Purification and inactivation of 3-hydroxyanthranilic acid 3,4-dioxygenase from beef liver.
Purification and inactivation of 3-hydroxyanthranilic acid 3,4-dioxygenase from beef liver.
复制标题
牛肝中 3-羟基邻氨基苯甲酸 3,4-双加氧酶的纯化和灭活。
DOI：
10.1016/s1357-2725(02)00347-3
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Silverman,RichardB
中科院分区：
文献类型：
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作者：
Nandi,Dhirendra;Lightcap,EricS;Koo,YumeeKim;Lu,Xingliang;Quancard,Jean;Silverman,RichardB
3-使用两个染料柱（汽巴克隆蓝和活性绿色19）和羟基磷灰石从牛肝中纯化羟基邻氨基苯甲酸3，4-双加氧酶（EC 1.13.11.6; HADO）至均一。从羟基磷灰石柱上或通过酶在羟基磷灰石之前的非变性色谱聚焦分离酶的两个活性峰。这两种活性形式移动时，他们受到非变性聚丙烯酰胺凝胶电泳，无论分离的方法不同的电泳迁移率。然而，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中，这些物种具有明显相同的迁移率，因此具有接近的分子量。通过基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱分析，它们的分子量分别为32，566和32，515 Da，表观pI值分别为5.60和4.98，表明它们仅在铁辅因子的存在或不存在方面存在差异。N-末端基团似乎被封闭，因为直接Edman降解不可能产生氨基末端序列。合成了一种新的酶失活剂6-氯-3-羟基邻氨基苯甲酸，并显示出时间依赖性失活。提出了一种可能的失活机制。
3-Hydroxyanthranilic acid 3,4-dioxygenase (EC 1.13.11.6; HADO) was purified to homogeneity from beef liver with the use of two dye columns (Cibacron Blue and Reactive Green 19) and hydroxyapatite. Two active peaks of enzyme were isolated from the hydroxyapatite column or by nondenaturing chromatofocusing of the enzyme prior to hydroxyapatite. The two active forms moved with different electrophoretic mobilities when they were subjected to nondenaturing polyacrylamide gel electrophoresis, regardless of the method of isolation. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), however, these species had apparently identical mobilities and have, therefore, close molecular mass. Analysis by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry gave them a molecular mass of 32,566 and 32,515Da, respectively, for the species with apparent pI values of 5.60 and 4.98, respectively, suggesting that they differ only in the presence or absence of the iron cofactor. The N-terminal group appears to be blocked as no amino-terminal sequence was possible from direct Edman degradation. A new inactivator of the enzyme, 6-chloro-3-hydroxyanthranilic acid, was synthesized and was shown to exhibit time-dependent inactivation. A possible mechanism for inactivation is proposed.