通过使用吩嗪甲硫酸酯和醋酸锌改进硝酸盐还原酶测定中亚硝酸盐的显色反应。
Improvements of the nitrite color development in assays of nitrate reductase by phenazine methosulfate and zinc acetate.
机构信息
Department of Agronomy, University of Illinois, and United States Regional Soybean Laboratory, Urbana, Illinois 61801.
出版信息
Plant Physiol. 1974 Jun;53(6):825-8. doi: 10.1104/pp.53.6.825.
Abstract
Nitrate reductase activity is most commonly assayed by measurement of product formation. Excess NADH and factor(s) present in the enzyme extract that interfere with the diazotization and azo color complex of nitrite cause a depression of apparent nitrate reductase activity. Two postassay treatments were found that markedly enhanced the extent of nitrite color formation and apparent nitrate reductase activity. The procedure involves stopping the reaction with zinc acetate (50 mumoles per ml of reaction mix), followed by removal of the precipitate by centrifugation. Presumably the zinc acetate removes extract factor(s) that interfere with color development, because it does not remove the NADH. Phenazine methosulfate (15 nmoles per ml of reaction mix) is added to aliquots of the supernatant and allowed to stand for 20 min at 30 C to oxidize the residual NADH before color development.
摘要
硝酸还原酶活性通常通过产物形成的测量来测定。酶提取物中过量的 NADH 和干扰亚硝酸盐的重氮化和偶氮色络合物的因素会导致明显的硝酸还原酶活性降低。发现两种后测定处理方法可以显著增强亚硝酸盐颜色形成和明显硝酸还原酶活性的程度。该程序包括用醋酸锌（反应混合物中每毫升 50 毫摩尔）停止反应，然后通过离心去除沉淀物。推测醋酸锌去除了干扰显色的提取物因素，因为它不会去除 NADH。吩嗪甲硫酸酯（反应混合物中每毫升 15 毫摩尔）被添加到上清液的等分试样中，并在 30°C 下放置 20 分钟，以使显色前氧化残留的 NADH。
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