Catalase Activity of IgG and κκ-IgG, λλ-IgG, and κλ-IgG Subfractions in HIV-Infected Patients and Healthy Donors.

BACKGROUND: Human immunodeficiency virus (HIV) infection is associated with pronounced oxidative stress, leading to the development of various virus-associated pathologies. A wealth of evidence suggests that, along with canonical enzymes of reactive oxygen species regulation, human blood contains antibodies with peroxidase, superoxide dismutase, and catalase activities. Here we show that the catalase activity of IgGs and their κκ-IgG, λλ-IgG, and κλ-IgG subfractions of HIV-infected individuals is significantly different compared to the healthy donors. METHODS: Protein G-Sepharose sorbent was used to resolve IgG from blood of healthy donors and HIV-infected patients by affinity chromatography. Subfractions of κκ-IgG, λλ-IgG, and κλ-IgG were separated from IgGs samples of each group by affinity chromatography on sorbents containing immobilized antibodies to κ or λ light human chains. The IgG catalase activity level was measured spectrophotometrically by evaluating the decrease in optical density (A240) due to hydrogen peroxide decomposition. RESULTS: The relative catalase activity of antibodies from HIV-infected patients (kcat = (1.41 ± 0.92) × 103 min-1, 95% CI: [1.01-1.81]) was statistically significant, 1.6 times higher (p = 0.014) compared to apparently healthy donors ((0.86 ± 0.49) × 103, 95% CI: [0.69-1.03]). The activity level of κκ-IgG HIV-infected patients ((0.44 ± 0.04) × 103 min-1) was 1.4 times higher than that of λλ-IgGs ((0.31 ± 0.025) × 103 min-1); the opposite was observed for κκ-IgGs from apparently healthy donors, which activity ((0.17 ± 0.015) × 103 min-1) was 3.1 times lower compared to λλ-IgGs ((0.53 ± 0.045) × 103 min-1). CONCLUSIONS: Thus, the data obtained may indicate that IgG with increased catalase activity may prevent harmful processes arising from oxidative stress in HIV-infected patients, acting as an additional natural molecular mechanism of regulation of hydrogen peroxide level.

Catalase Activity of IgGs of Patients Infected with SARS-CoV-2.

Coronavirus disease (COVID-19), caused by the SARS-CoV-2 coronavirus, leads to various manifestations of the post-COVID syndrome, including diabetes, heart and kidney disease, thrombosis, neurological and autoimmune diseases and, therefore, remains, so far, a significant public health problem. In addition, SARS-CoV-2 infection can lead to the hyperproduction of reactive oxygen species (ROS), causing adverse effects on oxygen transfer efficiency, iron homeostasis, and erythrocytes deformation, contributing to thrombus formation. In this work, the relative catalase activity of the serum IgGs of patients recovered from COVID-19, healthy volunteers vaccinated with Sputnik V, vaccinated with Sputnik V after recovering from COVID-19, and conditionally healthy donors were analyzed for the first time. Previous reports show that along with canonical antioxidant enzymes, the antibodies of mammals with superoxide dismutase, peroxidase, and catalase activities are involved in controlling reactive oxygen species levels. We here show that the IgGs from patients who recovered from COVID-19 had the highest catalase activity, and this was statistically significantly higher each compared to the healthy donors (1.9-fold), healthy volunteers vaccinated with Sputnik V (1.4-fold), and patients vaccinated after recovering from COVID-19 (2.1-fold). These data indicate that COVID-19 infection may stimulate the production of antibodies that degrade hydrogen peroxide, which is harmful at elevated concentrations.

Autoantibody-Abzymes with Catalase Activity in Experimental Autoimmune Encephalomyelitis Mice.

