Oxidation of Natural and Thermal Denatured Bovine Serum Albumin Hydrazyl Free Radicals in the Presence of Cyclodextrins GABRIELA IONITA 1 * and VICTOR SAHINI 2 1 Institute of Physical Chemistry, 202 Spl. Independentei, 77208 Bucharest, Romania; 2 Faculty of Chemistry, University of Bucharest 4–12 Regina Elisabeta Boulevard, R. 70346 Bucharest, Romania (Received: 6 October 2003; in final form: 25 April 2004) Key words: BSA, cyclodextrins, hydrazyl radicals, thermal denaturation Abstract The influence of certain thermal treatments on the reactivity of bovine serum albumin (denaturation followed by renaturation in three different cooling conditions) was studied monitoring the kinetics of oxidation of BSA with two water-soluble stable hydrazyl radicals. The results showed that the addition to the reaction mixture of a-cyclodextrin and b-cyclodextrin decreases the oxidation rate, probably due to the encapsulation of terminal amino acid rests by the cyclodextrins cavity. b-Cyclodextrin protects more efficiently the albumin probes than a-cyclodextrin. The denatured albumin probes are more reactive than natural albumin as a consequence of the reorienting of the hydrophobic rests of albumin molecule to their surface. Introduction Albumins represent a class of the transport proteins responsible for the transport, distribution and metabo- lism of many endogenous and exogenous ligands [1]. Particularly, the macromolecule of bovine serum albu- min (BSA) has a prolate ellipsoid form with axes of 40 and 140 A in solution [2]. In the native state, the BSA molecules take a conformation with the polar residues of amino acids oriented towards the outside of the mole- cule, and the nonpolar (hydrophobic) residues directed towards the inside of the molecule [3]. The structure of BSA explains its capacity to interact by hydrophobic or hydrophilic forces with other molecules (especially with those of biological significance). Increasing the temperature of BSA solutions, followed by different cooling procedures, has as effect the modifi- cations of native albumin conformation. We expect that thermal denaturation of BSA could be responsible for changes in some physical properties or chemical reactiv- ity. Our previous research showed that the chromato- graphic [4, 5], electrophoretic [4] or optical properties [6, 7] of albumin probes obtained after thermal treatment are considerably changed besides the natural BSA probe. Also, the interaction of BSA with some alkaline halide [5] or dyes [7] is modified after thermal treatment. The goal of this paper is to discuss the changes in the chemical reactivity of albumin, modified after different thermal treatments. In two other papers [8, 9] we studied the kinetics of oxidation of amino acids by stable free radicals in the absence or in the presence of natural cyclodextrins. We used the same hydrazyl water-soluble type radicals (the sodium salts of 2-p-phenylsulfonic- acid-2-phenyl-1-picrylhydrazyl (1) and 2,2¢-di-p-phenyl- sulfonic-acid-2-phenyl-1-picrylhydrazyl (2) see Figure 1a) to oxidize the albumin samples. The hydrophobic residues of amino acids from BSA structure are avail- able to the cyclodextrins cavity. Some of these residues could be oxidated by hydrazyl radicals and the kinetic investigation on albumin’s oxidation process, in the presence of cyclodextrin, was made in order to evidence, in an indirect way, the interaction between cyclodextrins and this protein. The ability of cyclodextrins to form inclusion com- pounds with organic molecules in water solutions, as a consequence of their toroidally shaped cavity, has attracted attention, and the literature reported many papers in the last 50 years. Our study deals with two cyclodextrins (CDs), a and b; these are the most common and they are composed by linking of 6 and 7D -glucopyranose unity, respectively. The majority of literature data present equilibrium studies on inclusion complexes of CDs, using various physico-chemical methods, as spectrascopic techniques [10], circular dichroism [11], NMR [12, 13], competitive inhibition of catalytic reactions [14], microcalorimetry [15], solu- bility [16], chromatography [17–19], potentiometry [20]. However, there are also many studies on the effects of CDs on the organic reaction which are explained by two models: ‘the enzyme model’ and ‘the extra reaction field model’, designed by the hydrophobic cavity of cyclo- dextrins [21]. * Author for correspondence. E-mail: ige@chimfiz.icf.ro Journal of Inclusion Phenomena and Macrocyclic Chemistry 50: 183–186, 2004. 183 Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.