Anal. Chem. 2007, 79, 5444-5448
Localization and Quantification of Carbon-Centered Radicals on Any Amino Acid of a Protein G. Mousseau,† O. P. Thomas,† S. Oppilliart,† A. Coirier,† A. Salcedo-Serna,† R. Thai,‡ F. Beau,‡ J.-P. Renault,§ S. Pin,§ J.-C. Cintrat,*,† and B. Rousseau*,†
CEA, iBiTec-S, Service de Chimie Bioorganique et de Marquage, Baˆ timent 547, SIMOPRO, Baˆ timent 152, and DSM/ DRECAM/Service de Chimie Mole´ culaire/CNRS URA 331, Baˆ timent 546, 91191 Gif sur Yvette Cedex, France
A general strategy to localize and quantify carbon-centered radicals within proteins is described. The methodology was first exemplified on amino acids and then on a peptide. This method is applicable to any protein system regardless of size, and the site of hydrogen abstraction by •OH on all residues within proteins is easily and accurately detected. Protein radicals are highly reactive species, present at low concentration, and consequently difficult to detect. However, their detection and quantification is mandatory for many crucial reasons: (i) to understand the factors and mechanisms that control their formation, localization, delocalization, and propagation,1 (ii) to study the reactivity and the mechanisms of metalloenzymes (catalase, peroxidases, ...) for which radicals are quite frequently involved,2 (iii) to unravel the reaction pathways of the deleterious effects in •OH-induced oxidation of proteins. This oxidative stress promotes pathological or toxicological processes and has been implicated in numerous diseases (Alzheimer, Parkinson).3 Despite the importance of protein radicals, few methods are available for their detection and characterization. These methods include radical observation by EPR, mass spectrometry detection of trapped or scavenged radicals, or 2H NMR.4-7 Each method * To whom correspondence should be addressed. E-mail:
[email protected] (J.-C.C.);
[email protected] (B.R.). † CEA, iBiTec-S, Service de Chimie Bioorganique et de Marquage. ‡ CEA, iBiTec-S, SIMOPRO. § DSM/DRECAM/Service de Chimie Mole´culaire. (1) Wright, P. J.; English, A. M. J. Am. Chem. Soc. 2003, 125, 8655-8665. (2) (a) Ivancich, A.; Jakopitsch, C.; Auer, M.; Un, S.; Obinger, C. J. Am. Chem. Soc. 2003, 125 (46), 14093-14102. (b) Kappler, U.; Bailey, S. J. Biol. Chem. 2005, 280, 24999-25007. (c) Wilson, J. C.; Wu, G.; Tsai, A.; Gerfen, G. J. J. Am. Chem. Soc. 2005, 127, 1618-1619. (d) Hetterscheid, D. G. H.; Kaiser, J.; Reijerse, E.; Peters, T. P. J.; Thewissen, S.; Blok, A. N. J.; Smits, J. M. M.; de Gelder, R.; de Bruin, B. J. Am. Chem. Soc. 2005, 127, 18951905. (3) (a) Barnham, K. J.; Masters, C. L.; Bush, A. I. Nat. Rev. Drug Discovery 2004, 3, 205-214. (b) Berlett, B. S.; Stadtman, E. R. J. Biol. Chem. 1997, 272, 20313-20316. (c) Stadtman, E. R. Annu. Rev. Biochem. 1993, 62, 797-821. (4) Davies, M. J.; Dean, R.T. Radical-Mediated Protein Oxidation; Oxford University Press: Oxford, U.K., 1997. (5) (a) Buettner, G. R.; Mason, R. P. Methods Enzymol. 1990, 186, 127-133. (b) Davies, M. J.; Hawkins, C. L. Free Radical Biol. Med. 2004, 36, 10721086.