The exact mechanisms of the evolution of multiple sclerosis are still unknown. At the same time, the development in C57BL/6 mice of experimental autoimmune encephalomyelitis (EAE, simulating human multiple sclerosis) happens as a result of the violation of bone marrow hematopoietic stem cell differentiation profiles integrated with the production of toxic auto-antibodies splitting the basic myelin protein, myelin oligodendrocyte glycoprotein (MOG), histones, and DNA. It has been shown that IgGs from the plasma of healthy humans and autoimmune patients oxidize many different compounds due to their peroxidase (H2O2-dependent) and oxidoreductase (H2O2-independent) activities. Here, we first analyzed the changes in the relative catalase activity of IgGs from C57BL/6 mice blood plasma over time at different stages of the EAE development (onset, acute, and remission phases). It was shown that the catalase activity of IgGs of 3-month-old mice is, on average, relatively low (kcat = 40.7 min-1), but it increases during 60 days of spontaneous development of EAE 57.4-fold (kcat = 2.3 × 103 min-1). The catalase activity of antibodies increases by a factor of 57.4 by 20 days after the immunization of mice with MOG (kcat = 2.3 × 103 min-1), corresponding to the acute phase of EAE development, and 52.7-fold by 60 days after the treatment of mice with a DNA-histone complex (kcat = 2.1 × 103 min-1). It is the acceleration of the EAE development after the treatment of mice with MOG that leads to the increased production of lymphocytes synthesizing antibodies with catalase activity. All data show that the IgGs' catalase activity can play an essential role in reducing the H2O2 concentration and protecting mice from oxidative stress.

Antibodies-Abzymes with Antioxidant Activities in Two Th and 2D2 Experimental Autoimmune Encephalomyelitis Mice during the Development of EAE Pathology.

The exact mechanisms of multiple sclerosis development are still unknown. However, the development of EAE (experimental autoimmune encephalomyelitis) in Th and 2D2 mice is associated with the infringement of the differentiation profiles of bone marrow hematopoietic stem cells which are bound with the production of compounds that are harmful for human autoantibodies-abzymes that hydrolyze myelin oligodendrocyte glycoprotein, myelin basic protein, and DNA. It showed that autoimmune patients' antioxidant IgG antibodies oxidise some compounds due to their peroxidase (H2O2-dependent) and oxidoreductase (H2O2-independent) activities more effectively than those in healthy humans can. It was interesting to identify whether the redox activities of the antibodies change during the development of autoimmune diseases. Here, we analyzed the change in these redox activities of the IgGs from the blood of Th and 2D2 mice, which corresponded to different stages of the EAE development. The peroxidase activity in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) in the Th (4-fold) and 2D2 (2-fold) mice IgGs, on average, is higher than the oxidoreductase activity is. The peroxidase activity of the Th (1.9-fold) and 2D2 (3.5-fold) mice IgGs remarkably increased during the 40 days of the spontaneous development of EAE. Forty days after the immunization of the MOG peroxidase activity, the IgGs of the Th and 2D2 mice increased 5.6-6.0 times when they were compared with those that presented no increase (3 months of age). The mice IgGs were oxidized with 3,3'-diaminobenzidine (2.4-4.3-fold) and o-phenylenediamine (139-143-fold) less efficiently than they were with ABTS. However, the temper of the change in the IgG activity in the oxidation of these substrates during the spontaneous and MOG-induced development of EAE was close to that which occurred for ABTS. All of the data show that the IgG peroxidase and oxidoreductase activities of EAE mice can play an important role in their protection from toxic compounds and oxidative stress.

Essential Protective Role of Catalytically Active Antibodies (Abzymes) with Redox Antioxidant Functions in Animals and Humans.

