Báo cáo khoa học: Re-evaluation of intramolecular long-range electron transfer between tyrosine and tryptophan in lysozymes Evidence for the participation of other residues doc

Re-evaluation of intramolecular long-range electron transfer between tyrosine and tryptophan in lysozymes Evidence for the participation of other residues Marilyne Stuart-Audette1, Yves

Re-evaluation of intramolecular long-range electron transfer between tyrosine and tryptophan in lysozymes

Evidence for the participation of other residues

Marilyne Stuart-Audette1, Yves Blouquit2, Moshe Faraggi3, Ce´cile Sicard-Roselli1, Chantal Houe´e-Levin1 and Pierre Jolle`s4

1 LCP, Centre Universitaire, Orsay Cedex, France; 2 Institut Curie-Section de Recherche, Centre Universitaire, Orsay Cedex, France;

3 Department of Nuclear Engineering, Ben Gurion University of the Negev, Israel; 4 Laboratoire de Chimie, Muse´um National d’Histoire Naturelle, and LNCP, Universite´ Paris 6, France

One-electron oxidation of six different c-type lysozymes

from hen egg white, turkey egg white, human milk, horse

milk, camel stomach and tortoise was studied by

gamma-and pulse-radiolysis.In the first step, one tryptophan side

chain is oxidized to indolyl free radical, which is produced

quantitatively.As shown already, the indolyl radical

subse-quently oxidizes a tyrosine side chain to the phenoxy radical

in an intramolecular reaction.However this reaction is not

total and its stoichiometry depends on the protein.Rate

constants also vary between proteins, from 120Æs)1 to

1000Æs)1at pH 7.0 and room temperature [extremes are hen

and turkey egg white (120Æs)1) and human milk (1000Æs)1)]

In hen and turkey egg white lysozymes we show that another

reactive site is the Asn103–Gly104 peptidic bond, which gets broken radiolytically.Tryptic digestion followed by HPLC separation and identification of the peptides was performed for nonirradiated and irradiated hen lysozyme.Fluorescence spectra of the peptides indicate that Trp108 and/or 111 remain oxidized and that Tyr20 and 53 give bityrosine Tyr23 appears not to be involved in the process.Thus new features of long-range intramolecular electron transfer in proteins appear: it is only partial and other groups are involved which are silent in pulse radiolysis

Keywords: gamma and pulse radiolysis; intramolecular long-range electron transfer; lysozyme; one-electron oxidation

The early suggestions [1,2] of intramolecular long-range

electron transfer (LRET) have now been verified by

numer-ous observations of LRET between donor/acceptor redox

centres with known separation distances in proteins, peptides

and other small rigid organic molecules.This property exists

in polymers and especially in biopolymers (proteins and

DNA).Thus these macromolecules are considered as

candidates for nanoelectronics components.In this view,

several studies were devoted to a better understanding of the

factors leading to modulation of LRET (for a review, see [3]

and references therein [4–6]).As part of a program to unravel

the mechanistic basis of LRET in proteins, systematic

investigations on electron transfer between tyrosine and

tryptophan in proteins and peptides have been conducted

[7–10].In this experimental system, developed first by Pru¨tz

et al.[11], pulse radiolytically generated azide radical and

dibromide radical anion preferentially oxidize the side chain

of tryptophan (Trp) to the indolyl radical (TrpÆ) in molecules that also contain tyrosine (Tyr)

NÆ

3þ Trp-X-Tyr ! TrpÆ-X-Tyr+N

3 þ Hþ ð1Þ The initiating oxidation (1) can be effected by other radiolytically generated species such as the dibromide (Br2) and dichloride (Cl2) radical anions [12].The trypto-phan neutral radical, with a midpoint reduction potential that is near 1 V at pH 7.0 [13], rapidly oxidizes the tyrosine side chain to the phenoxy radical (TyrOÆ) in the intra-molecular equilibration:

TrpÆ-X-Tyr$ Trp-X-TyrOÆ ð2Þ Both TyrOÆand TrpÆabsorb strongly in different parts of the visible region.One of the first observations of reaction (2) in proteins was obtained in hen egg white lysozyme (HEWL) [14–16]

