Ceruloplasmin. A scavenger of superoxide anion radicals.
Ceruloplasmin. A scavenger of superoxide anion radicals.
复制标题
铜蓝蛋白。
DOI：
10.1016/s0021-9258(18)50692-x
复制
发表时间：
1979
期刊：
影响因子：
--
通讯作者：
G. Weissmann
中科院分区：
文献类型：
--
作者：
I. Goldstein;H. Kaplan;H. Edelson;G. Weissmann
材料和方法部分纯化的人血浆铜蓝蛋白（III型）得自Sigma Chemical Co.，St. Louis，MO，为0.25 M NaCl、0.05 M乙酸钠（pH 7.0）中的5.0%溶液。还从Sigma获得邻联茴香胺二盐酸盐、硝基蓝四唑、氧化黄嘌呤（EC 1.2. 3.2）、铁细胞色素C、次黄嘌呤、核黄素和抗坏血酸钠。Bio-Phore聚丙烯酰胺凝胶和Chelex 100来自Bio-Rad Laboratories，里士满，CA。乙醛和氯仿来自Eastman Chemical Co.，纽约州罗切斯特。Sephacryl S-200来自Pharmacia Fine Chemicals，皮斯卡特维，NJ;磷脂酰胆碱来自Grand Island Biological Co.，Grand Island，NY和硬脂胺，来自K & K Laboratories Inc.，新泽西州普莱恩维尤。其他试剂级化学品来自Fisher Scientific Co.，费尔劳恩，NJ.血浆铜蓝蛋白的纯化-部分纯化的人血浆铜蓝蛋白在Sephacryl S-200上进行分子筛层析，使用磷酸盐（0.01 M）缓冲的0.14 M NaCl，pH 7.4作为洗脱剂。通过使用邻联茴香胺二盐酸盐在0.1 M乙酸盐缓冲液（pH 5.0）中作为底物，测定级分的血浆铜蓝蛋白氧化酶活性（18）。还通过测量610 nm和280 nm处的吸光度保存馏分（19）。在下述研究中仅使用吸光度比（610 nm/280 nm）超过0.040的组分。这些被认为是大于90%纯铜蓝蛋白（19）。根据610 nm处的吸光度系数Ef 'f，0.68（20）计算血浆铜蓝蛋白的浓度。一些级分通过Chelex 100柱进一步纯化，Chelex 100柱预先用磷酸盐缓冲的0.14 M NaCl，pH 7.4平衡。奥德血浆铜蓝蛋白的清除活性-血浆铜蓝蛋白对OZ-2的清除能力，从而抑制OZ-2介导的反应。通过测量黄嘌呤氧化酶对两种底物次黄嘌呤和乙醛（21-23）的需氧作用介导的铁细胞色素C和硝基蓝四唑还原的抑制来测定。在室温（21-23 ℃）下，在Beckman 25型记录式等离子体光度计中，分别在550 nm和560 nm处测量铁细胞色素c还原为铁细胞色素c和NBT'还原为甲.
MATERIALS AND METHODSPartially purified human ceruloplasmin (type III) was obtained from Sigma Chemical Co., St. Louis, MO, as a 5.0% solution in 0.25 M NaCl, 0.05 M sodium acetate, pH 7.0. Also obtained from Sigma were o-dianisidine dihyclrochloride, nitroblue tetrazolium, xanthine oxidaae (EC 1.2. 3.2), ferricytochrome c, hypoxanthine, riboflavin, and sodium ascorbate. Bio-Phore polyacrylamide gels and Chelex 100 were from Bio-Rad Laboratories, Richmond, CA. Acetaldehyde and chloroform were from the Eastman Chemical Co., Rochester, NY. Sephacryl S-200 was from Pharmacia Fine Chemicals, Piscataway, NJ; phosphatidylcholine from Grand Island Biological Co., Grand Island, NY, and stearylamine from K & K Laboratories Inc., Plainview, NJ. Other reagent grade chemicals were from the Fisher Scientific Co., Fairlawn, NJ.Purification of Ceruloplasmin-Partially purified human ceruloplasmin was subjected to molecular sieve chromatography on Se-phacryl S-200 employing phosphate (0.01 M)-buffered 0.14 M NaCl, pH 7.4, as the eluant. Fractions were assayed for ceruloplasmin oxidase activity by employing o-dianisidine dihydrochloride in 0.1 M acetate buffer, pH 5.0, as substrate(18). Fractions were also assaved by measuring absorbance at 610 nm and at 280 nm (19). Only those fractions with an absorbance ratio (610 nm/280 nm) exceeding 0.040 were employed in the studies described below. These were considered to be greater than 90% pure ceruloplasmin(19). Concentrations of ceruloplasmin were calculated according to the absorbance coefficient at 610 nm, Ef’f, 0.68 (20). Some fractions were purified further by passage through columns of Chelex 100, previously equilibrated with ahosnhate-buffered 0.14 M NaCl, pH 7.4. Od.-scavenging Activity of C&doplasmin-The ability of ceruloplasmin to scavenge 02-s and thereby inhibit reactions mediated by OZ-. was assayed by measuring inhibition of reduction of ferricytochrome c and nitroblue tetrazolium mediated by the aerobic action of xanthine oxidase on two substrates, hypoxanthine and acetaldehyde (21-23). Reduction of ferricytochrome c to ferrocvtochrome c and of NBT’ to formazan were measured at 550 nm and 560 nm, respectively, at room temperature(21-23 C) in a Beckman model 25 recording ipectrophotometer.