Continuous spectrophotometric assay for ascorbate oxidase based on a novel chromophoric substrate, 2-aminoascorbic acid.
Continuous spectrophotometric assay for ascorbate oxidase based on a novel chromophoric substrate, 2-aminoascorbic acid.
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DOI：
10.1006/abio.1993.1150
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发表时间：
1993-04
影响因子：
2.9
通讯作者：
K. Wimalasena;S. Dharmasena
中科院分区：
文献类型：
--
作者：
K. Wimalasena;S. Dharmasena
最近，我们基于新的发色电子供体2-氨基抗坏血酸[K.Wimalasena和D.S.Wimalasena(1991)anal]建立了一种灵敏的连续分光光度分析抗坏血酸依赖的哺乳动物酶-多巴胺-β-单加氧酶的方法。生物化学。197,353-361]。我们现在报道抗坏血酸氧化酶(EC 1.10.3.3，L-抗坏血酸：O2氧化还原酶)也催化2-氨基抗坏血酸氧化成发色的2，2‘-腈-2(2’)-脱氧-L-抗坏血酸(红色素)。该反应动力学行为良好，表现出与氧和氧化产物(红色色素)有关的氧化酶催化反应的预期化学计量比，表明2-氨基抗坏血酸是该酶的良好替代底物。抗坏血酸氧化酶是一种对其天然底物抗坏血酸非常有效的酶。尽管与抗坏血酸相比，2-氨基抗坏血酸是该酶的弱底物，但表观初始速率动力学参数表明，在本实验条件下，该红色素的高消光系数表明，该酶的这种新的反应活性可用于设计一种灵敏、方便、连续的抗坏血酸氧化酶分光光度分析方法。虽然这种方法比现有的氧监测方法更方便，但它对固定化形式的酶活性的测量的适应性可能有助于开发用于工业或临床应用的生物液中抗坏血酸的自动检测技术。此外，该酶的这种新的反应活性可用于检测该酶的底物专一性和作用机制。
Recently, we reported the development of a sensitive continuous spectrophotometric assay for the ascorbate-dependent mammalian enzyme dopamine beta-monooxygenase based on the novel chromophoric electron donor 2-aminoascorbic acid [K. Wimalasena and D.S. Wimalasena (1991) Anal. Biochem. 197, 353-361]. We now report that ascorbate oxidase (EC 1.10.3.3, L-ascorbate:O2 oxidoreductase) also catalyzes the oxidation of 2-aminoascorbic acid to chromophoric 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment). The reaction is kinetically well behaved, displaying the expected stoichiometry for an oxidase-catalyzed reaction with respect to oxygen and the oxidation product (red pigment), demonstrating that 2-aminoascorbic acid is a well-behaved alternative substrate for the enzyme. Ascorbate oxidase is a very efficient enzyme toward its natural substrate, ascorbic acid. Although 2-amino-ascorbic acid is a significantly weak substrate for the enzyme in comparison to ascorbic acid, as indicated by the apparent initial rate kinetic parameters, the high extinction coefficient of the red pigment under our assay conditions suggests that this novel reactivity of the enzyme could be used to design a sensitive, convenient, and continuous spectrophotometric assay for ascorbate oxidase. While this assay is more convenient than the existing oxygen monitor assay, its adaptability to measure the activity of the enzyme in the immobilized form may be helpful in the development of technologies for the automated detection of ascorbic acid in biological fluids for industrial or clinical applications. In addition, this novel reactivity of the enzyme may be used to examine the substrate specificity and the mechanism of action of the enzyme.