Jouve H M, Pelmont J, Gaillard J
Arch Biochem Biophys. 1986 Jul;248(1):71-9. doi: 10.1016/0003-9861(86)90402-9.
Two different fractions were present in crystalline bovine liver catalase, and could be resolved using dye-ligand affinity chromatography with Red-A Matrex gel containing Procion HE 3B. The major part (alpha) was not adsorbed on this gel. The second fraction (beta) was firmly adsorbed to the gel, and could be eluted either by high salt or by NADPH in the micromolar range. Elution of catalase beta was also obtained with NADH, NADP+, and ADP at higher concentration. Fractions alpha and beta displayed no detectable difference in specific activity, stability to heat, and light absorption data. It is suggested that the difference in behavior between alpha and beta is related to the binding of NADPH to the mammalian catalase [H. N. Kirkman and G. F. Gaetani (1984) Proc. Natl. Acad. Sci. USA 81, 4343-4347], and that the beta fraction corresponds to the enzyme molecules that have at least one free site for NADPH binding. Modifications of catalase molecules in the presence of dithioerythritol (DTE) were examined using light absorption and EPR data. Thiol induced changes that corresponded to the formation of catalase complex II. They were partially reversed by NADPH at very low level, and the dinucleotide appeared to be oxidized in this process. DTE-treated bovine catalase was totally adsorbed on the Red-A Matrex columns, and could be eluted as fraction beta. Similar spectral changes in the presence of DTE and NADPH were displayed by a bacterial catalase from Proteus mirabilis. This enzyme was also able to oxidize NADPH, but was not adsorbed by Red-A Matrex. This work suggests that dye-affinity chromatography provides a very convenient tool for isolating dinucleotide-depleted catalase from bovine liver, facilitating further study of the physiological function of this cofactor within the enzyme.
结晶牛肝过氧化氢酶中存在两种不同的组分，使用含有Procion HE 3B的Red - A Matrex凝胶通过染料 - 配体亲和色谱法可将它们分离。主要部分（α）不吸附在这种凝胶上。第二部分（β）牢固地吸附在凝胶上，可用高盐或微摩尔浓度的NADPH洗脱。在较高浓度下，NADH、NADP⁺和ADP也能洗脱过氧化氢酶β。α和β组分在比活性、热稳定性和光吸收数据方面没有可检测到的差异。有人提出，α和β在行为上的差异与NADPH与哺乳动物过氧化氢酶的结合有关[H. N. Kirkman和G. F. Gaetani（1984年）美国国家科学院院刊81，4343 - 4347]，并且β组分对应于至少有一个NADPH结合自由位点的酶分子。使用光吸收和电子顺磁共振数据研究了在二硫苏糖醇（DTE）存在下过氧化氢酶分子的修饰。巯基诱导的变化对应于过氧化氢酶复合物II的形成。它们在非常低的水平下被NADPH部分逆转，并且在此过程中二核苷酸似乎被氧化。经DTE处理的牛过氧化氢酶完全吸附在Red - A Matrex柱上，并可作为β组分洗脱。奇异变形杆菌的一种细菌过氧化氢酶在DTE和NADPH存在下也表现出类似的光谱变化。这种酶也能够氧化NADPH，但不被Red - A Matrex吸附。这项工作表明，染料亲和色谱法为从牛肝中分离出二核苷酸耗尽的过氧化氢酶提供了一种非常方便的工具，有助于进一步研究这种辅因子在酶内的生理功能。
