Acta Chim. Slov. 2000, 47, 111-119. 111 KINETICS OF OXIDATION OF AMINO ACIDS BY SOME FREE STABLE HYDRAZYL RADICALS‡ GABRIELA IONITA,a VICTOR Em. SAHINI,b GHEORGHE SEMENESCU a and PETRE IONITA c a University of Pitesti, Dept. of Physical Chemistry, Gh. Doja Street 41, 0300 Pitesti, Roumania University of Bucharest, Faculty of Chemistry, Bd. Carol I 13, 70346 Bucharest, Roumania c Institute of Physical Chemistry, Spl. Independentei 202, Bucharest, Roumania Received 7.5.1999 Abstract The kinetics of amino acids (Ser, Pro, Leu, Trp, Thr, Phe, Met, His) oxidation by sodium salts of 2-p-phenylsulfonic acid-2-phenyl-1-picrylhydrazyl (I) and 2,2-di-p-phenylsulfonic acid-2-phenyl-1-picrylhydrazyl (II) at isoelectric point of amino acids has been studied over the temperature range 298 - 318 K. The rate studies were made under pseudo-first order conditions with an excess of amino acid over the oxidant. The kinetics was followed by monitoring the disappearance of I and II, spectrophotometrically, at 520 nm and 514 nm, respectively. The activation parameters were determined from rate constant dependence on temperature. The amino acids with aromatic structure (His, Trp, Phe) were oxidised more rapidly than the others. A mechanistic pathway for amino acids oxidation was proposed and discussed, similarly with their enzymatic degradation, which have as final products a-keto-acids and ammonia. In this aim was followed a mechanistic and structural investigation grounded on isokinetic theory. Keywords: kinetics; free radical; hydrazyl; amino acids; BSA Introduction Amino acids represent for organism forerunners of essential biomolecules such as proteins, hormones, enzymes, etc; also, they may serve as energy source, losing their amino group by two pathways: transamination or oxidative deamination. The steps of oxidative deamination are resumed in the following scheme [1-3]: H I [O] -NH3 R----C----COOH 2H^ R----C----COOH NH R----C----COOH NH 2 NH O Scheme 1. Oxidative deamination of amino acids G. Ionita, V. Em. Sahini, G. Semenescu, P. Ionita: Kinetics of Oxidation of AminoAcids by Some… 112 Acta Chim. Slov. 2000, 47, 111-119. 1 R\^\ NO 2 k^\ T_^/V 1 N— —N— products (2) The same reactions can be written for the (NaSO3)2DPPH free radical (with kinetic constants k2 and k2 , of course); so the experimental kinetic constant is kexp = k1 + k1, where k1 was determined in a separate experiment (k1 = 0.76 * 10-5 s-1, k2 = 1.24 * 10-5 s-1). The influence of the pH over these kinetic constants were small; thus, in the 5-8 units pH range the variation was smaller then 10%; also, it is well known that another hydrazyl free radical, DPPH (2,2-diphenyl-1-picrylhydrazyl), is stable at 6-8 area of pH only [12]. On notice that the compound I and II have similar reactivity. Table 1 shown the kinetic constant determined for amino acids oxidation by free radicals I and II. Table 1. Kinetic constant for amino acid oxidation at 306 K by NaSO3DPPH (k1) or (NaSO3)2DPPH (k 2) and the correlation coefficient r. k 1 x 105 (s-1) r 1 k 2 x 105 (s-1) r 2 3.21 0.989 2.29 0.989 9.40 0.992 6.62 0.981 1.20 0.998 1.43 0.988 3.57 0.999 2.77 0.986 1.19 0.989 1.76 0.986 1.82 0.994 2.17 0.989 2.00 0.987 1.78 0.996 13.00 0.990 6.74 0.982 Amino acid pI13 Phe 5.91 His 7.64 Leu 6.04 Met 5.74 Pro 6.30 Ser 5.68 Thr 5.86 Trp 5.88 In many cases, the rate constants k1 and k2 are close, except values for Trp, His, and Phe. For these amino acids, the aromatic structure of the R moiety (RCH(NH2)COOH) and moreover the presence of a supplementary nitrogen atom for Trp and His can influence the reactivity of amino acids [1, 2]. In order to find out the redox potential of the compounds I and II, cyclic voltammetry was performed for the redox couple S-DPPH- (anion) / S-DPPH (radical), where S stands for SO3Na or (SO3Na)2, eq. 3. G. Ionita, V. Em. Sahini, G. Semenescu, P. Ionita: Kinetics of Oxidation of AminoAcids by Some… Acta Chim. Slov. 2000, 47, 111-119. 