Identification, by electron paramagnetic resonance spectroscopy, of free radicals generated from substrates by peroxidase.
Identification, by electron paramagnetic resonance spectroscopy, of free radicals generated from substrates by peroxidase.
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通过电子顺磁共振波谱法鉴定过氧化物酶从底物产生的自由基。
DOI：
10.1016/s0021-9258(18)64640-x
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发表时间：
1960
期刊：
影响因子：
--
通讯作者：
L. Piette
中科院分区：
文献类型：
--
作者：
I. Yamazaki;H. S. Mason;L. Piette
用近藤法和森田法(6)从日本萝卜中提纯过氧化物酶，用Kenten-Mann法(7)结晶。本研究中使用的样品的RZ=2.7；抗坏血酸在0.1M醋酸盐缓冲液(pH 4.8，25“)中的消失率KQ为2.6×104，与已报道的马萝卜过氧化物酶的值2×10~4相对应。以伊士曼柯达白标对苯二酚和Mallinckrodt抗坏血酸为底物。按Hartree(9)方法制备的乙醚=9270(308MP)中的二羟基富马酸E~I和无重金属的醋酸酯缓冲液由FK Anan博士提供。在整个实验过程中，使用去离子的蒸馏水作为溶剂，以尽量减少痕量金属的自氧化影响。PH为4.8、6.0和7.0的缓冲液分别为0.1M醋酸盐、柠檬酸盐和磷酸盐混合物。使用新鲜试剂等级30%Hz02(Baker和Adamson)，并在每次实验前立即小心准备新鲜底物溶液，以最大限度地减少自氧化。所使用的电子顺磁共振波谱仪是一台瓦里安V-4500x波段仪器，配有6英寸磁铁和电源。该光谱仪采用100kC的场调制，外置扫描线圈，霍尔兹腔为空载&=7700。样品池是一个0.9×0.5×0.01英寸(7.5×2毫升)的石英试管，放置在野外，以避免水中的介质损失。用于动力学测量的流动系统是由犹他州立大学的Robert Burger博士设计的。该系统是仿照Roughton(10)和Chance(11)的系统。
Materials and MethodsPeroxidase was purified from Japanese turnips by the method of Kondo and Morita (6) and crystallized by the Kenten-Mann procedure (7). The sample used in this study had RZ= 2.7; kq for the rate of ascorbic acid disappearance in 0.1 M acetate buffer, pH 4.8 at 25”, was 2.6 X 104, corresponding to the value 2 X lo4 already reported for horse-radish peroxidase (8). Eastman Kodak white label hydroquinone and Mallinckrodt ascorbic acid were used as substrates. Dihydroxyfumaric acid, E,~ I. in ether= 9270 (308 mp), prepared according to the method of Hartree (9), and heavy-metal-free acetate buffer, were kindly supplied by Dr. FK Anan. Deionized, distilled water was used as solvent throughout the experiments to minimize autoxidative effects of trace metals. The buffers at pH 4.8, 6.0, and 7.0 were 0.1 M acetate, citrate, and phosphate mixtures, respectively. Fresh reagent grade 30% Hz02 (Baker and Adamson) was used, and care was taken to prepare fresh substrate solutions immediately before each experiment to minimize autoxidation. The electron paramagnetic resonance spectrometer used was a Varian V-4500 x-band instrument with associated 6inch magnet and power supply. The spectrometer was operated with 100 kc field modulation, with external sweep coils, and with an Holz cavity of unloaded &= 7700. The sample cell was a quartz cuvette 0.9 X 0.5 X 0.01 inch (7.5 X low2 ml) positioned in the fields in order to avoid dielectric losses in water. The flow system used for kinetic measurements was designed by Dr. Robert Burger of Utah State University. The system was modeled after that of Roughton (10) and Chance (11).