About twenty secondary substrates were screened for the detection of peroxidase by the print technique following thin-layer isoelectric focusing. The best results were obtained with o-toluidine, guaiacol, o-phenylenediamine, and o-dianisidine. Detection of horseradish peroxidase was optimal when the paper was buffered at pH 5, and impregnated with a methanolic solution containing 1% of urea-peroxide and 1-2% of the secondary substrate. Uniform staining of the enzyme zones (compared to metachromatic staining with many other secondary substrates) and high color stability on storage was obtained. o-Toluidine and guaiacol were less sensitive by a factor of 10-100 than o-phenylenediamine and o-dianisidine, which belong to the most sensitive chromogens. With the two latter substrates as little as 0.001-0.01 μg of peroxidase could be detected by the print technique. o-Toluidine and guaiacol gave almost perfect white backgrounds with contrasting zones, whereas o-phenylenediamine and o-dianisidine yielded a moderately stained, but still acceptable, background. Peroxidase detection by the print technique described has been found suitable also for other thin-layer separation methods, e.g., thin-layer gel filtration. © 1972.
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