可用于区分 3'和 6'唾液酸连接的组成。
