Title: Modificatie-effecten van hydroxylradicalen op de zinkbinding en aminozuursamenstelling van humane metallothioneïnes
Other Titles: Modification effects of hydroxyl radicals on zinc binding and amino acid composition of human metallothioneins
Authors: Goossens, Lieven; M9407371;
Issue Date: 10-Sep-2013
Abstract: Metallothioneins (MTs), a low mass class of metalloproteins, are characterized by a high thiolate sulphur- and metal content. MTs are involved in metal homeostasis and heavy metal detoxification, and are efficient scavengers of free radicals. This study investigates the reactions of human fetal liver metallothioneins with bioradicals (hydroxylradicals and NO) with respect to metal release and modification of their amino acid composition. MT was isolated from human fetal liver by the method of Clough et al. (1986), involving extraction of the metallothioneins from liver tissue, gel chromatography, negative affinity chromatography and anion exchange chromatography, resulting in MT-0, MT-1 and MT-2 isoforms, devoid of fatty acid binding protein (FABP). The purity of the isoform fractions, i.e. absence of FABP and other protein impurities, was confirmed by multiple electrophoresis and blotting methods. The effects of NO (radicals) on MT were investigated as a preliminary study. The results confirmed and expanded the data obtained by Aravindakumar et al. (2000). Two efficient systems were developed to generate hydroxyl radicals either chemically or by physical means. The chemical Fenton reaction, based on the reaction of ferrous sulphate with hydrogen peroxide, yielded the highest quantity of hydroxyl radicals. The presence of the radicals was demonstrated and quantified by the ‘TBA-test’ and by electron paramagnetic resonance. Physically generated hydroxyl radicals were obtained by radiolysis of aqueous solutions of MT by gamma irradiation obtained from a 137CsCl source unit. As the main part of this study we investigated the modification effects on metallothioneins caused by hydroxyl radicals generated either chemically or by gamma irradiation. Amino acid analysis prior to and past treatment of MT with hydroxyl radicals generated by the chemical Fenton reaction demonstrated that the contents of cysteine/half-cystine was reduced dramatically following treatment with hydroxyl radicals. The number of serine residues increased considerably. Serine is one of the first oxidation products of the treatment of cysteine with hydroxyl radicals. The repeatedly demonstrated increase of the MT glycine content was also remarkable. These results were interpreted as complete destruction of the side chains of certain amino acids to the protein backbone due to the high reactivity of the hydroxyl radicals, hence leading to an increased number of glycine residues.The number of free sulfhydryl groups of the native protein was reduced following treatment of MT samples with hydroxyl radicals, without formation of disulfide bonds. The side chains of MT’s cysteines are either oxidized or destructed, but definitely not permanently converted to cystine.In our experimental conditions, Fenton reaction generated hydroxyl radicals caused a zinc release of about 95%. Hydrogen peroxide as such in the same experimental conditions also caused an extensive zinc release. It was causedby direct reaction of hydrogen peroxide with the metallothionein thiol groups.As an interesting side line on this study we also concluded that the MT-0 concentration is systematically underestimated if determined by AAS measurements. Secondly, we investigated the irradiation dose- and atmosphere-related effects of hydroxyl radicals on zinc release and amino acid modifications when aqueous solutions of purified MT-1 were subjected to gamma irradiation by doses ranging from 85 to 1500 Gy. We compared four irradiation conditions concurrently: aerobic conditions, anaerobic argon atmosphere, and dinitrogen monoxide atmosphere on native MT-1; and anaerobic argon atmosphere on metal-depleted MT-1. Irrespective of the pretreatment of the MT samples, we always observed a fairly rapid increase of serine content by 15 to 20% as the main irradiation effect. Under strictly anaerobic argon atmosphere however, the serine content eventually reached a plateau value of over 100% increase at 750 Gy dose. Under all atmosphere conditions the cysteine/half-cystine content decreased simultaneously with the rapid serine formation. Irradiation also led to increased cysteic acid formation except under argon atmosphere. At the higher irradiation doses, cysteic acid was partially reconverted into cystine; this is attributed to the reducing effect of the hydrated electrons generated as a by-product of radiolysis of aqueous solutions. The concentration of methionine decreased gradually, whereas in all but apo-MT-1 conditions, minute amounts of homocystine and cystathionine were formed. In addition a few other very limited amino acid modifications were observed. Amino acids with aliphatic side chains remained unchanged under all irradiation doses and atmospheric conditions.A zinc release of 27% was observed when the samples were subjected to 30 minutes of irradiation (250 Gy) in aerobic conditions; irradiation for 60 minutes (500 Gy) or more caused a zinc release of 100%, showing that initially a slow metal displacement occurred, followed by a facilitated fast and more extensive metal release. When aqueous solutions of zinc-MT-1 are treated with hydroxyl radicals, formed during gamma irradiation in doses ranging from 85 to 1500 Gy, at first one or a few cysteine/half-cystine residues are converted into serine residues with a limited simultaneous zinc release. As a consequence, MT-1 loses its rigid structure organized around the two distinct metal clusters, triggering a chain reaction of more zinc being rapidly released, hence exposing more cysteine residues to the oxidizing effects of the hydroxyl radicals present. Cysteines are converted into serine and cysteic acid. In anaerobic argon conditions further conversion occurs only into serine. Side effects include partial destruction of methionine, and formation of homocystine, cystathionine, and minute amounts of hydroxyproline, hydroxylysine and ornithine under selected irradiation conditions.In case higher concentrations of hydroxyl radicals react with MT, as in the experiments with Fenton reaction generated radicals, the main effects are not restricted to formation of serine or cysteic acid, but complete destruction of some amino acid side chains to the aplha-carbon atom occurs, leading to formation of glycine. Presumably the hydrogen peroxide, present in the Fenton reaction system, is also involved.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Biochemistry, Molecular and Structural Biology Section

Files in This Item:
File Status SizeFormat
Doctoraat Lieven Goossens.pdf Published 19125KbAdobe PDFView/Open Request a copy

These files are only available to some KU Leuven Association staff members


All items in Lirias are protected by copyright, with all rights reserved.