However previous pulse radiolysis studies indicated a low yield of tyrosyl radical compared to that of initial oxidant and of tryptophanyl radical [14].Since the final products coming from this process were never characterized, the stoichiometry of LRET was never evaluated.The aim of the experiments described in this paper was thus to verify whether the stoichiometry in LRET (equality of tyrosyl vs tryptophanyl radicals) was respected, to identify some of the aromatic amino acids responsible of LRET, and possible other processes involved

Correspondence to C.Houe´e-Levin, LCP, UMR 8000 baˆt.350,

Centre Universitaire, 91405 Orsay Cedex, France.

Fax: +33 1 69 15 30 53, Tel : +33 1 69 15 55 49,

E-mail: chantal.houee-levin@lcp.u-psud.fr,

Web site: http://www.lcp.u-psud.fr

Abbreviations: HEWL, hen egg white lysozyme; TEWL, turkey egg

white lysozyme; Trp Æ , the indolyl radical of tryptophan; TrpH Æ+ , the

protonated indolyl radical of tryptophan; TyrOÆ, the phenoxy radical

of tyrosine; LRET, long-range electron transfer.

(Received 7 April 2003, revised 6 June 2003, accepted 7 July 2003)

The c-type lysozymes (129–130 amino acids in most

cases) having similar sequences (for the sequences see [17]),

three-dimensional structures (obtained by crystallography

and two-dimensional NMR in solution) and biochemical

activity [18–22] provide an excellent model system for

exploring the effect of amino acid substitution on the

intramolecular electron transfer

This approach complements other studies, directed at

understanding the electron transfer process, which involves

changing the protein sequence by site-directed mutagenesis

and the use of chemically modified proteins.However in this

work we show that another process i.e a peptide bond

fragmentation, is initiated by LRET, which means that

there might be some leakage in electron transfer.These

points are crucial for understanding of redox processes

in biology and also for an eventual development of

nano-electronics

Experimental procedures

Chemicals (sodium azide, potassium phosphate, sodium

hydroxide) were of the highest quality available (Prolabo

Normatom or Merck Suprapure).Nitrous oxide was

delivered by ALPHA GAZ; its purity was higher than

99.99% HEWL, EC 3.2.1.17 (crystallized three times,

dialyzed and lyophilized), and turkey egg white lysozyme

(TEWL) were supplied by Sigma.Other lysozymes were

prepared using known procedures [23].The purity of the

protein preparations was tested by SDS/PAGE according

to Sha¨gger and von Jagow [24] and by reversed-phase

HPLC.All proteins were used after dialysis against buffer

The solution concentration was verified by the protein

absorbance at 278 nm [25].Water was obtained from an

Elga Maxima device (resistivity¼ 18.2 MX)

In gamma radiolysis, protein solutions (1.4· 10)4M) in

10 mM potassium phosphate buffer (pH 7.0) and 0.1M

sodium azide (NaN3) in order to produce the azide radical,

were saturated with N2O.Protein solutions were prepared

shortly prior to irradiation.The samples were submitted to c

irradiation in a panoramic IL60PL (Cis Bio International)

60Co source at a dose rate, determined by Fricke dosimeter,

of approximately 1.0 GyÆs)1

For pulse radiolysis, radicals were generated by the

introduction into an aqueous solution of a 200-ns pulse of

high-energy electrons ( 4 MeV) from a linear accelerator

located at the Curie Institute, Orsay France [26]