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has advantages and limitations; the latter are mainly related to sensitivity and difficulties in EPR spectral interpretation. Moreover, although the information obtained from radiolytic modification coupled with mass spectrometry can be obtained on 65% of the sequence of a typical protein, since only 14 of the 20 amino acids are useful probes,6 a more general strategy is still needed to gain more accurate data. We report here a method that allows the localization and quantification of carbon-centered radicals within proteins and on any amino acid using a tritium atom reporter. EXPERIMENTAL SECTION General Information. L-Amino acids, D,L-leucine-2,3,3,4,5,5,5,6,6,6-d10, dichlorophenylphosphine, kemptide, Dowex 50 W×8, and dansyl chloride were from Sigma-Aldrich. Platinum(IV) oxide (PtO2) was from Merck, tritium gas (98.9% tritium, 0.7% deuterium, 0.4% hydrogen) was from Tritec (Switzerland), and tritiated water (37 GBq‚mL-1) was from Amersham. OH radicals were generated by either β-rays, γ-rays, or an electron beam. In all cases, the energy deposited in the samples (dose expressed in Gy or J‚kg-1) was determined with a chemical dosimeter commonly used in radiation chemistry: the Fricke dosimeter.8 β-Rays were obtained from tritiated water (18.5 GBq‚mL-1, 61 Gy‚h-1). γ-Rays were produced by an IBL637 irradiator (137Cs, 57 Gy‚min-1). The 10 MeV electron beam was provided by the ALIENOR facility in Saclay (Laboratoire de Radiolyse).9 The pulse duration was 10 ns (15 Gy‚pulse-1), and the repetition rate was 0.4 Hz. The energy deposited in the samples by β-rays, γ-rays, or electron beam corresponds, in our gas-saturated conditions, to an OH radical concentration of about 35 µmol‚dm-3‚h-1, 33 µmol‚dm-3‚min-1, and 8 µmol‚dm-3‚pulse-1, respectively. 1H and 3H NMR were recorded at, respectively, 300 and 320 MHz on an AC 300 MHz Bruker spectrometer. Chemical shifts (6) Xu, G.; Chance, M. R. Anal. Chem. 2005, 77, 4549-4555. (7) (a) Nukuna, B. N.; Goshe, M. B.; Anderson, V. E. J. Am. Chem. Soc. 2001, 123, 1208-1214. (b) Goshe, M. B.; Chen, Y. H.; Anderson, V. E. Biochemistry 2000, 7, 1761-1770. (c) Goshe, M. B.; Anderson, V. E. Radiat. Res. 1999, 151, 50-58. (8) Fricke H.; Hart E. J. In Radiation Dosimetry; Attix, F. H., Roesch, W. C., Eds.; Academic Press: New York and London, 1966; Vol. 2 pp 167-232. (9) Mialocq, J.-C.; Hickel, B.; Baldacchino, G.; Juillard, M. J. Chim. Phys. Phys.Chim. Biol. 1999, 96, 35-43. 10.1021/ac070751k CCC: $37.00
© 2007 American Chemical Society Published on Web 06/19/2007
were measured in parts per million relative to the residual proton signal from deuterium oxide (D2O; Euriso-top France) 4.67 ppm (1H). Lyophilized horse heart metmyoglobin (Sigma M-1882) was dissolved in pure water and dialyzed against water. Apomyoglobin was prepared according to a previously published method10 and then lyophilized. Apomyoglobin samples were prepared by dissolving the protein in ultrapure water. The concentration was determined by UV absorption (280nm ) 13 500 M-1 cm-1)11 and adjusted to a final concentration of 10 mM. Peptide and protein were purified after labeling on an analytical HPLC (Agilent Zorbax C18, 300 Å, 250 mm × 4.6 mm), and dansylamino acids were purified on an Agilent Zorbax C18, 80 Å, 250 mm × 4.6 mm column with a Merck Hitachi L-7100 pump using a Degazys DG-1310 UnifLows degassing system, a Shimadzu SPD10A VP UV-vis detector, and a Radioflow LB509 detector with Quickszint flow 302 scintillation