Neue Vorstellungen zum Problem der Isoperoxidasen anhand der Trennung durch Disk-Elektrophorese und isoelektrische Fokussierung

Summary 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 G_I (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 G_I can be separated by pI's of the molecules. Maybe the heterogeneity of G_I bands after DE indicates the presence of conformational isomers (conformers). — Because of the reduced number of bands in G_I 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 G_II (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 G_I, G_II, G_III, and G_IV.

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Abkürzungen IEF Isoelektrische Fokussierung - DE Disk-Elektrophorese - pI Isoelektrischer Punkt