115 S-DPPH + e- ^^ S-DPPH- (3) The experimental oxidation potentials obtained were 1.667 V and 1.576 V, respectively. As was state above, the reactivity of these compounds are comparable. The rate constant dependence upon temperature was also studied. Using Arrhenius equation the activation parameters were determined for each amino acid - free radical pair (eq. 4-6) [8]. k = Ae a (4) . kT AS*/R A = e— e (5) h E =AH*+RT (6) The activation parameters obtained from Arrhenius lines are shown in Table 2 and Table 3. The results shown in these tables may explain the variation of the rate constants (see also Table 1). Table 2. Activation parameters from amino acids oxidation by free stable radical I Amino acid Ea (kJ/mol) lnA AS* (J/molK) AH* (kJ/mol) Fen 81.88 23.744 -55.85 79.387 His 9.32 -4.503 -290.54 6.827 Leu 42.12 -3.835 -285.00 39.62 Met 108.22 33.193 22.70 105.72 Pro 18.36 -1.166 -262.81 15.86 Ser 20.65 -0.426 -256.67 18.15 Thr 21.105 -0.353 -256.06 18.61 Trp 14.21 11.00 -161.05 11.71 Table 3. Activation parameters from amino acids oxidation by free stable radical II Amino acid Ea (kJ/mol) lnA AS* (J/(molK) AH* (kJ/mol) Fen His 60.41 11.96 14.97 4.05 -128.55 -286.61 57.94 9.49 G. Ionita, V. Em. Sahini, G. Semenescu, P. Ionita: Kinetics of Oxidation of AminoAcids by Some… 116 Acta Chim. Slov. 2000, 47, 111-119. Leu 73.62 18.39 -100.10 71.15 Met 37.00 5.44 -207.75 34.53 Pro 49.86 9.78 -171.60 47.39 Ser 37.89 5.48 -207.40 35.42 Thr 68.22 16.84 -113.01 65.75 Trp 31.74 4.85 -212.60 29.27 The activation energy Ea is smallest for His, Thr and Trp, explaining the higher oxidation rate of these amino acids. An oxidation mechanism of amino acids by free radicals NaSO3DPPH and (NaSO3)2DPPH is proposed in Scheme 2. R H Rad* R—C-----COOH-------«> -RadH NHo -CO NH2 -RadH -NH3 NHo JH .R- Rad COOH------^ -RadH NHo 4> r----CHCOOH Rad> -RadH NH R- -COOH-HOH> R- -NH 3 COOH NH Scheme 2. Pathway of oxidation of amino acids by free hydrazyl radicals (noted as Rad). Scheme 2 shown that in a first step it is possible to obtain three types of amino acid radicals, with the unpaired electron on oxygen, carbon or nitrogen, the second one being probably more stable. These radicals are achieved by a hydrogen atom transfer between amino acid and NaSO3DPPH or (NaSO3)2DPPH radical. In the first case the oxidative deamination of amino acid occurs, with evolution of ammonia and aldehyde (which can be further oxidised to acid). In the second and the third cases the initially formed amino acid radical is further oxidised to the imino acid, which in water presence yields ammonia and keto acid, as it was presented in experimental part. The hydrazones identified by theirs melting point derived only from the corresponding a-keto acids, but also some unidentified compounds of red colour were observed. We do not exclude first pathway, because hydrazyl radicals are known as a good hydrogen abstractor from G. Ionita, V. Em. Sahini, G. Semenescu, P. Ionita: Kinetics of Oxidation of AminoAcids by Some… Acta Chim. Slov. 2000, 47, 111-119. 117 amines [4]. The oxidation of some amino acids by Fremy’s salt (potassium nitrosodisulfonate) or by alkaline hexacyanoferrate (III) unfold in this way [1, 2]. For His and Trp the nitrogen from R moiety can be also involved in such processes. The attempt to trapped the short-lived free radicals (see Scheme 1) with a spin-trapper (t-butyl-a-phenylnitrone or 2-methyl-2-nitrosopropane) at room temperature failed; only at 77 K this kind of radicals could be evidenced, as literature data indicated [1]. Some isokinetic aspects. Isokinetic theory was applied in this paper for mechanistic investigation. One of the