Fieldes M A
Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada.
Electrophoresis. 1992 Jan-Feb;13(1-2):82-6. doi: 10.1002/elps.1150130116.
When plant tissue extracts are electrophoresed on polyacrylamide gels and the gels are stained for malate dehydrogenase by the standard NAD-dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [1]. The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)-mediated reduction of NBT. This reduction was enhanced by H2O2 and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, HRP also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS-mediated reduction of NBT and for the peroxidase-catalyzed NADH-dependent formation of H2O2. This peroxidase-catalyzed reaction has been proposed for the plant peroxidases involved in lignification.
当植物组织提取物在聚丙烯酰胺凝胶上进行电泳，然后通过标准的依赖烟酰胺腺嘌呤二核苷酸（NAD）的脱氢酶反应对凝胶进行苹果酸脱氢酶染色，最终形成还原型硝基蓝四氮唑（NBT）时，除了预期的有色条带外，还观察到无色条带。当染色条件有利于凝胶的普遍背景染色时会出现无色条带，并且在先前的一项研究中已表明，这些无色条带是由过氧化物酶同工酶引起的[1]。本研究使用辣根过氧化物酶（HRP）研究了过氧化物酶产生无色条带的机制。普遍的背景染色是由硫酸甲酯吩嗪（PMS）介导的NBT还原引起的。这种还原作用被过氧化氢（H2O2）增强，被HRP抑制。过氧化物酶显然催化了还原型PMS的过氧化氧化反应，从而抑制了含有产生无色条带的过氧化物酶同工酶的凝胶区域中NBT的普遍还原。然而，相比之下，HRP似乎也催化了还原型NAD的过氧化氧化反应，但该反应增加了NBT的还原。将结合其他人提出的关于PMS介导的NBT还原以及过氧化物酶催化的依赖NADH的H2O2形成的机制来讨论这些结果。有人提出这种过氧化物酶催化的反应与参与木质化过程的植物过氧化物酶有关。