During the life of aerobic organisms, the oxygen resulting from numerous reactions is converted into reactive oxygen species (ROS). Many ROS are dangerous due to their high reactivity; they are strong oxidants, and react with various cell components, leading to their damage. To protect against ROS overproduction, enzymatic and non-enzymatic systems are evolved in aerobic cells. Several known non-enzymatic antioxidants have a relatively low specific antioxidant activity. Superoxide dismutases, catalase, glutathione peroxidase, glutathione S-transferase, thioredoxin, and the peroxiredoxin families are the most important enzyme antioxidants. Artificial antibodies catalyzing redox reactions using different approaches have been created. During the past several decades, it has been shown that the blood and various biological fluids of humans and animals contain natural antibodies that catalyze different redox reactions, such as classical enzymes. This review, for the first time, summarizes data on existing non-enzymatic antioxidants, canonical enzymes, and artificial or natural antibodies (abzymes) with redox functions. Comparing abzymes with superoxide dismutase, catalase, peroxide-dependent peroxidase, and H2O2-independent oxidoreductase activities with the same activities as classical enzymes was carried out. The features of abzymes with the redox activities are described, including their exceptional diversity in the optimal pH values, dependency and independence on various metal ions, and the reaction rate constants for healthy donors and patients with different autoimmune diseases. The entire body of evidence indicates that abzymes with redox antioxidant activities existing in the blood for a long time compared to enzymes are an essential part of the protection system of humans and animals from oxidative stress.

Increase in Autoantibodies-Abzymes with Peroxidase and Oxidoreductase Activities in Experimental Autoimmune Encephalomyelitis Mice during the Development of EAE Pathology.

The exact mechanisms of multiple sclerosis (MS) development are still unknown, but the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice is associated with the violation of bone marrow hematopoietic stem cells (HSCs) differentiation profiles associated with the production of harmful for human's autoantibodies hydrolyzing myelin basic protein, myelin oligodendrocyte glycoprotein (MOG35-55), and DNA. It was shown that IgGs from the sera of healthy humans and autoimmune patients oxidize many different compounds due to their H2O2-dependent peroxidase and oxidoreductase activity in the absence of H2O2. Here we first analyzed the change in the relative redox activities of IgGs antibodies from the blood of C57BL/6 mice over time at different stages of the EAE development. It was shown that the peroxidase activity of mice IgGs in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is on average 6.9-fold higher than the oxidoreductase activity. The peroxidase activity of IgGs increased during the spontaneous development of EAE during 40 days, 1.4-fold. After EAE development acceleration due to mice immunization with MOG35-55 (5.3-fold), complexes of bovine DNA with methylated bovine serum albumin (DNA-metBSA; 3.5-fold), or with histones (2.6-fold), the activity was increased much faster. The increase in peroxidase activity after mice immunization with MOG35-55 and DNA-metBSA up to 40 days of experiments was relatively gradual, while for DNA-histones complex was observed its sharp increase at the acute phase of EAE (14-20 days). All data show that IgGs' redox activities can play an important role in the protection of mice from toxic compounds and oxidative stress.

Substrate specificity of IgGs with peroxidase and oxidoreductase activities from sera of patients with systemic lupus erythematosus and multiple sclerosis.

The analysis of IgGs to protect humans from oxidative stress through oxidation of harmful compounds was carried out. We have compared here for the first time peroxidase (in the presence of H2 O2 ) and oxidoreductase (in the absence of H2 O2 ) activities of IgGs from sera of healthy humans and patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS). In addition, substrate specificity of SLE and MS IgG preparations in the oxidation of different compounds was analyzed: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3'-diaminobenzidine (DAB), homovanillic acid (HVA), o-phenylenediamine (OPD), α-naphthol, 3-amino-9-ethylcarbazole (AEC), p-hydroquinone (pHQ), and adrenaline. IgGs of healthy humans and SLE and MS patients oxidized DAB, ABTS, and OPD due to their peroxidase and oxidoreductase activities, while other compounds were substrates of IgGs only in the presence of H2 O2 : adrenaline was not oxidized by both activities of IgGs. The average SLE IgGs peroxidase activity increased statistically significant in comparison with abzymes from healthy humans in the order (-fold): OPD (1.2) <  DAB (1.7) < α-naphtol (2.2) ≤ AEC (2.4) < ABTS (4.5) < 5-ASA (10.6), while with oxidoreductase activity: OPD (1.8) ≤ DAB (2.1-fold) < ABTS (5.0). Only HVA was oxidized by IgGs with peroxidase activity of healthy donors faster than by SLE (1.3-fold) and MS abzymes (2.4-fold). In the oxidation of several substrates, only three IgGs of MS patients were used. The data speak of a tendency to increase the peroxidase and oxidoreductase activities of MS IgGs in comparison with healthy donors, but to a lesser extent: OPD (1.1 to 1.2-fold) ≤ ABTS (1.2 to 1.8-fold). It was shown that development of SLE and MS leads to increase in peroxidase and oxidoreductase activities of IgGs toward most of classical substrates. Thus, abzymes can serve as an additional factor of reactive oxygen species detoxification protecting of patients with SLE and MS from some harmful compounds somewhat better than healthy peoples.