cocktail. Data were acquired via Borwin chromatography software. Sequencing was done on an ABI 492 HT/Procise peptide sequencer from Applied Biosystems (Foster City, CA) with acquisition on Procise 1.1 and model 610A 1.1 software. All products are commercially available (Perkin-Elmer), and PTHamino acids were collected using a Gilson FC 203B fraction collector. The collected samples (100 µL) were diluted in 4 mL of Zinsser Analytic Unisafe 1 scintillation cocktail, and counting was carried out with a Wallac 1409 liquid scintillation counter using the 1414 WinSpectral program. Radioactive polyacrylamide gels were analyzed on a β-imager (Biospace, Paris, France) for 1 h. Data were acquired via an intensified CCD camera, a BV Acquisition and BV Zoom software. The lowest activity detected was 0.007 cpm‚mm-2 for 3H (one disintegration every 150 min‚mm-2). Generation of D,L-Leucine-d10 Radicals in the Presence of Phenylphosphinic Sodium Salt (BPASS). A solution (1 mL) of leucine-d10 (2.5 mM), BPASS (2.5 mM) in 10 mM phosphate buffer pH 7.2 was degassed by N2O bubbling for 1 h. The resulting solution was submitted to γ-irradiation (57 Gy‚min-1) for 45 min to generate leucine-d10 radicals. D2O (0.5 mL) was added after evaporation for 1H NMR monitoring of the incorporation of hydrogen within leucine-d10. The efficiency of BPASS was evaluated by measuring the increase in the residual signal of leucined10 CD2H (0.8 ppm) by comparison with the signal of an internal standard (4.3 ppm), the phosphate buffer. Hydrogen incorporation was 11%. The kinetics of the P-H/P-D exchange in BPASS were monitored by 1H NMR at pH 7 in D2O and proved to be low enough to conduct our transfer experiment with 3H-BPASS without loss of tritium. Preparation of 3H-BPASS. In a glovebox, a 10 mL twonecked flask was charged with anhydrous PtO2 (25.2 mg, 0.11 mmol) and dry THF (1 mL), and the mixture was frozen in liquid nitrogen. The reaction mixture was degassed under vacuum, and an atmosphere of tritium gas was admitted (1 bar). When THF was thawed, PtO2 was reduced to 3H2O over a period of 30 min. PhPCl2 (10 µL, 74 µmol) was then added using a microsyringe, and the reaction mixture was stirred for 1 h. The suspension was (10) Rossi-Fanelli, A.; Antonini, E.; Caputo, A. Biochim. Biophys. Acta 1958, 30, 608-615. (11) Colonna, G.; Irace, G.; Parlato, G.; Aloj, S. M.; Balestrieri, C. Biochim. Biophys. Acta 1978, 532, 354-367.
frozen in liquid nitrogen, and the tritium atmosphere was evacuated and trapped on a La/Ni/Mn bed and replaced by N2. The reaction mixture was buffered with solid Na2CO3 (20 mg, 0.19 mmol) over 10 min, and then a NaHCO3 (16 mg, 0.19 mmol) aqueous solution (pH 7.5) was added. The resulting mixture was filtered through 45 µm Millex-FH, and the flask and filter were rinsed with THF. The resulting solution was evaporated on a rotary evaporator. The residue was dissolved in 2 mL of water and then evaporated to dryness. The above procedure was repeated three times to remove all exchangeable tritium atoms. The solid was dissolved in water (10 mL), and an aliquot was taken and submitted to liquid scintillation counting to give an activity of 2.98 GBq‚mL-1. The crude mixture was then purified by semipreparative HPLC performed on a thermohypersil Keystone Hypurity C18 5 µm, 250 mm × 10 mm column, 10 mM phosphate buffer, 2 mL‚min-1, using a double detection system (Merck L-4200 UVvis detector and Berthold LB2040 nuclear spectrometer radioactive detector) to give pure 3H-BPASS (Rt ) 23.2 min, radiochemical