ISSN:
1432-119X
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
Notes:
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, λ E(max) pH 7 = 397.5 nm, ε 22 °C, pH 7 397.5 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00511078
Permalink