Metal ions-dependent peroxidase and oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats

Abstract

We present evidence showing that a small fraction of electrophoretically homogeneous IgGs from the sera of healthy Wistar rats is bound with several different Me2+ ions and oxidizes 3,3'-diaminobenzidine through a peroxidase activity in the presence of H2O2 and through an oxidoreductase activity in the absence of H2O2. During purification on Protein A-Sepharose and gel filtration, the polyclonal IgGs partially lose the Me2+ ions. Therefore, in the absence of external metal ions, the specific peroxidase activity of IgGs from the sera of different rats varied in the range 1.6-26% and increased up to 13-198% after addition of Fe2+ or Cu2+ ions as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of HRP is 24-fold lower than its peroxidase activity, while oxidoreductase and peroxidase activities of IgGs are comparable. Oxidoreductase activities of different IgGs in the absence of external metal ions varied from 22 to 800%, and in the presence of Fe2+ or Cu2+ ions, from 37 to 1100% in comparison with the HRP oxidoreductase activity (100%). Chromatography of the IgGs on Chelex-100 leads to the adsorption of a small IgG fraction bound with metal ions and to its separation to many different subfractions demonstrating various affinities to the chelating resin and increased levels of the specific oxidoreductase and peroxidase activities. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defense mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activity of antibodies may play an important role in the protection of organisms from oxidative stress and toxic compounds.

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

“…10/30 column in the acidic buffer destroying non-covalent protein complexes as in (Ikhmyangan et al, 2005(Ikhmyangan et al, , 2006a. After each chromatography the IgG fraction was collected to cooled tubes containing 50 ml of 0.5 M K-phosphate (pH 8.5) and then additionally neutralized with this buffer and dialysed against 20 mM K-phosphate (pH 7.0).…”

“…During purification on Protein A-Sepharose and gel filtration, the pIgGs partially lose the Me 2þ ions. Therefore, the specific peroxidase and oxidoreductase activities of IgGs depending on a preparation, increase $8-120-fold after addition of external Cu 2þ or Fe 2þ (Ikhmyangan et al, 2006a). Chromatography of the IgGs on Chelex-100 leads to the adsorption of a small IgG fraction containing metal ions and to its separation into many different subfractions demonstrating various affinities to the chelating sorbent and increased levels of the specific oxidoreductase and peroxidase activities (Ikhmyangan et al, 2006a).…”

“…These Abs were isolated by affinity chromatography on Protein A-Sepharose but no criteria for assigning the peroxidase activity to Abs were applied and this activity was not proven to be intrinsic to Abs. Later it was shown that intact IgGs from the sera of healthy Wistar rats in the presence (H 2 O 2 -dependent peroxidase) and in the absence of H 2 O 2 (peroxide-independent oxidoreductase) similarly to horseradish peroxidase (HPR) oxidize effectively 3,3 0 -diaminobenzidine, o-phenylendiamine, phenol, p-dihydroquinone, a-naphthol and NADH but, in contrast to HRP, cannot oxidize adrenalin (Ikhmyangan et al, 2005(Ikhmyangan et al, , 2006a. To prove that these peroxidase and oxidoreductase activities are an intrinsic property of the IgGs and are not due to co-purifying enzymes, several strict criteria were applied, including an in situ assay involving incubation of an SDS-PAGE gel after removing of SDS in a standard reaction mixture containing DAB (Ikhmyangan et al, 2005(Ikhmyangan et al, , 2006a.…”

“…Later it was shown that intact IgGs from the sera of healthy Wistar rats in the presence (H 2 O 2 -dependent peroxidase) and in the absence of H 2 O 2 (peroxide-independent oxidoreductase) similarly to horseradish peroxidase (HPR) oxidize effectively 3,3 0 -diaminobenzidine, o-phenylendiamine, phenol, p-dihydroquinone, a-naphthol and NADH but, in contrast to HRP, cannot oxidize adrenalin (Ikhmyangan et al, 2005(Ikhmyangan et al, , 2006a. To prove that these peroxidase and oxidoreductase activities are an intrinsic property of the IgGs and are not due to co-purifying enzymes, several strict criteria were applied, including an in situ assay involving incubation of an SDS-PAGE gel after removing of SDS in a standard reaction mixture containing DAB (Ikhmyangan et al, 2005(Ikhmyangan et al, , 2006a. It is known that plant, bacterial and mammalian peroxidases, oxidoreductases, oxidases and dismutases are mostly metal ion-dependent enzymes (Feuers et al, 1993;Mates et al, 1999aMates et al, , 1999b.…”

Diversity of DNA‐hydrolyzing antibodies from the sera of autoimmune‐prone MRL/MpJ‐lpr mice

“…Rabbit IgGs against DNase I, DNase II, RNase A, DNA, and RNA (Krasnorutskii et al, 2008a(Krasnorutskii et al, , 2008b(Krasnorutskii et al, , 2008c(Krasnorutskii et al, , 2008d(Krasnorutskii et al, , 2009, similarly to human AI Abzs (Nevinsky et al, 2002a;Nevinsky and Buneva, 2002bCatalytic antibodies, 2005), were inactive in the presence of EDTA, while very small subfractions of human milk IgGs and sIgAs are Me 2þ -independent (Nevinsky et al, 2000b). We have recently shown that mammalian IgGs lose most bound metal ions during the standard procedure of purification to electrophoretic homogeneity (Polosukhina et al, 2006;Ikhmyangan et al, 2006). DNase pIgGs from the sera of MRL-lpr/lpr mice usually possess low activity after purification due to remaining intrinsically bound metal ions.…”