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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.

The first example of rabbit Abzs with superoxide dismutase activity was revealed in 1988 [36]. However, it was suggested that this activity might be due to traces of canonical enzymes with this activity. The existence of this activity was confirmed later; polyclonal and monoclonal Abs from various sources were shown efficiently to reduce singlet oxygen ( [37,38]. The superoxide dismutase activity of IgGs is linked with Fc, but not with their Fab-fragments [37,38]. However, because it is still the activity of antibodies, they can also be assigned to specific type of abzymes. These results suggest a possible protective function of these Abs and raise the question of whether a need in detoxification of ( 1 O 2 * ) can play a critical role in the evolution of the immunoglobulin fold. These Abzs show a mechanism through which oxygen can be reduced and recycled in phagocyte action, thereby enhancing the microbicidal action of the immune system [37,38]. Even more surprising was the discovery of higher eukaryotes abzymes catalysing the formation of ozone used by cells during phagocytosis [39]. Superoxide dismutase, catalase, 22 -independent oxidoreductase and 22 -dependent peroxidase activities of polyclonal IgGs of healthy Wistar rats were analysed [39][40][41][42][43][44]. About 83% of Abs demonstrated superoxide dismutase activity, while only 17% of preparations possess catalase activity [44], but all IgGs oxidized 3,3 -diaminobenzidine (DAB) in the presence and the absence of hydrogen peroxide [40][41][42][43][44][45]. 22 -independent oxidoreductase and 22 -dependent peroxidase activities were higher in the presence of different metal ions [44].
IgGs from patients with viral hepatitis B or C and healthy donors were shown to possess comparable peroxidase activities [46,47]. Later more detail analysis of healthy human IgGs was performed [48]. After dialysis of IgGs against EDTA, the relative peroxidase activity dependently of individual IgGs decreased from 100 to approximately 10-85%, while oxidoreductase activity from 100 to 14-83%. Separation of Abs on Chelex non-charged and charged with Cu 2+ ions results in separation of IgGs to many different subfractions having various levels of the specific oxidoreductase and peroxidase activities [48]. Among different metal ions, external Cu 2+ ions were the best activators of these abzymes.
In the present report, we characterize for the first time the substrate specificity of the human abzymes with peroxidase and oxidase activities.

Purification and characterization of IgGs
We have obtained electrophoretically homogeneous individual IgGs from sera of nine healthy volunteers as in [48]. All nine IgGs were electrophoretically homogeneous. Figure 1 demonstrates SDS-PAGE analysis of an equimolar mixture of nine IgGs (IgG mix ). One can see that IgG mix demonstrates a single band of the typical 150 kDa IgG before (lane 1) and two bands corresponding to the L and H and chains (lane 2) after Abs reduction with DTT (silver staining; figure 1a). It was previously shown that all IgG preparations purified by affinity chromatography on Protein G-Sepharose with following FPLC gel filtration oxidize DAB and do not contain any canonical enzymes [48]. This new set of nine IgGs was used for analysis of substrate specificity in the oxidation of several different typical substrates of various H 2 O 2 -dependent peroxidases and H 2 O 2 -independent oxidoreductases (designated as peroxidases and oxidoreductases, respectively). To prove that oxidoreductase and peroxidase activities of IgG mix are its intrinsic properties several strict criteria were validated similar to [48]. IgG mix was electrophoretically homogeneous (figure 1a). It was shown that similar to [48] after FPLC gel filtration of IgG mix in the acidic buffer (pH 2.6), the peaks of peroxidase and oxidoreductase activities tracked exactly with intact IgG mix . Sepharose bearing immobilized mouse Abs against human IgGs completely absorbed both activities and their peaks coincided with IgG mix eluted by acidic buffer (data not shown). To exclude possible traces of contaminating canonical peroxidases and oxidoreductases, the IgG mix was subjected to in situ SDS-PAGE, and its peroxidase and oxidoreductase activities were detected by the gel incubation using the standard mixture containing DAB. Yellow-brown bands were detected only in the position of intact IgG mix (figure 1b). As SDS usually dissociates all complexes of protein, the revealing of the peroxidase and oxidoreductase activities in the gel fragments corresponding only to the IgG mix and the absence of any other bands of the activities or proteins, provides direct evidence that IgG mix possesses these activities.
It is noted above that the superoxide dismutase activity is exhibited by the Fc fragments of antibodies [37,38]. We compared the relative peroxidase and oxidoreductase activities of F(ab) 2 and Fc of IgG mix . It was shown that F(ab) 2 fragments possess the peroxidase and oxidoreductase activities approximately 21to 22-fold higher than Fc fragments in the presence or absence of metal ions (e.g. figure 1c). Consequently, these activities are mainly determined by the variable regions of the abzymes.

