Towards versatile metal associating substrates for the determination of peroxidatic activity/hydrogen peroxide by chemical designing of Schiff base derivatives.
Novel chromogenic N-arylmethyl-aniline-substrates of the general formula R-NH-CH2C6H5-n-Xn (X = OH, NHR) for the localization of peroxidatic activity/hydrogen peroxide were synthesized in two steps from starting amines and aromatic aldehydes. When using 1,2-dinucleophiles (e.g. diaminobenzidine) as starting material there may be limitations resulting from dominant altemative reaction courses (amino-imines vs bis-imines) or tautomerism (amino-imines vs benzimidazolines). This has been investigated in a model study on 1,2-phenylendiamine. All substrates were evaluated for application in histochemistry, electrophoresis, colorimetry and electron microscopy. Thus, 1/ endogenous peroxidatic activity in native cryotome sections of Wistar rats was stained. One selected reagent was used for immuno-histochemical demonstration of vimentine and applied for laser microscopy at 543 nm as well. 2/ Electro-blotted dilution series of horseradish peroxidase were stained and reagents ranked according to their sensitivity. 3/ In test tube experiments precipitation behavior, color and solubility of precipitates was investigated. 4/The chromogens are capable of forming electron opaque final reaction products by way of increased osmiophilicity of the specific reaction product or/and by complexation of electron dense metals as demonstrated by electron microscopical investigations. As a result, two novel reagents derived from 1,2-phenylendiamine and 2-aminophenol are recommended especially for electron microscopy: The discrimination between internum and extemum of specific granules after osmium tetroxide treatment is resolved clearly as compared with results obtained with the standard Kamovsky protocol.
Krieg
R
R
Institute of Anatomy II, Friedrich Schiller University Jena, Germany. rkreig@mti-n.mti.uni-jena.de
Oehring
H
H
Halbhuber
K J
KJ
D000818:Animals; D001596:Benzylamines / Q000138:chemical synthesis / Q000737:chemistry; D002863:Chromogenic Compounds / Q000138:chemical synthesis* / Q000737:chemistry*; D015195:Drug Design; D005260:Female; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008297:Male; D008670:Metals / Q000378:metabolism*; D015394:Molecular Structure; D010545:Peroxides / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012545:Schiff Bases / Q000138:chemical synthesis* / Q000737:chemistry*
D001596:Benzylamines; D002863:Chromogenic Compounds; D008670:Metals; D010545:Peroxides; D012545:Schiff Bases; D006861:Hydrogen Peroxide