Malaria is a life-threatening disease induced by parasites of the Plasmodium falciparum species, exacerbated by resistant strains of Plasmodium and lacks effective vaccines and drugs. Survival, growth and multiplication of malaria parasites are strongly dependent on redox balance and oxidative stress, hydrogen peroxide (H₂O₂) redox signaling in pathogen-host cell interactions and involved in the target action mechanism of various antimalarial drugs [1] [2] . Moreover, degradation of hemoglobin as a result of P. falciparum induced oxidative stress during its intraerythrocytic life cycle, leads to the generation, significantly higher amount of H₂O₂, with the ability to selectively oxidize reactive cysteine residues in protein, and albumin as particularly. Thus, H₂O₂-dependent redox-signaling mechanism and molecular targets of changes H₂O₂-mediated signaling in P. falciparum cellular damage and tissues injury can contribute to deciphering parasite-host cell interactions and mechanisms of drug action and resistance. Moreover, it was suggested that oxidation modification of plasma proteins, predominantly albumin and fibrinogen, and as a result hyperproduction of its advanced oxidation protein products (AOPP), as one of the inflammatory markers, could be a candidate for biomarkers of malaria progression, anemia development [3] . One of the pathways of development of new drug for rational therapy and to overcome of the spread of parasites resistance is elaborated the strategy management based on susceptibility of the Plasmodium and others parasities, viruses and infections insights into disturbances, the redox balance and oxidative insults induction. Continuous strengthening of oxidative stressors substances during its intraerythrocytic cycle internal prooxidants derived in cytoplasm, degraded the hemoglobin to peptides and amino acids, and releases heme (Fe²⁺) which converts into hemin (Fe³⁺), acted as a long-range and fast acting signaling molecule for generation of superoxide anion that dismutase’s to H₂O₂ [4] [5] . Peroxidative actions depended on soluble free hemin which then accumulated of chloroquine into the digestive vacuole of Plasmodium and resulting in oxidative damage to membranes, proteins, and DNA, etc. [6] [7] . Furthermore, generation of H₂O₂, the most important reactive oxygen species (ROS) in cells controls protein functions of redox-sensitive proteins and enzymes, usually by selectively oxidizing of cysteine (Cys) reactive residues. Antioxidant properties of HSA with normal serum concentration about 35 - 50 g/L and 70% - 80% of one redox active Cys 35 (from 34 Cys residues in BSA) in reduced/sulfhydryl form [8] [9] assumed that H₂O₂ diffused from the source to the target protein, where it collides with the target thiol and directly introducing a mildly pro-oxidative state (hydrogen peroxide at a low physiological concentration of 10 nM on the susceptibility of protein (albumin) to glycation, particularly at physiological) [10] . This oxidation-driven H₂O₂-induced oxidation hypothesis was also tested on reduced albumin (mercaptalbumin) on the assumption that commercially source native albumin would be already partly oxidized and whether in vitro modification of albumin by low level of radicals lead to loss thiol group content. Moreover, the well-known antimalarial drug, chloroquine increases oxidative effects in the parasite through increased soluble free hemin and the most active pro-oxidant as 8-hydroxyquinoline (a phenolic compound), thus did not promote oxidation. Increased peroxidation produced by chloroquine depended on hemin and it was evidenced in presence of H₂O₂ [5] . However, neither molecular targets nor regulatory mechanisms and dynamics changes of H₂O₂-mediated signaling in P. falciparum have been clearly understanding, including the differences in the mechanisms of H₂O₂ or other agents oxidative and/or carbonyl stress modified in key transporting and more abundant protein of plasma, human serum albumin, and its relation to functioning of HSA efficacy. Overall, present work defined the different posttranslational modifications of main antioxidant molecule in human blood albumin, generated by thiol oxidation and revise its properties and possible biological function, pseudo-esterase activity, and advanced oxidation protein products (AOPP) formation, as oxidative stress biomarker.