purity ) 98%). The elutant was removed under vacuum, the desired compound was dissolved in water (20 mL), and the amount of product was assigned by UV and liquid scintillation counting (specific activity ) 514 GBq‚mmol-1). The final solution was diluted to a concentration of 37 MBq‚mL-1 (720 mL) and buffered to pH 7.2 with phosphate buffer to a final concentration of 10 mM. 1H and 3H NMR of 3H-Phenylphosphinic Sodium Salt. A solution of 3H-BPASS (200 µL, 370 MBq) with D2O (100 µL) was analyzed by 3H NMR. 3H NMR (D 0, 320 MHz) δ (ppm): 7.54 (d, 1J 2 P-3H ) 525 Hz). 1H NMR (D 0, 300 MHz) δ (ppm): 7.45 (d, 1J 2 P-H ) 561 Hz, 1H, PH), 7.4-7.7 (m, 5H, Ph). Protocol for Amino Acid-Based Radical Identification. In a septum-capped 1 mL vial containing phosphate buffer pH 7.8, 10 mM (890 µL), two amino acids (X + Leu) (2 × 5 µL of 20 mM in ultrapure water), except for tyrosine (100 µL of 1 mM Tyr + 5 µL of 20 mM Leu with 795 µL of phosphate buffer) and a 3HBPASS solution (100 µL of 1 mM and 514 MBq‚mL-1) were added, and the resulting solution was degassed by N2O bubbling for 15 min. The solution was submitted to an electronic bombardment (25 pulses of 15 Gy at a frequency of 0.4 Hz). The mixture was then lyophilized and dissolved in water (100 µL). The sample was passed through a strongly acid cation exchange resin Dowex 50 W×8 (conditioned with 1 mL of 1 N HCl and washed three times with 100 µL of 10-2 N HCl), washed with 10-2 N HCl (4 mL), and eluted with NH4OH 10-1 N (8-12 mL depending on the pKa of the amino acid). The amino acid fractions were pooled and evaporated to dryness. The amino acids were dansylated with DsCl (25 µL of a 20 mM solution in acetone) in NaHCO3 pH 8 (0.5 M, 25 µL) for 1 h at 37 °C and then submitted to an acidic treatment (TFA 10% v/v) for 15 h to remove labile tritium, and the resulting mixture was lyophilized. Samples were then analyzed by HPLC (Agilent Zorbax C18, 80 Å, 4.6 mm × 250 mm; elutant A, 0.1 M NH4HCO3/ACN 90/10; elutant B, 0.1 M NH4HCO3/ACN 55/45 using different gradients (Supporting Information Figures S5-S23). Specific activity (Bq‚nmol-1) was determined by dividing the radioactive peak area by the UV peak area. Radioactive peak area was standardized by injecting a known quantity of radioactivity, Analytical Chemistry, Vol. 79, No. 14, July 15, 2007
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whereas UV peaks were standardized by injecting a quantified amount of each dansylated amino acid (either commercially available or synthesized in our laboratory). For each amino acid, specific activity was normalized using leucine as an internal standard. The results are reported in Figure S1 (see the Supporting Information). Protocol for Peptide-Based Radical Identification. A septum-capped 1 mL vial containing ultrapure water (235 µL) was placed in a glovebox. Tritiated water was added to a final activity of 18.5 GBq‚mL-1 (555 µL of 33.3 GBq‚mL-1), and the resulting solution was degassed by N2O bubbling for 1 h. The needle was removed from the solution, and under a gentle flow of N2O, a solution of peptide (LRRASLG) (10 µL of 10 mM in ultrapure water) was added followed by 3H-BPASS solution (144 µL of 1 mM and 514 MBq‚mL-1). A volume of 100 µL was taken at 2.25, 4, 6, and 19.75 h and passed through a SepPack light C18 (Waters) and eluted with 5 × 1 mL of water (approximately 95% of 3HBPASS was removed) followed by 1 mL of ACN/H2O/TFA 70/ 30/0.1. To the last fraction, 400 µL of TFA was added, and the mixture was kept for 24 h at