Substrate specificity of healthy human sera IgG antibodies with peroxidase and oxydoreductase activities.

We have carried out an analysis of whether blood IgG antibodies can protect humans from oxidative stress by oxidizing different harmful compounds. A somewhat unexpected result was obtained. We show here for the first time that healthy human sera IgGs with the peroxidase (in the presence H2O2) efficiently oxidize different compounds: 3,3'-diaminobenzidine (1; DAB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (2; ATBS), o-phenylenediamine (3; OPD), homovanillic acid (4; HVA), α-naphthol (5), 5-aminosalicylic acid (6; 5-ASA) and 3-amino-9-ethylcarbazole (7; AEC), but seven of nine IgG preparations from different volunteers cannot oxidize p-hydroquinone (8: pHQ). The average apparent kcat values in the H2O2-dependent oxidation by human IgGs decreased in the following order (min-1): ATBS (73.7) ≥ DAB (66.3) > AEC (38.0) ≥ HVA (19.8) ≥ α-naphthol (8.6) > OPD (0.62) ≥ 5-ASA (0.48) > pHQ (0.24). In the absence of H2O2 (oxidoreductase activity), the relative average kcat values decreased in the following order (min-1): DAB (52.1) ≥ ATBS (50.5) > OPD (0.25). The peroxidase average activity of human IgGs was higher than the oxidoreductase one: 1.2-, 1.5- and 2.5-fold for DAB, ATBS and OPD, respectively. It should be assumed that antibodies can oxidize in addition to the large number of other different compounds analysed by us. As a whole, the specific wide repertoire of polyclonal human IgGs oxidizing various compounds could play an important role in protecting humans from oxidative stress and serve as an additional natural system destroying H2O2 and different toxic mutagenic and carcinogenic compounds.

IgG abzymes with peroxidase and oxidoreductase activities from the sera of healthy humans.

We present the evidence showing that small fractions of electrophoretically homogeneous immunoglobulin G (IgGs) from the sera of healthy humans and their Fab and F(ab)2 fragments oxidize 3,3'-diaminobenzidine through a peroxidase activity in the presence of H2 O2 and through an oxidoreductase activity in the absence of H2 O2 . During purification on protein G-Sepharose and gel filtration, the polyclonal IgGs partially lose the Me(2+) ions. After extensive dialysis of purified Abs against agents chelating metal ions, the relative peroxidase activity decreased dependently of IgG analyzed from 100 to ~10-85%, while oxidoreductase activity from 100 to 14-83%. Addition of external metal ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. Chromatography of the IgGs on Chelex non-charged with Cu(2+) ions results in the adsorption of a small IgG fraction bound with metal ions (~5%), while Chelex charged with Cu(2+) ions bind additionally ~38% of the total IgGs. Separation of Abs on both sorbents results in IgG separation to many different subfractions demonstrating various affinities to the chelating resin and different levels of the specific oxidoreductase and peroxidase activities. In the presence of external Cu(2+) ions, the specific peroxidase activity of several IgG subfractions achieves 20-27 % as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of these fractions is ~4-6-fold higher than that for HRP. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defence mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activities of human IgGs could also 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.

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.