Substrate specificities of human IgGs
IgGs of different donors oxidized several compounds in the presence and/or in the absence of H 2 O 2 . The affinity of all potential substrates to antibodies was relatively low, and the achievement of their concentrations close to saturation in most cases was impossible due to the low solubility of the compounds. Taking this into account, the kinetic curves were obtained using for each substrate feasible maximal concentrations giving no precipitates during the reaction. To estimate the activities quantitatively, we have found the concentration for each IgG preparation corresponding to the pseudofirst order of the reaction conditions within the linear regions of the time courses and Ab concentration curves. Figure 2   For a more precise definition of a possible affinity of various compounds for abzymes the K m and V max (k cat ) values characterizing Ab-dependent oxidation of good substrates were measured in the case of one preparation, IgG-4 (figure 4). All data obtained are summarized in
Interestingly, the k cat of IgGs from sera of 11 Wistar rats in DAB oxidation in the presence of hydrogen peroxide varied from 1.8 × 10 2 to 2.9 × 10 3 min −1 (average value 9.6 × 10 2 min −1 ), while in the absence of H 2 O 2 from 91 to 3.6 × 10 3 min −1 (7.2 × 10 2 min −1 ) [41]. Thus, the average peroxidase activity of Wistar  rat antibodies is approximately 11.4-fold lower than that of HRP, while oxidoreductase activity is 1.6-fold higher; for several individual Wistar rats, it is 3.8-8.0 times greater than that for HRP [41]. HRP oxidizes DAB with and without H 2 O 2 approximately 173-and 8.6-fold, respectively, faster than polyclonal human IgGs (according to the average values for IgGs, table 2). Interestingly, polyclonal rat IgGs demonstrate 14.5-fold higher average k cat value in DAB oxidation in the presence and 13.8-fold greater activity in the absence of hydrogen peroxide compared to those for human IgGs. The k cat values for polyclonal human and Wistar rat IgGs were calculated using their total concentrations. Since the specific activities were calculated using the total concentration of polyclonal IgGs and only a small fraction of IgGs possesses peroxidase-or oxidase-like activities [40][41][42][43], the specific activities of monoclonal IgGs possessing these activities may be significantly higher compared with total IgGs analysed by us (tables 2-4). As shown earlier, abzymes with several enzymatic activities are the earliest indicators of development of different AI diseases, while some of them are absent in the blood of healthy donors [11][12][13][14][15]. It was shown that DNase and RNase abzymes of AI patients present a 'cocktail' of Abs directly to DNA and RNA and anti-idiotypic Abs against active centres of DNase I, DNase II, RNase and other enzymes hydrolysing nucleic acids [11][12][13][14][15]. Abzymes of AI patients with proteolytic and oligosaccharidehydrolysing activities are Abs against different proteins and oligosaccharides [11][12][13][14][15]. For now it is not clear what antigens in healthy donors can stimulate the formation of abzymes with oxidoreductase and peroxidase activities.
An ever-increasing number of investigations suggests that AI diseases originate from defects in the HSCs [49]. It is known that apoptotic cells are the primary source of antigens and immunogens in SLE and other AI diseases, which trigger the recognition, perception, processing and/or presentation of apoptotic autoantigens by antigen-presenting cells, and can cause AI processes [50]. In addition, the appearance of abzymes in SLE and encephalomyelitis AI mice is associated with significant changes in profile of differentiation and level of proliferation of mice bone marrow HSCs as well as with increase in the level of lymphocyte proliferation in different organs [21,34,35]. However, production of abzymes   may also be not associated with defects of the HSCs. Immunization of healthy non-AI mice with DNA or other antigens also leads to the production of catalytic antibodies [21,34]. But this process is not associated with the changes in the profile of differentiation of mice bone marrow HSCs; it is the result of further differentiation of previously differentiated cells in bone marrow and suppression of the level of apoptosis of lymphocytes in different organs [21,34]. Most probably, in healthy humans the production of abzymes with peroxidase and oxidoreductase activities is not associated with any changes in the profile of differentiation of bone marrow HSCs. This process may be stimulated by different toxic, mutagenic and carcinogenic compounds falling into human organisms and stimulating lymphocytes' additional differentiation and increase in their proliferation in different organs. In addition, anti-idiotypic Abs against active centres of various enzymes can also possess different catalytic activities [11][12][13][14][15]. Thus, one cannot exclude a possibility of the formation of different catalase and oxidoreductase abzymes may be due to production of anti-idiotypic Abs against canonical catalase, glutathione peroxidase, superoxide dismutase and other enzymes oxidizing different substrates in parallel with formation of Abs to different dangerous compounds. Tables 3 and 4 demonstrate the catalytic diversity of abzymes towards various substrates in the case of different humans. One cannot exclude that it may be a consequence of the fact that various harmful substances enter the organisms of different people, which can lead to the production of abzymes with a different substrate specificity. As was mentioned above, HRP can oxidize different compounds mostly in the presence of H 2 O 2 and to a much lesser extent in its absence. At this time, it is not clear whether the same or different molecules of human IgGs oxidize substrates in the presence and in the absence of hydrogen peroxide. However, the same IgG preparations oxidize three substrates in the presence and absence of hydrogen peroxide, while another five only in the presence of H 2 O 2 (tables 2 and 3). Therefore, one can propose that in the case of the same IgG preparations substrate specificity of peroxidase H 2 O 2 -dependent activity may be advanced comparing with their oxidoreductase activity.        Here we demonstrate for the first time that the peroxidase and oxidoreductase activities of human IgGs can effectively oxidize not only DAB but also other typical substrates of various enzymes with oxidative activities. Sera of humans contain Abzs with superoxide dismutase activity. Therefore, Abzs with superoxide dismutase activity can reduce oxygen from  that substrate specificity of human IgGs peroxidase may be more expanded than its oxidoreductase specificity. Taken together, we suggest that the specific repertoire of polyclonal human Abs can serve as an additional natural system of reactive oxygen species detoxification and Abs can destroy hydrogen peroxide, mutagenic, toxic and carcinogenic compounds.