Quantitative Enzyme Activity Determination with Zeptomole Sensitivity by Microfluidic Gradient-Gel Zymography

We describe a sensitive zymography technique that utilizes an automated microfluidic platform to report enzyme molecular weight, amount, and activity (including kcat and Km) from dilute protein mixtures. Calf intestinal alkaline phosphatase (CIP) is examined in detail as a model enzyme system, and the method is also demonstrated for horseradish peroxidase (HRP). The 40 min assay has a detection limit of 5 zmol (∼3 000 molecules) of CIP. Two-step pore-limit electrophoresis with enzyme assay (PLENZ) is conducted in a single, straight microchannel housing a polyacrylamide (PA) pore-size gradient gel. In the first step, pore limit electrophoresis (PLE) sizes and pseudoimmobilizes resolved proteins. In the second step, electrophoresis transports both charged and neutral substrates into the PLE channel to the entrapped proteins. Arrival of substrate at the resolved enzyme band generates fluorescent product that reveals enzyme molecular weight against a fluorescent protein ladder. Additionally, the PLENZ zymography assay reports the kinetic properties of CIP in a fully quantitative manner. In contrast to covalent enzyme immobilization, physical pseudoimmobilization of CIP in the PA gel does not significantly reduce its maximum substrate turnover rate. However, an 11-fold increase in the Michaelis constant (over the free solution value) is observed, consistent with diffusional limitations on substrate access to the enzyme active site. PLENZ offers a robust platform for rapid and multiplexed functional analysis of heterogeneous protein samples in drug discovery, clinical diagnostics, and biocatalyst engineering.

本文报道一种高灵敏度酶谱法（zymography）技术，该技术借助自动化微流控平台，可从稀蛋白混合物中检测酶的分子量、含量及活性（包括催化常数kcat与米氏常数Km）。本研究以小牛肠碱性磷酸酶(CIP)作为模型酶体系进行了详细探究，同时也验证了该方法对辣根过氧化物酶(HRP)的适用性。该耗时40分钟的测定方法对CIP的检测限可达5泽摩尔(zmol)（约3000个分子）。两步孔径限制电泳联用酶测定法（PLENZ）在单一直型微通道中完成，该通道内嵌聚丙烯酰胺(PA)孔径梯度凝胶。第一步通过孔径限制电泳(PLE)完成分离蛋白的分子量筛分与伪固定。第二步中，电泳将带电与中性底物均输送至PLE通道内的固定化蛋白位点处。底物抵达分离的酶条带后会产生荧光产物，结合荧光蛋白分子量梯即可反推酶的分子量。此外，PLENZ酶谱法可实现CIP动力学特性的全定量检测。与共价酶固定化方式不同，CIP在PA凝胶中的物理伪固定并未显著降低其最大底物周转率。但观测到米氏常数较游离溶液体系升高11倍，这与底物到达酶活性位点过程中存在的扩散限制效应相符。PLENZ为药物发现、临床诊断及生物催化剂工程领域中异质性蛋白样品的快速多重功能分析提供了可靠的技术平台。