检测瓜氨酸和非对称二甲基精氨酸的化学发光方法

非对称二甲基精氨酸（ADMA）是内源性一氧化氮合酶抑制剂，被公认是一种与心血管疾病、糖尿病、性功能障碍和肾功能衰竭等多种疾病密切相关的危险因子. 本文通过化学发光法检测瓜氨酸或ADMA经鸟氨酸氨基甲酰转移酶（ArcB）和氨基甲酰磷酸激酶（ArcC）偶联生成的ATP，并对该方法检测的灵敏度和动态范围进行了初步评价.1）从绿脓杆菌中克隆了鸟氨酸氨基甲酰转移酶和氨基甲酰磷酸激酶，转化大肠杆菌实现高效可溶性表达，用镍柱亲和纯化得到融合蛋白，TLC薄层层析法定性和测氨法定量验证了融合蛋白活性.2）用化学发光法检测了瓜氨酸或ADMA经相应酶偶联反应后的产物ATP，并且对实验进行了优化，结果表明两者都能在偶联酶作用下催化释放ATP，相应的底物浓度检测下限为01 μmol/L，检测结果接近正常生理血清中ADMA的浓度，且远低于正常生理血清中瓜氨酸浓度. 用正常尿液样品检测结果表明,该方法可行，为下一步血清和血浆样品的检测奠定了基础.

Luminescence Assay for L-citrulline and Asymmetric Dimethylarginine

Asymmetric dimethylarginine（ADMA） is an endogenous inhibitor of nitric oxide synthase. ADMA is a well-recognized player in cardiovascular diseases, diabetes mellitus, erectile dysfunction, and certain forms of kidney disease. A sensitive homogenous enzymatic assay for ADMA and L-citrulline was developed by measuring the luminescence generated from the oxidation of luciferin in the presence of luciferase and ATP, which was quantitatively released when L-citrulline was converted to L-ornithine by ornithine carbamoyl transferase（ArcB）and carbamate kinase（ArcC）.1）Recombinant ArcB and ArcC from the Pseudomonas aeruginosa（Schroeter）were expressed in E.coli, and purified by Nickle-NTA affinity chromatography. Two enzymes were shown to be well active as verified by analyzing citrulline-to-ornithine conversion through thin-layer chromatography and ammonia generated during the enzymatic reaction. 2）Under optimized condition, the lower detection limits for both L-citrulline and ADMA were down to 01 μmol/L, close to the physiological level of ADMA in plasma and well below the one for L-citrulline. Preliminary results indicated that this assay could be well applied to measuring ADMA and L-citrulline in urine samples and further optimization was needed for plasma and serum samples.