In both cases, the predominant primary radical products,

eaq– and OHÆ, formed by ionizing particle (high energy

electrons or photons) interactions with water [27], were

converted to the azide radical, N3, by reactions with N2O

at saturation and 0.1 molÆL)1azide anion [27].The pH of

the solution was adjusted with 5–20 mmolÆL)1phosphate

buffer.In order to prevent foam formation (denaturation of

the protein) saturation with N2O was carried out by

introducing the gas on top of the stirred cold (4C)

solution for 60 min.The total radical concentration was

 2 lmolÆL)1 determined by methyl viologen dosimetry

[28].These low concentrations were used to minimize

possible second-order decays of TyrOÆand TrpÆ.The protein

was present at 0.14 mmolÆL)1(2 mgÆmL)1), except when we

determined the concentration dependence of the rate

constant for reaction (1).For this experiment the protein

concentration was varied between 0.05 and 0.5 mmolÆL)1 All reactions, monitored by changes in the UV–visible region, were functionally first order, and the rate constants were obtained by nonlinear least-squares fitting to the absorbance data.The numbering of the amino acid residues

in each lysozyme protein follows that of the Protein Data Bank (PDB) of the Brookhaven National Laboratories Irradiated protein solutions were analysed by SDS/ PAGE and by HPLC b-mercaptoethanol was added to the irradiated samples to reduce the disulphide bridges before electrophoretic analysis on a SDS-containing gel Proteins bands were stained with Coomassie blue R-250 Stained gels were scanned using an Ultrascan XL laser microdensitometer (LKB).The proportion of material present in each band was determined using the intensity of the vertical cut corrected for the bandwidth

Dimers were separated from the monomers by HPLC, using a PorosR1 (4· 100 mm) column.A gradient of 35–60% (trifluoroacetic acid 0.1%/acetonitrile 70%) in trifluoroacetic acid over 30 min (at a flow rate of 1.5 mLÆmin)1) was used.Detection was at 214 nm

Evaluation of the fraction of dimerized proteins was achieved by calculation of the portion of the total area under the curve occupied by the dimers peak.The obtained value was divided by two

MALDI-TOF MS was performed at The Institute of Nuclear Physics, Orsay, France.N-terminal sequencing was done at IBCP, Lyon, France

For aminoethylation, proteins were kept 2 h in a solution containing 6M urea, 1M Tris pH 8.6, 10 mM EDTA; b-mercaptoethanol (50 mM) was added to reduce the disulfide bridges.Ethylene imine (Fluka) was added (20 times expected thiol concentration) and the solution was incubated in the dark for 40 min before 20 lLÆmL)1 of 2-mercaptoethanol was added.Salts were removed using reversed-phase HPLC and the samples were dried using a Speed-vac Savant apparatus

Trypsin hydrolysis was performed in ammonium hydro-genocarbonate (100 mgÆmL)1) aqueous solution.Protein concentration was 2 mgÆmL)1, 3% w/w of trypsin (Worth-ington) was added.The solution was kept overnight at

30C.Peptides were separated on a column Vydac C4 (4.6· 250 mm).Solution A was a solution of trifluoroacetic acid (0.1%), solution B was a solution of 0.1% trifluoro-acetic acid and 70% acetonitrile (gradient 0–100% B in

90 min, flow rate 1 mLÆmin)1).Detection was at 214 nm Gel permeation chromatography was performed at 5C with buffer 0.2MNa2SO4, 0 1Mphosphate pH 6.5, sapo-nine 0.005% Precipitation of proteins was first achieved with ammonium sulphate.The column (3· 50 · 90 cm) was filled with gel TSK Toyopearl (HW40S or HW50S) or Ultrogel ACA50

Results

Pulse radiolysis Pulse radiolysis studies of amino acids and peptides have shown that below pH 9.0 the tryptophan side chains, compared with other amino acid residues, react rapidly with the azide radical so that one-electron indole oxidation is the predominant reaction in peptides and proteins containing

both tryptophan(s) and tyrosine(s) [7–14].This initial, rapid

increase of absorbance between 350 nm and 600 nm with a

maximum at 510 nm is due to Trp formation (Fig.1).From

the linear dependence of the pseudo-first order rate

constants on protein concentration an average second-order

rate constant of (9.5 ± 0.9)· 108LÆmol)1Æs)1(25C) for

TEWL was calculated.Similar second-order rate constants

were obtained for the other lysozymes (Table 1)

