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Determination of Disulfide Radical pKa

The determination of the free radical pA"a is obtained by plotting the free radical average extinction coefficient sT at fixed wavelength (such as 420 nm), as a function of pH (Fig. 3). The extinction coefficients of the basic and of the acidic forms (respectively, eA and eB) are measured in acidic and basic solutions, respectively, and thus the only adjustable parameter is KA in Eq. (13).

_ eA x 10~ph eB + Ka £J ~ 10-PH + KA + 10-PH + KA (B)

Typical values of pKA lie between ~4.5 and 6 for small molecules and for proteins. Thioredoxin behavior is markedly different23 because its pKA is less than 3. This abnormal behavior is suppressed by site-directed mutagenesis of Trp-35.

Final Products and Chain Length

Final products can be detected after pulse radiolysis; however, it is often easier to prepare larger amounts of irradiated solutions by steady state y radiolysis. Both conditions differ mainly by the dose rate, which is several orders of magnitude higher in pulse radiolysis.

Final compounds include reduced protein (with two thiol functions) and dimers linked by intermolecular disulfide bridges. For lysozyme we also found a fragment.22 For immunoglobulin G (IgG), the combination of reduction-dimeriza-tion leads to mixtures of heavy and light chains, with varous kinds of heavy-light combinations.13 For thioredoxin, only the reduced protein is found, but mutations of Trp-35 or of Asp-30 allow the formation of dimers. It is possible that thioredoxin reduces its own dimer and that mutants are not active enough to do so.

The chain length also varies according to the protein. Typical values are 10-20 in basic medium. It increases with acidity, because of the easier formation of thiyl radicals, and it can reach values as high as 200 for lysozyme or glutathione at pH 4. Hence the low values obtained for thioredoxin (1 in basic and neutral medium, <20 at pH 4.5)24 reflect (1) the absence of formation of thiyl radicals in neutral and basic medium and (2) a reduction potential and/or accessibility much lower than that of other thiyl radicals in acidic medium.

Specificity of Thioredoxin

Our results show several differences in the behavior of thioredoxin sulfur free radicals compared with that of other sulfur free radicals.23'24 First, the disulfide radical is much more acidic, and this acidity seems to be controlled by Trp-35. Asp-30 also plays a role, although less clear, in the proton transfer. Second, the thiyl

23 C. El Hanine, D. Conte, J.-P. Jacquot, and C. Houee-Levin, Biochemistry 39, 9295 (2000).

24 C. El Hanine, D. Conte, J.-P. Jacquot, and C. Houee-Levin, Res. Chem. Intermediates 25,313 (1999).

radical is much less oxidant. The two cysteines responsible for the redox properties of thioredoxin (Cys-36 and Cys-39) have different properties. Cys-36 is solvent accessible,25 whereas Cys-39 has a p/sfa that is at least 4 units higher than physiological pH,26-28 and is relatively unreactive. Our results would indicate a preferential localization of the thiyl radical on Cys-39, and thus a nonsymmetric bond opening of the disulfide radical. Thiyl radicals are responsible for the pro-oxidant side of sulfur compounds. Although thioredoxin and glutathione play similar roles in antioxidant defense, thioredoxin would be a much better antioxidant because of its weak pro-oxidant properties. It would be interesting to know whether this specificity is also found in other members of the thiol-disulfide oxidoreductase family.

Acknowledgment

I thank the Curie Institute (Dr. V. Favaudon) for the use of the accelerator.

25 P. T. Chivers and R. T. Raines, Biochemistry 36, 15810 (1997).

26 P. T. Chivers, K. E. Prehoda, B. F. Volkman,B. M. Kim, J. L. Markley,andR. T. Raines,Biochemistry 36, 14985 (1997).

27 J. F. Andersen, D. A. Sanders, J. R. Gasdaska, A. Weichsel, G. Powis, and W. R. Montfort, Biochemistry 36, 13979 (1997).

28 D. M. LeMaster, P. A. Springer, and C. J. Unkefer, J. Biol. Chem. 272, 29998 (1997).

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