approximation made in results interpretation was the attempt to apply the active complex theory for the obtaining of the kinetic parameter from oxidation of amino acids [8, 14, 15]. The linear correlation between rate of the reaction at different temperature shown that a common point of intercept does not exist between Arrhenius lines, which means that an effect of compensation between activation entropy and enthalpy does not exist, however by plotting DH versus DS for both series of reactions it is possible to achieve an apparent linear relationship (see Fig. 2, r = correlation coefficient) [16]. DS(JfrnolK) DS(JAroirç Fig. 2. Plot of DH versus DS for the compound I and II The correct representation between activation parameters DH and DG (DG = DH - TDS ) shown the absence of some compensation effect between DH and DS , which means that derived parameters DH and DS are statistically dependent which cannot be used for further regression analysis in aims to find any isokinetic relationship (Fig. 3) [8, 16]. G. Ionita, V. Em. Sahini, G. Semenescu, P. Ionita: Kinetics of Oxidation of AminoAcids by Some… 118 Acta Chim. Slov. 2000, 47, 111-119. EO- 70- ¦ 50-î ^40- ¦ -1-30- 1 20- r=0.67 10- 1 40 60 60 ICC 120 1J0 34 E6 E6 100 102 DG(kJAnol) DG(kJAral) Fig. 3. Plot of DH versus DG for the compound I and II In conclusion, analysis of the obtained results can supply information about the mode of action on reaction conditions, for changing the factors which determine the active complex formation. The mechanistic pathway of amino acids oxidation occurs probably by an intermediate of amino acid radical type, which leads further to keto acid. This study presents also some importance in understanding of natural ageing and oxidative stress processes (mainly due to the generation of free radicals in vivo). References Paper presented in part at “The 9th Conference on Physical Chemistry”, Galati, Romania, 1998, 2.38. [I] D. Laloo and M. K. Mahanti, J. Chem. Soc. Dalton Trans. 1990, 311-313. [2] A. G. Raso, P. M. Deya and J. M. Saa, J. Org. Chem. 1986, 51, 4285-4287. [3] M.P. Alvarez-Macho, Rev. Roum. Chim. 1993, 38, 999-1006. [4] G. H. Schenk, Tetrahedron Lett. 1967, 193-198. [5] G. Schenk and D. Brown, Talanta 1967, 14, 257-261. [6] A. M. Rao and U. C. Singh, Biochem. Biophys. Acta 1982, 711, 134-139. [7] P. Ionita, M. T. Caproiu and A. T. Balaban, paper submitted to Eur. J. Org. Chem. [8] W. Linert, Chem. Soc. Rev. 1994, 23, 429-438. [9] G. V. Putirszkaja and T. Siladi, Acta Chim. (Hung.) 1972, 72, 329-339. [10] J. Volke and F. Liska, Electrochemistry in Organic Synthesis, Springer-Verlag, Berlin, 1992. [II] G. Stanciuc, N. Zarna, N. Spataru, T. Constantinescu, A. Caragheorgheopol, H. Caldararu and A.T. Balaban, Rev. Roumaine Chim. 1996, 41, 755-761. [12] G. J. Papariello and M. A. M. Janish, Anal. Chem. 1965, 37, 899-902. [13] Handbook of Chemistry and Physics, 59th Edition, CRC Press, Florida, p.C-756, 1979. [14] W. Linert and R. F. Jameson, Chem. Soc. Rev. 1989, 18, 447-501. [15] W. Linert, Chem. Phys. 1987, 116, 381-393. [16] R. R. Krug, Ind. Eng. Chem. Fundam. 1980, 18, 50-59. Povzetek Raziskali smo kinetiko oksidacijo amino kislin pri izoelektrični točki in temperaturah med 298 do 318 K. Reakcije smo zasledovali spektrofotometrično pri 520 in 514 nm, odvisno od uporablčjenega oksidanta. Ugotovili smo, da oksidacija amino kislin z aromatskimi obroči (His, Trp, Phe) poteka hitreje kot ostalih. Predlagamo možen mehanizem oksidacije G. Ionita, V. Em. Sahini, G. Semenescu, P. Ionita: Kinetics of Oxidation of AminoAcids by Some… Acta Chim. Slov. 2000, 47, 111-119. 119 amino kislin, po katerem pdobno, kot pri encimatski razgradnji, nastane a-keto kislina in amoniak. G. Ionita, V. Em. Sahini, G. Semenescu, P. Ionita: Kinetics of Oxidation of AminoAcids by Some…