room temperature. The solution was evaporated to dryness, 100 µL of H2O was added, and the resulting solution was purified by HPLC (column, Agilent Zorbax C18, 300 Å, 4.6 mm × 250 mm; elutant A, H2O/0.1% TFA; elutant B, ACN/ 0.1% TFA using a gradient from t ) 0 A/B (98/2) to t ) 15 min A/B (80/20); Tr ) 10 min). An aliquot of the above purified samples was then submitted to automatic sequencing (ABI Procise 492HT), the PTH amino acids were automatically collected, and the specific activity of each amino acid was measured by UV quantification and scintillation counting (Wallac 1409 liquid scintillation counter). Protocol for Protein-Based Radical Identification. A septumcapped 1 mL vial containing 10 mM phosphate buffer pH 7.2 in ultrapure water (335 µL of 30 mM) was placed in a glovebox. Tritiated water was added to a final activity of 18.5 GBq‚mL-1 (555 µL of 33.3 GBq‚mL-1), and the resulting solution was degassed by N2O bubbling for 1 h. The needle was removed from the solution, and under a flow of N2O, a solution of protein (10 µL of 10 mM in ultrapure water) was added followed by 3H-BPASS solution (100 µL of 1 mM solution and 514 MBq‚mL-1). After the desired reaction time (3, 6, and 9 h), 250 µL was taken, and 20 µL was immediately frozen for 12% SDS-PAGE analysis. The gel was transferred onto a PVDF membrane, and radioactive counting was carried out by β imaging. The remaining reaction mixture was passed through a SepPack light tC2 and eluted with 5 × 1 mL of water (to remove about 95% of 3H-BPASS) followed by 1 mL of ACN/H2O/TFA 70/30/0.1. The last fraction was evaporated to dryness, 100 µL of H2O was added, and the solution was analyzed by HPLC: column, Agilent Zorbax C18, 300 Å, 4.6 mm × 250 mm; elutant A, H2O/TFA 0.1%; elutant B, ACN/TFA 0.1% using a gradient from t ) 0 A/B (100/0) to t ) 30 min A/B (50/ 50) and then purified using the same conditions (see Supporting Information Figure S25). The protein in 50 mM NaHCO3 pH 8 was digested for 24 h with trypsin 1/100 (w/w), and TFA was then added to a final concentration of 25% (v/v). The mixture was kept for 4 h at room temperature, lyophilized, analyzed (Figure 5), and purified (column, Agilent Zorbax C18, 300 Å, 4.6 mm × 250 mm; elutant A, H2O/0.1% TFA; elutant B, ACN/0.1% TFA using a gradient from t ) 0 A/B (100/0) to t ) 60 min A/B (50/50)). 5446 Analytical Chemistry, Vol. 79, No. 14, July 15, 2007
Figure 1. Strategy for rapid localization of protein-based radicals.
Figure 2. Tritium transfer from 3H-BPASS to leucine radical. Table 1. Amino Acid Tritium Incorporation specific activity (Bq‚nmol-1)
amino acids
1 < s.a. < 500 500 < s.a. < 1000 1000 < s.a. < 1500 s.a. > 1500
G, S, N, A, P, Q Y, D, T, E, H, M C, V, F, W I, R, L, K
An aliquot of the above-purified peptide fragments was then submitted to automatic sequencing (ABI Procise 492HT), the PTH amino acids were automatically collected, and the specific activity of each amino acid was measured as previously described for the peptide. Finally, 91.5% of the apomyoglobin sequence was identified and mapped. It should be mentioned that one peptide (4856) was not collected, two lysine residues (K63 and K78) were lost during digestion, and owing to some peptide lengths too little A94, T95, and K96 were collected to give a relevant specific activity. The 3D apomyoglobin model was then redesigned using Swiss-Pdb Viewer (v3.7 SP5) based on the following color code for each amino acid: yellow, >100 Bq‚nmol-1; green, 50-100 Bq‚nmol-1; light blue, 20-50 Bq‚nmol-1; blue,
