Open AccessCharacterization of Cd, Zn‐Thionein (Metallothionein) Isolated from Rat and Chicken Liver
Author(s) -
Weser Ulrich,
Rupp Heinz,
Donay Falk,
Linnemann Frithjof,
Voelter Wolfgang,
Voetsch Walter,
Jung Günther
Publication year - 1973
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1973.tb03111.x
Subject(s) - chemistry , metallothionein , circular dichroism , size exclusion chromatography , sephadex , cysteine , chromatography , chloroform , metal ions in aqueous solution , cystine , nuclear chemistry , metal , zinc , biochemistry , enzyme , organic chemistry
The preparation of pure metallothionein from Cd‐pretreated rats and chicken was performed using short‐time treatment with chloroform/ethanol followed by ion‐exchange and gel chromatography. The protein contained 7 g‐atoms metal ions per 12000 g protein. The Zn to Cd ratio was 1:2.4 ± 0.1. The Cd, Zn‐thionein remained homogeneous during Sephadex G‐25, G‐50 and G‐75 gel filtration, during polyacrylamide disc‐gel electrophoresis and in the analytical ultracentrifuge. The high content of cysteine residues (approx. one‐third of the total residues) was consistent with the cysteine content of metallothionein isolated from human or equine tissues. The Cd, Zn‐thionein was of considerable temperature stability. The physicochemical properties were examined by ultraviolet spectroscopy, circular dichroism (CD) measurements and X‐ray photoelectron spectroscopy. The millimolar absorption coefficient of the native protein was at 250 nm ɛ 250 = 80.6 mM −1 · cm −1 . Virtually no aromatic amino acids were present. The ɛ 250 value of the apoprotein prepared by metal displacement using HCl was ɛ 250 = 13.2 mM −1 · cm −1 . When the ultraviolet spectrum of the apoprotein was recorded at pH 6.6 a distinct peak was detectable at 255 nm (ɛ 250 = 18.7 m M −1 · cm −1 ). The existence of inter‐ or intramolecular disulphide formation was confirmed by CD measurements employing the polyethyleneglycol esters of di‐ tert ‐butyloxy‐carbonyl‐ l ‐cystine and tert ‐butyloxycarbonyl‐ l ‐cysteine as model compounds for high‐molecular‐weight and water‐soluble cysteine compounds, respectively. From CD data it was concluded that Cd, Zn‐thionein exists mainly as a random‐coil peptide. Some indication of the binding of Cd and Zn in the native protein with ⊖ S–R moieties was obtained from ultraviolet data. Final proof of the exclusive coordination of these metal ions with cysteine sulphur was successful using both circular dichroism measurements and especially X‐ray photo‐electron spectroscopy. The last method proved most convenient for determining the binding energies of the core electrons of Cd (Cd 3d 3/2 = 411.0 eV and Cd 3d 5/2 = 404.4 eV), Zn (2 p 3/2 = 1021.0 eV) and S (2p = 161.7 eV). The corresponding binding energies for S in the cystine complexes with Zn 2+ and Cd 2+ were 163.0 eV and 162.8 eV, respectively.