Info

"Also called G values.

''Data taken from J. W. T. Spinks and R. J. Woods, "Introduction to Radiation Chemistry," 3rd Ed. John Wiley & Sons, New York, 1990.

"Also called G values.

''Data taken from J. W. T. Spinks and R. J. Woods, "Introduction to Radiation Chemistry," 3rd Ed. John Wiley & Sons, New York, 1990.

concerning the interaction of ionizing radiation with matter are given in the literature.19 Briefly, y rays from 60Co, or high-energy electrons, interact similarly with an aqueous solution. Because the energy of the radiation is much higher than that of electrons in molecules, both rays extract an electron from the major compound, that is, water. The result is the creation of a homogeneous solution of OH, H, and eaq free radicals in the nanosecond time scale after interaction of ionizing radiation with matter. Some hydrogen peroxide is also created. The absorbed dose is the amount of energy absorbed per unit mass and is expressed in grays (1 Gy = 1 J kg-'). The quantities of the radiolytic species are known and are proportional to the dose. Table I gives the yields of radiolytic compounds.

Water free radicals may then react with solutes or with themselves (by recombinations, disproportionation, or dimerization). In an aqueous solution containing formate ions in high quantity (10 2 mol liter-1 or higher), and in an atmosphere of nitrous oxide, the most probable reaction of OH and H radicals is with formate. Hydrated electrons react with N2O. The kinetic scheme is the following:

Thus all water free radicals are transformed into the reductant COO'- radical. Under the conditions of concentration described above, and in the pH range 4-10, its yield G(COO' ) is thus equal to

G(COO -) = g(H) + g(OH) + g(e~) = 0.62 fimol J"1

In this expression g(H), g(OH), and g(e~) denote the respective G values given in Table I. The whole process [reactions (10)—(12)] is finished in less than 1 /usee after interaction of the ionizing radiation.

19 J. W. T. Spinks and R. J. Woods, "Introduction to Radiation Chemistry," 3rd Ed. John Wiley & Sons, New York, 1990.

Lamp

Cell

Cell

Monochromator + Photomultiplier

Fig. 1. Schematic drawing of a pulse radiolysis setup. The solution to be irradiated is in the cell. The electron beam (coming from the accelerator) is usually perpendicular to the analyzing light beam.

Monochromator + Photomultiplier

Shielding Oscilloscope

Fig. 1. Schematic drawing of a pulse radiolysis setup. The solution to be irradiated is in the cell. The electron beam (coming from the accelerator) is usually perpendicular to the analyzing light beam.

What Are y and Pulse Radiolysis?

Pulse radiolysis is a fast kinetic method, whereas y radiolysis is a steady state method. In pulse radiolysis, pulses of high-energy electrons (2-10 MeV; in our setup MeV) are delivered to the irradiation cell from a linear accelerator (ours is located at the Curie Institute, Orsay, France). A schematic drawing of such a setup is given in Fig. 1.

The pulse duration is usually 2-200 nsec long. The sample is contained in a quartz cell (100-400 /xl in our case). The modifications of the solution are followed by absorption (the most frequent method). A UV-visible beam is delivered by a lamp (Xe-Hg "super-quiet" or Xe arc) perpendicularly to the electron beam. The residual light is analyzed by a conventional system (monochromator, photomultiplier, oscilloscope, and computer). The equipment that we use has been described.13 The doses per pulse (2-60 Gy) are calibrated by the thiocy anate dosimeter (10~2 M sodium thiocy anate, nitrous oxide atmosphere).19 The data collected are absorption spectra of free radicals and kinetics of their formation and decay. In this work, it is thus possible to distinguish between disulfide radicals in anionic or protonated form13 and thus to measure the pK.cl of this free radical (if it is above ~4). If the lifetime of the thiyl radical is long enough, it can be observed.13

In steady state y radiolysis, the ionizing radiation (from 60Co or 137Cs) is delivered at a low rate compared with that of pulse radiolysis; however, for this system the chemistry is the same. Free radicals can be assumed to reach steady states that last as long as the irradiation. This method is convenient for preparing large quantities of final products and thus allows further analysis. For instance, the number of reducible disulfide bonds can be determined, and it is possible to observe whether products other than reduced protein (with thiol functions) and dimers linked by intermolecular disulfide bonds are created. The chain length, expressed as the ratio of the yield of thiol functions versus that of reductant G(-SH)/G(COO'~), can be measured. Its value would depend on the pKd of the disulfide radical and on the reduction potential of the thiyl/thiol couple (Scheme 2). Thus it allows an evaluation of the one-electron reduction potential of the thiyl/thiol redox couple compared with that of the C02 /HCOO couple (1.07 V at pH 720).

Experimental Procedures

Reagents

Radiolysis is sensitive to metal impurities, and thus salts for radiolysis should be as pure as possible. Also, many buffers such as Tris or carbonate react with water free radicals; therefore their use should be avoided except in small concentrations. Whenever possible, phosphate buffer should be used.

Sodium formate and potassium hydrogen phosphate are of the highest quality available (Normatom [Prolabo, Fontenay sous Bois, France] or Suprapure [Merck, Fontenay sous Bois, France]). 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB) is provided by Sigma (L'Isle d'Abeau, France). Nitrous oxide is delivered by Air Liquide (Grigny, France). Its purity is higher than 99.99% (>20 ppm 02). Water is purified by an Elga Maxima system (Plessis-Robinson, Hauts-de-Seine, France) (resistivity, 18.2 Mfi).

All proteins should be as pure as possible. Purification by dialysis is recommended to eliminate organic buffers, conservators, and transition metal cations.

1. Unless otherwise stated, samples to be irradiated are made up in 20 mM phosphate, 100 mM sodium formate buffer adjusted to the required pH with sulfuric acid or sodium hydroxide, and saturated with N20. The doses per pulse are -5-40 Gy ([C02~] % 3-25 ¡iM). The protein concentration is 10-100 ¡xM. The optical path is 1 or 2 cm. The irradiated volume is 200-400 /i l per pulse.

2. Radiolysis results are interpreted with the help of other methods. In addition to radiolysis, we use nuclear magnetic resonance, high-performance liquid chromatography, electrophoresis, etc.

3. The free sulfhydryl group concentration is determined by optical titration with DTNB at pH 8.0 (100 mMTris-HCl buffer) using e4io„m = 13-6 mM"1 cm"1 for the 3-carboxylato-4-nitrothiophenolate anion21 (verified by us, using glutathione).

4. Thiol groups in the reduced protein are alkylated before electrophoresis by reaction with 10 mM iodoacetamide (10 min, room temperature).

20 P. Wardman, J. Phys. Chem. Ref. Data 18, 1637 (1989).

21 G. L. Ellman, Arch. Biochem. Biophys. 74,443 (1958).

5. For y radiolysis, the samples are subjected to irradiation in a panoramic IL60PL (CIS Bio International, Gif sur Yvette, France) 60Co source at a dose rate, determined by Fricke dosimeter,19 of approximately 1.0 Gy sec-1.

0 0

Post a comment