231 ISSN 1607-6729, Doklady Biochemistry and Biophysics, 2017, Vol. 474, pp. 231–235. © Pleiades Publishing, Ltd., 2017. Original Russian Text © M.G. Gorobets, L.A. Wasserman, A.D. Vasilyeva, A.V. Bychkova, P.G. Pronkin, A.E. Bugrova, M.I. Indeykina, N.G. Shilkina, M.L. Konstantinova, A.S. Kononikhin, E.N. Nikolaev, M.A. Rosenfeld, 2017, published in Doklady Akademii Nauk, 2017, Vol. 474, No. 6, pp. 751–755. Modification of Human Serum Albumin under Induced Oxidation M. G. Gorobets a *, L. A. Wasserman a , A. D. Vasilyeva a , A. V. Bychkova a , P. G. Pronkin a , A. E. Bugrova a , M. I. Indeykina a,c , N. G. Shilkina a , M. L. Konstantinova a , A. S. Kononikhin a, b, c , E. N. Nikolaev a, c, d , and M. A. Rosenfeld a Presented by Academician A.L. Buchachenko December 5, 2016 Received February 12, 2017 Abstract—For the first time, by using the complex of physicochemical methods (mass-spectrometry, differ- ential scanning calorimetry, spectrofluorimetry, method of spectral and fluorescent probes, dynamic light scattering, and UV spectrophotometry), the oxidation-mediated modification of chemical and spatial struc- ture of albumin has been studied. All albumin structural regions are subjected to oxidation, methionine and aromatic amino acids primarily involved in oxidation. The albumin melting shows a decrease in thermal sta- bilization of the structure and changing of aggregation upon oxidation. The change in physical and chemical properties of albumin under different quantity of the oxidizer has been analyzed. DOI: 10.1134/S1607672917030218 It is known that proteins circulating in blood plasma are among the major targets for reactive oxy- gen species (ROS). Under the action of ROS, proteins undergo oxidative modification leading to disruption of their structures and functions. Oxidized damaged proteins accumulate in the course of aging and in var- ious pathological conditions [1]. Human serum albumin (HSA) is a dominant (by weight) blood plasma protein (approximately 60% of the total proteins). It was established that HSA can trap more than 70% of blood plasma ROS [2]. It can bind metals of variable valency, bilirubin (which, after binding to albumin, becomes able to inhibit lipid per- oxidation and exhibits other antioxidant properties [3]), and homocysteine (which can oxidize lipopro- teins [2]). The albumin molecule contains 17 disulfide bonds and one free cysteine residue (Cys34), as well as six methionine residues (Met87, Met123, Met298, Met329, Met446, Met548), which have an increased ability to react with ROS and, due to the high concen- tration of albumin in blood plasma, function as natural radical scavengers [2]. The antioxidant properties of albumin strongly decrease in some diseases, such as diabetes mellitus [2]. In liver and kidney diseases and in aging, oxidized forms of albumin accumulate in blood, which allows it to be considered as a biological marker of a pathological oxidative stress [2, 4]. The above features of albumin allow an approximate eval- uation of the amount of ROS generated in blood by the degree of oxidative modification of albumin [5]. Thus, the study of modifications of albumin oxidized in vitro is necessary for understanding the possible oxidative modifications of albumin in vivo. In this work, we studied the modification of the chemical and spatial structure of albumin as result of its induced oxidation. Human serum albumin (A1653, Sigma-Aldrich, United States) was oxidized with ozone as described earlier [5]. The amount of ozone in the reactor was varied in the range of 2.0–9.7 × 10 –7 mol (0.5–2.0 arb. units). Physicochemical properties of native and oxidized albumin samples were investigated by differential scanning calorimetry with the DASM-4 instrument (SKB NP, Russia) in the temperature range of 40– 120°C at a scan rate of 2°C/min at a constant pressure of 2 atm.; by dynamic light scattering with a Zetasizer Nano-S system (Malvern, United Kingdom); and by UV spectrophotometry with SF-2000 spectrophotom- BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY a Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, 119334 Russia b Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia c Moscow Institute of Physics and Technology, Dolgoprudny, Moscow oblast, Russia d Skolkovo Institute of Science and Technology, Skolkovo, 143025 Russia *e-mail: maria.g.gorobets@gmail.com; mary-gorobec@yandex.ru