A complex antioxidant system of the organism resists free radical oxidation. Blood plasma proteins with antioxidant properties are components of this system [3]. Among them, a key role is assigned to human serum albumin (HSA). This protein interacts with free radicals, undergoes oxidative modification and protects the body. Albumin oxidative modification causes complete or partial loss of its diverse functional activity, which in itself can produce a variety of effects. However, during this process HSA acquires new properties and, possibly, new functions. Besides, albumin modified by oxidation can be an effective marker of OS.

Considering the increased interest in HSA role in free radical oxidation, we thought it important to summarize all the data collected and to examine this protein from two perspectives: as a source of and as a target for free radicals.

Serum albumin as a source of free radicals

In blood plasma there are two main proteins responsible for copper binding and transportation that at the same time prevent copper damaging effect on other plasma proteins, blood cells and surrounding tissues [4]. Ceruloplasmin contains copper in its active site. Reactions involving this protein are not accompanied by the formation of any radicals. The second largest copper pool in blood plasma is associated with human serum albumin that contains a high affinity site for copper, namely, the N-terminal tripeptide Asp-Ala-His (Cu²⁺/Ni²⁺-binding motif) [5]. Under normal conditions less than 1 % of total albumin is copper-bound, but this amount is enough to generate a big number of radicals in blood. In some pathological states, for example, Wilson’s disease or arthritis, the level of albumin bound copper can be considerably higher (2 to 5 times) [6, 7, 8].

Y.A. Gryzunov et al. [9] thoroughly investigated the conditions and reasons related to the change in the radical producing (pro-oxidant) activity of HSA-copper ions complex. Those authors studied, firstly, the effect of cystein-34 amino acid residue (Cys-34) modification on catalytic activity of the complex, and, secondly, the result of non-esterified fatty acid binding to albumin. To monitor the pro-oxidant activity of the complex, the rate of ascorbate radical formation was measured by the electron paramagnetic resonance assay (EPR), since ascorbate is one of the main interceptors of free radicals in blood plasma. At copper-albumin ratios below 1 : 1, the bound copper was almost redox-inactive, as long as Cys-34 was in a reduced state. We will term the inactive complex Cu/HSA-SH. Alkylation, nitrosylation and oxidation of thiol groups induced the catalytic radical producing activity of the Cu/HSA complex. This activity was more than an order of magnitude lower than the activity of free copper ions not bound to albumin. However, the evidence of such activity itself is important. Using ultrafiltration, it was shown that it is the complex with copper:protein stoichiometry of 1 : 1 and not free copper ions, generated accidentally during Cu/HSA-SH processing, that exhibits such activity.

In that work [9] it was also established that being catalytically inactive, Сu/HSA-SH complex displayed radical producing activity as a result of protein conformational changes when bound to free fatty acids, given that albumin did not contain any fatty acid impurities. Both conformational changes measured by probe fluorescence (fig. 1, probes I and II) and catalytic activity reached their maximum at a fatty acid to protein molar ratio of 3 : 1 for oleic acid and 2 : 1 for linoleic acid. Parallel to fatty acids

binding and profound conformational changes caused by this process, oxidation of Cys-34 SH-groups and a simultaneous increase in redox activity of copper-albumin complex were observed. The authors concluded that fatty acids regulate anti- and pro-oxidant properties of Сu/HSA complex by changing Cys-34 redox status.

The process described above includes the following stages (fig. 1):

1. binding of fatty acids in protein domains I, II and III;
2. albumin conformational changes (measured by the fluorescence of probes I and II);
3. activation of catalytic (redox) activity of the Cu complex in the binding site;
4. Cys-34 SH-group oxidation;
5. oxidation of other molecules by dissolved oxygen facilitated by Сu/HSA and accompanied by free radicals formation (oxidative stress).

Thus, pro-oxidant properties of HSA complex with copper ions are implemented only after protein SH-group has been oxidized or a thiol group has interacted with nitrogen monoxide (NO).

Serum albumin as a target for free radicals

A lot of data confirm that HSA antioxidant activity is determined by at least three factors: 1) its binding of variable valency metals, such as copper; 2) its reactions with free radicals (a radical trap); 3) the formation of products with antioxidant properties during its oxidative modification.

If HSA is added to blood lipoproteins that are quickly oxidized in the presence of copper ions, then lipid peroxidation in lipoproteins is inhibited [10], but not blocked completely, because copper retains its catalytic activity in the complex with albumin. It means that HSA is an antioxidant because it forms a complex with copper ions. However, this complex alone cannot be a pro-oxidant, which depends on the amount of albumin-bound copper in blood plasma and this complex activity. As stated previously, binding of NO and fatty acids, as well as a chemical modification of Cys-34 thiol group, makes Cu/HSA complex catalytically active. In contrast, native HSA completely inhibits catalytic activity of copper ions.

In serum albumin, Cys-34 SH-group is a primary radical interceptor; because of this group HSA constitutes the majority of reactive thiols in blood plasma [11, 12]. Cys-34 oxidation results in the formation of sulfenic acid (RSOH) that is later oxidized to sulfinic (RSO₂H) or sulfonic (RSO₃H) acids [13]. As mentioned before, SH-groups serve as a defense mechanism against free radical oxidation [14, 15], their concentration in blood plasma lowers considerably when OS increases, which occurs in various diseases [16, 17, 18] and aging [19]. Using high performance liquid chromatography (HPLC) with fluorescence detection, K. Oettl et al. studied HSA redox state as a potential systemic marker of OS in patients with various diseases (cataract, glaucoma, age related macular degeneration, diabetes mellitus, diabetic retinopathy and hypertension), with or without complications and with consideration of possible effects on age [20].

Another amino acid sensitive to free radical attacks is methionine. HSA contains 6 methionine residues. Its oxidation by various oxidizing agents leads to the formation of methionine sulfoxide (MetSO). However, changes in HSA properties induced by free radicals are tricky to interpret. To look at enzymes from this perspective, one can refer to the work of R. Levine et al. [21], who found that preferential oxidation of unprotected methionine residues of enzymes had little effect on the biological functions of glutamine synthetase. At the same time a supposition was made that methionine residues redox cycle in biological systems can be a factor of defense against reactive oxygen species and prevent other functionally important changes in protein structure.