This initial, rapid increase of absorbance is followed by a

slower decrease of the 510 nm band due to TrpÆreduction

This TrpÆreduction is accompanied by tyrosine oxidation to

TyrOÆ, seen from the simultaneous absorbance increase at

410 nm (Fig.1) The time-dependent spectral changes

during this one-electron transfer from tyrosine to TrpÆ

resemble the changes obtained with peptides containing

both tryptophan and tyrosine; i.e absorbance maxima at

 510 and 410 nm, and isosbestic points at  430 nm and

 370 nm [7,8].These isosbestic points, together with the

fact that the apparent first-order reaction rate constant is the

same whether measured at 410 or 510 nm, suggest that

electron transfer from tyrosine to TrpÆ occurs in a single

step.It was also established that this transfer is in fact first

order by demonstrating that the apparent rate constant is

independent of protein concentration from 5· 10)5 to

5· 10)4molÆL)1.Thus, electron transfer between a tyro-sine residue and a tryptophanyl radical in all the lysozymes studied appears to be an intramolecular process.The monomolecular rate constants at pH 7.0 and 25C are summarized in Table 1.In the seconds time range there is a final slow radical decay.This reaction was always second-order representing a radical–radical recombination reaction (e.g dimerization of TyrOÆradical).Thus, the reaction (2) equilibrium is reached before appreciable radical loss occurs via a slow decay [under the condition of our experiments the slowest intramolecular electron transfer half-life for HEWL and TEWL was 5 ms at pH 7.0) The determination of the intramolecular first-order rate constant is not compromised

by a reaction overlap with radical decay

We can estimate the TrpÆand TyrOÆyields in the lysozymes from the magnitudes of the absorbance changes at 510 and

410 nm and the assumption that the extinction coefficients

of the protein bound radicals and those measured from amino acids peptides are the same: for TrpÆ, these are 1800 and 300 LÆmol)1cm)1at 510 and 410 nm, respectively; for TyrOÆ, 70 and 2600 LÆmol)1Æcm)1at, respectively, 510 and

410 nm [29,30].The estimated TrpÆ yields calculated for the initial oxidation of the proteins above pH 4.5 by the inorganic radicals were quantitative.The estimated overall TyrOÆyields after reaction (2) varied with the protein and could be as low as 40% compared to the 90% loss of Trp after the completion of this reaction.We confirm that the process is nonstoichiometric and that the amount of LRET depends on the primary structure of the protein

Final products The final products were detected and quantified after pulse and steady-state gamma radiolysis, by SDS/PAGE and by HPLC (typical electrophoresis gels are shown on Fig.2) Localization of dimers

For all lysozymes studied, dimers are found and their quantity increases with the dose.The G-values of dimeri-zation measured by HPLC or by SDS/PAGE do not depend much on the origin of the protein [31]

The presence of bityrosine could be detected by absorp-tion [32] and fluorescence, with an excitaabsorp-tion wavelength of

325 nm; its emission signal is centred at 410 nm.Using gel permeation liquid chromatography, lysozyme dimers were partially separated from the monomers.It was observed that the fluorescence signal for bityrosine comigrates with the fractions containing the dimerized proteins

Fig 1 Pulse radiolysis study of TEWL oxidation by azide radicals.

Protein 1.4 · 10)4ÆmolÆL)1, phosphate buffer 10 m M pH 7.0, [N 3 ] ¼

10)2ÆmolÆL)1, N 2 O atmosphere ( 1 atm), dose 4 Gy.Optical path

2 cm.

Table 1 Rate constants of LRET, number and location of Tyr and Trp residues, for the various lysozymes.

Lysozyme source

(No.of amino acids)

Number of Trp/

molecule (position)

Number of Tyr/

molecule (position) k Lys+N3 mol)1Ædm 3 Æs)1 k LRET s)1

Hen egg white (129) 6 (28, 62, 63, 108, 111, 123) 3 (20, 23, 53) (7.9 ± 0.8) · 10 8

120 ± 10 Turkey egg white (129) 6 (28, 62, 63, 108, 111, 123) 4 (3, 20, 23, 53) (9.5 ± 0.9) · 10 8 120 ± 10 Horse milk (129) 5 (28, 62, 63, 108, 111) 4 (23, 34, 54, 123) (7.0 ± 0.7) · 10 8 230 ± 20 Camel stomach (130) 6 (3, 28, 34, 64, 109, 112) 6 (20, 38, 45, 54, 63, 124) (6.3 ± 0.7) · 10 8