All materials (reagent) were purchased in form of analytical grade, and all solutions were prepared in deionized and/or bidistilllated water. Serum Albumin/ALB Protein, Human (P. pactoris) (HSA) was purchased from Med Chem Express MCE) USA. HSA dissolved in sodium phosphate buffer (1 M, pH 7.4) and 0.02% NaN₃ as a preservative. 1,4-Dithiothreitol (DTT), 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB, 99%), Sigma, 2,4-Dinitrophenylhydrazine (DNPP), hydrogen peroxide 30% (Meck Mellipore), Ascorbic acid and tert-butylhydroperoxide (t-BHP) were purchased from Merck, Germany. The salts of Phosphate buffered saline (PBS) were of analytical grade (8.77 g/L NaCl, 1.28 g/L Na₂HPO₄, 1.36 g/L KH₂PO₄, adjusted with 50% NaOH to pH 7.2 - 7.4). Highly purified water was obtained by deionization and filtration with a Millipore purification apparatus (18.2 MΩ·cm at 25˚C).

Albumin, human or bovine, is protein responsible for maintaining the redox state of the plasma, regulating osmotic pressure and distributing fluids and transporting function of various ligands. Albumin in its redox form, mercaptoalbumin (HMA), exerts antioxidant effect by binding of many types of biological active molecules and ions to prevent ROS (such as H₂O₂, $O_2^{-}$, HOCl, ONOO⁻ and etc.) and which capable of trapping several reactive species by one active free sulfhydryl groups of Cys 34, for example. Markers of HMA oxidative damage, significantly decreased antioxidant activity greater in the presence of chloramine T > tBHP > H₂O₂. Early it was shown H₂O₂, one of the major oxidants generated in neutrophils, and a substrate of myeloperoxidase, did not, unlike HOCl, convert HSA into chaperone like holdases, and thus occurs potentially less detrimental positive feedback loop for neutrophil protection and highly immunogenic foreign antigens formation [25] .

