Ko Francis H, Monbouquette Harold G
Biomedical Engineering Interdepartmental Graduate Program, University of California, Los Angeles, Los Angeles, California 90095, USA.
Biotechnol Prog. 2006 May-Jun;22(3):860-5. doi: 10.1021/bp050365t.
Beta-galactosidase (beta-gal) is shown to be a versatile new reporter enzyme in both photometric and electrochemical enzyme-multiplied assay techniques (EMATs). The well-known beta-gal substrate analog, o-nitrophenyl beta-d-galactopyranoside, yields the visibly colored, o-nitrophenol product upon hydrolysis, whereas the substrate, p-aminophenyl beta-D-galactopyranoside, gives rise to an electrooxidizable product, p-aminophenol. These beta-gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for beta-gal-based EMAT detection of estradiol (as the estradiol-bovine serum albumin (E-BSA) conjugate). The EMAT system is composed of the reporter enzyme, beta-gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the beta-gal-estradiol conjugate up to approximately 75%. Reporter enzyme inhibition is relieved significantly by addition of < or =2 ng/mL of estradiol (as E-BSA), which competes for binding with the antibody. Thus, the presence of analyte (E-BSA) is reported by the enzyme (beta-gal), which amplifies the ligand-protein dissociation event by turning over its substrate repeatedly. The electrochemical version of EMAT, based on amperometric detection of p-aminophenol, is responsive to added estradiol within minutes. These results show that beta-gal may serve as a useful alternative to glucose-6-phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems.
β-半乳糖苷酶（β-gal）在光度法和电化学酶倍增分析技术（EMATs）中被证明是一种多功能的新型报告酶。著名的β-gal底物类似物邻硝基苯基-β-D-吡喃半乳糖苷在水解后会产生可见颜色的邻硝基苯酚产物，而底物对氨基苯基-β-D-吡喃半乳糖苷则会产生可电氧化的产物对氨基酚。这些β-gal底物使得基于β-gal的EMAT检测雌二醇（作为雌二醇-牛血清白蛋白（E-BSA）偶联物）的光度法和电化学信号转导方案得以实现。EMAT系统由共价连接雌二醇的报告酶β-gal和雌激素抗体组成，雌激素抗体可将β-gal-雌二醇偶联物的酶活性抑制约75%。加入≤2 ng/mL的雌二醇（作为E-BSA）可显著缓解报告酶的抑制作用，因为雌二醇会与抗体竞争结合。因此，分析物（E-BSA）的存在由酶（β-gal）报告，该酶通过反复转化其底物来放大配体-蛋白质解离事件。基于对氨基酚安培检测的EMAT电化学版本在几分钟内对添加的雌二醇有响应。这些结果表明，β-gal可能是葡萄糖-磷酸脱氢酶的一种有用替代物，葡萄糖-磷酸脱氢酶目前在市售的EMAT系统中用作报告酶。