500 ± 50 Tortoise (130) 5 (28, 64, 109, 112, 124) 6 (3, 20, 23, 45, 54, 63) (7.8 ± 0.8) · 10 8 300 ± 30 Human milk 5 (28, 34, 64, 109, 112) 6 (20, 38, 45, 54, 63, 124) (4.7 ± 1.0) · 10 8 1000 ± 100

The fractions containing the HEWL dimers and samples

of irradiated solutions were hydrolysed by trypsin and amino

groups were ethylated.Then fragments were separated by

HPLC and identified by their amino acid sequences.The

relative population of the peptide fractions from the

irradiated samples were compared to the corresponding

ones obtained with the native lysozyme.The proportions of

tyrosine 23- and 53-containing fractions were found to be

significantly lower in the dimer-rich preparations.(Fig 3).A

fraction of each peptide was analysed by fluorescence, a very

sensitive technique.The bityrosine signal was observed in all

peptides containing tyrosine 23 or 53 but not in peptides

containing tyrosine 20 alone.According to these results,

tyrosines 23 and 53 could be involved in the dimerizations

whereas no sign of tyrosine 20 involvement was seen

Oxidized tryptophans

An increase in 315 nm absorbance was also observed in

nondimerized fractions demonstrating that at least another

oxidation product was formed.Tryptophan solutions,

irradiated in the presence of N3, also show a dose-dependent

increase in 315 nm absorbance (M.Stuart-Audette,

C.Sicard-Roselli, C.Houe´e-Levin, unpublished results)

Knowing that the electron transfer between Trp and Tyr is

partial, we have been seeking for tryptophan oxidation

products.Further fluorescence studies shown the presence

of a different emission spectrum upon excitation at 380 nm

The signal is compatible with the presence of

N-formyl-kynurenine [33]

The peptides obtained by hydrolysis of irradiated

solu-tion, for which the amino acid composition of peptides

was determined, were screened for the fluorescence signal

obtained when an excitation wavelength of 380 nm is used

The signal was observed in peptides containing tryptophans

108 and 111.Because none of the peptide contains only

tryptophan 108 or 111, it was not possible to identify which

of the two (if not both) is oxidized and not repaired.The relative proportions of the irradiated fractions, at different doses, were compared to the ones obtained with the native HEWL.Tryptophan 108- and 111-containing peptides decreased rapidly as doses were increased.No indication

of tryptophan 28, 62, 63 and 123 modifications was noted Fragments

In addition to dimerization, HEWL and TEWL appear to undergo fragmentation.The N-terminal sequences of the first 10 amino acids of the longest moieties are identical to that of the native proteins.MS of HEWL gave a small peak corresponding to 11 286 Da and another one corresponding

to 3047.5 Da (Fig 4) For TEWL, we find 3055.7 Da for the smaller moiety.This allows us to determine the site of fragmentation at the peptidic bond between Asn103 and Gly104 for both proteins.In addition, N-terminal sequen-cing of the smaller moiety indicated that Gly residue is intact

It is interesting to note that TEWL, which has the same Asn103–Gly104 sequence, gets also fragmented at the same site as HEWL, whereas human milk lysozyme and tortoise lysozyme, which do not get fragmented upon oxidation, do not have the Asn103–Gly104 sequence (Fig.2).The yields of fragmentation (Table 2) vary for these proteins

Discussion

In this work, we performed a comparative study of the one-electron oxidation of six proteins from the c-type family of lysozymes; these proteins differ by several amino acids but their three-dimensional structures are very similar Pulse radiolysis results show that the rate constants of one-electron oxidation by azide radicals are of the same order of magnitude.For all proteins, tryptophanyl radicals

Fig 2 SDS/PAGE analysis in reducing con-ditions of irradiated lysozymes (A) Hen egg white, (B) turkey egg white, (C) tortoise egg white, (D) human milk lysozymes.D, Dimer;

L, lyoszyme; F, fragment.