Prolonged and persistent Plasmodium falciparum infection with or without symptoms involves a strong interrelationship between oxidative stress and pathogenesis, and oxidative stress strengthening could result in adverse malaria outcomes and may lead to complications. The malarial parasite augments oxidative stress to weaken the host and exerts antioxidant effects against host defense mechanisms. However, the anti-malarial drugs induce oxidative insult to reduce parasitic load and exert antioxidant activity in hosts and/or pro-oxidant activity in parasites. Using cell imaging with the cytosolically and mitochondrially expressed H₂O₂ biosensors biotechnology, in asexual blood stages of stably transfected P. falciparum NF54-attB parasites [26] , were provided results, that the mode of action of some antimalarial drugs and some of the novel antimalarial compounds includes disturbance of H₂O₂ homeostasis and in addition, degradation of haemoglobin leads to the generation of H₂O₂ within the malaria parasite and in blood. Carbonyl (CO) groups (aldehydes and ketones) are produced on protein side chains (especially of Pro, Arg, Lys, and Thr) when they are oxidized. These moieties are chemically stable, which is useful for both their detection and storage. The key pathophysiological relevance of the non-oncotic properties of blood albumin as a major target-protein for oxidative modifications [27] - [32] and stronger than ascorbic acid free radical’s scavenger properties. Free radical’s induces post-translation alterations that in sum expressed as AOPP, can further induced ROS production in cells and tissues, which coupled to, and promotes, inflammation and immune cell activation, thus sustaining the existence of a feedback loop and circulated in the body oxidized albumin acts as a damage-associated molecular pattern, with immunogenic activity [33] . From the table, it can be seen that the activity of H₂O₂-HSA is the same as that of native HSA (p > 0.1). In contrast, the activities of CT-HSA were much reduced and to the same level (p > 0.1). HSA was mildly oxidized by treatment with a H₂O₂ (H₂O₂-HSA), t-BPH (t-BPH-HAS) or chloramine-T (CT-HSA) in in vitro oxidation model. According to the detailed study of Finch et al. [34] , the SH-group of ³⁴Cys and Met residues have been oxidized in the case of H₂O₂-HSA. According to these authors, chloramine-T also oxidizes Cys and Met residues. Tested the effect of chloramine-T on the Met residues of albumin preparations by CNBr fragmentation and SDS-PAGE showed that Met residues were oxidized to methionine sulfoxide in CT-HSA and to a lesser extent in H₂O₂-HSA. It was reported that the catalytic activity of HSA is exhibited by the reactive residue Tyr 411 present in subdomain IIIA with different catalytic cleavage mechanisms. When ligand molecules bind to the subdomain region, they may interfere with the catalytic activity of HSA. The t-BPH, as greater hydrophobicity agent than hydroperoxide, penetrates/conjugates more easily with Cys 34 located in 10 angstrom wide hydrophobic pocket in HSA, and thus occurs more damaging of this region of albumin. Early was shown that, t-BPH in contrast to H₂O₂, completely inhibited peroxiredoxin-4 activity of HSA, however, both ligands/oxidants resulted in the same oxidative damage leading to sulfinic acid formation in Cys residues [35] . Moreover, was demonstrated that AOPP-HSA possesses the ability to activate isolated monocytes in vitro as predictor of respiratory burst, is dependent of the chlorinated nature of the antioxidants and initiated by CT and hypochlorite, but not H₂O₂ in range 10 – 100 μ [22] [25] . Plasma AOPP levels were predictive for anaemia and oxidative stress markers for clinical malaria infection in children [3] . Hydrogen peroxide at sublethal concentrations affects S.aureus general fitness and leads to a downregulated genes, predominantly included in pyrimidine [36] . Moreover, obtained data that treated of mercato-HSA with H₂O₂ practically did not change the catalytic activity towards p-nitrophenyl acetate, which is predominantly associated with the conformation state of subdomain IIIA of HSA close proximity to ⁴¹⁰Arg and ⁴¹¹Tyr [37] , unlike induced by CT pronounced decrease in pseudo-esterase activity could indicate that hydroperoxide-inducible alterations in mercapto-albumin caused conformational changes which increase the distance between the ⁴¹⁰Arg and ⁴¹¹Tyr active groups [38] in less extent than chlorination modification. From the other side, on the model of oxidative modified HSA induced by the changes in microenviroutment could lead to disturbances in port-translational modification of HSA, which act as the primary carrier for various exogenous and endogenous compounds and free radicals, and alter the pharmacokinetic properties of several drugs. Beside the already known and pharmacologically used antimalarial drugs such as quinolines and artemisinins, the development of new medicines with dual-action increases oxidative stress in malaria parasites by elevating intracellular H₂O₂ levels and decreases the damage oxidative associated complications for blood proteins whchi remain the actual problem and medical parasitology and pharmacology [27] , the most important reactive oxygen species (ROS) in cells.

The implications of hyperproduced free radicals in physiopathogenesis of various infectious diseases and its complications, and malaria as particularly, in which erythrocytes infected with P. falciparum produced OH^• radicals and H₂O₂ about twice as much compared to normal erythrocytes [27] , gave ground to suggest a crucial role of oxidative stress in clinical manifestation and systemic complications of parasite-induced diseases. Long regarded as a toxic byproduct that can damage macromolecules including DNA, proteins and lipids [28] , H₂O₂ is increasingly recognized as an important cellular signaling molecule with regulatory functions [29] [30] . H₂O₂ selectively oxidizes reactive cysteine residues and thereby controls functions of redox-sensitive protein—HSA. The esterase-like property seems especially useful in converting prodrugs to active drugs in plasma and its activities significantly decrease under treatment of HSA with t-BPH and CT, but not hydrogen peroxide. Thus, research efforts aim to expand reduced HAS’s ability to interact with more different drugs in order to improve the delivery of various pharmacological drugs remained as important problem in rational medicine and diagnostics.

Authors wish to acknowledge MedChemExpress Company (MCE, USA) for supporting that this work was completed successfully.

The authors did not receive support from any organization for the submitted work.

The authors date that the experimental protocol of the study was revised and approved by the Interinstitutional Animal Care and Use Committee of the Tbilisi State Medical University and International Centre of Introduction of New Biomedical Technology, Tbilisi, Georgia (No. 11-819012; date: March 11, 2021).