A heme-nonapeptide tracer for electron microscopy |
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Authors: | Plattner Helmut Wachter Elmar Gröbner Peter |
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Affiliation: | (1) Department of Pharmacology, University of Innsbruck, Peter Mayr Strasse 1, A-6020 Innsbruck, Austria;(2) Department of Physiological Chemistry and Physical Biochemistry, University of München, D-8000 München, Germany;(3) Department of Biochemistry and Experimental Cancer Research, University of Innsbruck, A-6020 Innsbruck, Austria |
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Abstract: | Summary A heme-nonapeptide (H-9-P)1, applicable to electron microscopic cytochemistry via peroxidase-like activity, was prepared by passing horse heart cytochrome c through a column with Sepharose and covalently attached trypsin. After purification by column chromatography (Sephadex G50 Superfine, Biogel P-2) a maximal yield of 50% and purity of >99% was achieved. A concise schedule allows for inexpensive preparation of H-9-P with standard laboratory equipment. H-9-P has the following properties: Its structure is (14) Cys-Ala-Gln-Cys-His-Thr-Val-Glu-Lys (22) with heme attached to Cys (14) and (17). MW=1630, pI=4.95, E(max)pH 7= 397.5 nm, 22 °C, pH 7397.5 nm = 1.11 × 105 [Liter/Mole x cm]. With the use of a diaminobenzidine-H2O2-medium — as applied for cytochemistry — we determined spectrophotometrically a pHopt=12.5 and an apparent K5 = 3.14 × 10– 3 [M]. Glutardialdehyde leads to considerable de-activation and, according to SDS-polyacrylamide-gel-electrophoresis, to diffuse crosslinking accompanied by a shift of the active pH-region towards neutral pH values. An attempt was made to optimize the cytochemical assay. The peroxidase-like activity of H-9-P is well comparable to that of other heme-tracers; only horseradish peroxidase has a higher turnover number. When injected to mice or added to cell suspensions, even high concentrations of H-9-P did not entail any signs of toxicity.Abbreviations AAA amino acid analysis - AHC ammoniumhydrogencarbonate - BSA bovine serum albumin - Cyt c cytochrome c - DAB 5,3 -diaminobenzidine - GA glutardialdehyde - H-8-P heme-octapeptide - H-9-P heme-nonapeptide - H-11-P heme-undecapeptide - HR-POX horseradish peroxidase - MW molecular weight - PAGE polyacrylamid-gel-electrophoresis - pI isoelectric point - SDS sodiumdodecylsulphate - SG-TLC silicagel-thin-layer-chromatographyThis work was supported by the Österreichische Forschungsfonds![rdquo](/content/pk558km7118p7602/xxlarge8221.gif) |
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