Mäder M, Bopp M
Botanisches Institut der Universität Heidelberg, Hofmeisterweg 4, D-6900, Heidelberg, Federal Republic of Germany.
Planta. 1976 Jan;128(3):247-53. doi: 10.1007/BF00393236.
Isoelectric focusing (IEF) of peroxidases of different organs and tissues of Nicotiana tabacum L. was performed on thin-layers of Sephadex and polyacrylamide. Isoelectric points (pI's) of peroxidase bands were measured by special electrodes. - The two types of layers showed very similar results. Reproductibility of pI's was better on polyacrylamide. This method is also easier to practise and requires less time than IEF on Sephadex (3 h versus 18). Thus for analytical purposes the acrylamide-technique is preferable, but if it is necessary to regain the separated enzymes it is better to perform IEF on Sephadex. - When IEF-patterns of peroxidase are compared with the disk electrophoresis (DE) patterns of the same tissue, important differences are observed. The 6 bands of GI (fast migrating, anodic group of DE) are reduced to 2 on the strongly acidic side of IEF (independent of the tissue studied). That means only 2 proteins in GI can be separated by pI's of the molecules. Maybe the heterogeneity of GI bands after DE indicates the presence of conformational isomers (conformers). - Because of the reduced number of bands in GI after IEF there is no difference in the patterns of many tissues (flowers, leaves, shoots, pith) as there is after disk electrophoresis. In the case of GII (slow migrating, anodic group of DE) on the other hand, there are always 4 different bands after isoelectric focusing in the lower acid region instead of 3 after disk electrophoresis. Disk electrophoresis of peroxidase-groups separated by isoelectric focusing shows the same patterns as direct disk electrophoresis of the extract. The methods produce no artifacts. -Comparison of these results with the peroxidase-patterns of tobacco found by other workers and by other techniques leads to the conclusion that there exist at least 4 "isoenzymes" of peroxidase corresponding to the 4 groups GI, GII, GIII, and GIV.
在葡聚糖凝胶和聚丙烯酰胺薄层上对烟草不同器官和组织的过氧化物酶进行了等电聚焦（IEF）。通过特殊电极测量过氧化物酶带的等电点（pI）。- 两种类型的薄层显示出非常相似的结果。聚丙烯酰胺上pI的重现性更好。这种方法也比在葡聚糖凝胶上进行IEF更容易操作且所需时间更少（3小时对18小时）。因此，出于分析目的，丙烯酰胺技术更可取，但如果需要回收分离的酶，在葡聚糖凝胶上进行IEF更好。- 当将过氧化物酶的IEF图谱与同一组织的圆盘电泳（DE）图谱进行比较时，观察到重要差异。GI（DE中快速迁移阳极组）的6条带在IEF的强酸性侧减少到2条（与所研究的组织无关）。这意味着GI中只有2种蛋白质可以通过分子的pI分离。也许DE后GI带的异质性表明存在构象异构体（构象体）。- 由于IEF后GI中的带数减少，许多组织（花、叶、茎、髓）的图谱与圆盘电泳后没有差异。另一方面，对于GII（DE中缓慢迁移阳极组），等电聚焦后在较低酸性区域总是有4条不同的带，而圆盘电泳后是3条。通过等电聚焦分离的过氧化物酶组的圆盘电泳显示出与提取物直接圆盘电泳相同的图谱。这些方法不会产生假象。- 将这些结果与其他研究人员通过其他技术发现的烟草过氧化物酶图谱进行比较，得出的结论是，过氧化物酶至少存在4种“同工酶”，对应于4个组GI、GII、GIII和GIV。