are obtained first, and the stoichiometry N3Æ/TrpÆ is

observed.This oxidation is always followed by a reaction,

in which TrpÆdisappears and TyrOÆis formed.The limiting

step is intramolecular, as it was already shown for peptides

and proteins.The rate constants differ by a factor of 8

with the protein (Table 1)

For all proteins the LRET is not stoichiometric.In

addition the TyrOÆ yield varies with the protein.These

apparently low TyrOÆyields could be due to side reactions in

which TrpÆ is lost by some other path.Although TrpÆis a

relatively good oxidant because of its reduction potential,

there has been no report of TrpÆoxidizing any amino acid

other than tyrosine.For example, one could suggest that the

disulfide bond could be the electron donor to the indolyl

tryptophan radical.However, the reduction potential of

the couples TrpH+Æ/Trp (pH < 4.5) and TrpÆ/Trp (neutral

pH) are, respectively, 1.15 V and 1.05 V [13], whereas that of

the RSSR+Æ/RSSR is 1.3 V (D Armstrong, University of

Calgary, Canada, personal communication).This difference

in reduction potentials makes the oxidation of a disulfide

bond by the indolyl radical (protonated or unprotonated)

highly improbable.Nevertheless, we have studied this

reaction by observing the decay of the indolyl radical in the

presence and absence of the disulfide model compound

(lipoate anion) and found no effect (data not shown)

For the first time in this kind of study, we tried to

determine the nature of the final products in HEWL and

TEWL.We were expecting mostly dimers, since it is known

that the fate of tyrosyl radicals is dimerization.Surprisingly,

we found in addition to dimers, oxidized forms (without

great change of the molecular mass) and fragments.The site

of fragmentation is the peptide bond between Asn103 and

Gly104.The fragmentation is highly sequence specific: if

Asn–Gly sequence is not present, the protein is not cleaved

The N-terminal amino acid of the shorter fragment could be

detected by sequencing, showing that the amino group is

intact.Since no hydrolysis is observed without radiolytic

oxidation, the fragmentation process appears to be linked to

the oxidation.To our knowledge, for the first time, the

participation of residues other than aromatic in oxidative processes is demonstrated.Tryptophanyl radicals might reduce the peptide bond which would then undergo fragmentation, in agreement with [34].Indeed, we found that Trp108 and/or Trp111, which are the closest to the Asn103–Gly104 sequence, are oxidized.We have investi-gated the possibilities of creation of a free radical site, positive or negative, in this sequence by methods of quantum chemistry [35].The aim was to see if any of this

Fig 4 MALDI-TOF mass spectra of fragments from irradiated lyso-zymes Lysozymes were reduced by 2-mercaptoethanol and fragments were isolated by HPLC.(A) Hen egg white, (B) turkey egg white lysozymes.

Fig 3 Separation of tryptic fragments of HEWL after

aminoethyla-tion Detection at 214 nm *New fragments formed by irradiaaminoethyla-tion.

Nonirradiated; irradiated (100 Gy).

Table 2 Yields (G-values) of polypeptide chain fragmentation for hen, turkey and tortoise lysozymes.

Lysozyme

Fragmentation yield (molÆJ)1)

Percent of azide radicals (yield 5.5 · 10)7ÆmolÆJ)1)

Hen (1.42 ± 0.03) · 10)8 2.5 Turkey (0.68 ± 0.03) · 10)8 1.2

free radical would lead to peptide bond elongation, a

prerequisite for bond breakage.Such a modification was

found for the radical anion.This would be in agreement

with intramolecular reduction of this sequence

Attempts to identify the position of oxidized residues

were performed with HEWL.Tryptic hydrolysis followed

by identification of the fragments and fluorescence

detec-tion, indicated that: (a) Tyr53 and Tyr23 are oxidized; (b)

Trp108 and/or Trp111 are oxidized.Typ53 is very close to

Trp62 and Trp63.Bobrowski et al.[36] have shown that

residues of HEWL Trp62 are part of the initial process

yielding 50% of the observed LRET.They proposed that

these radical residues are reduced by Tyr53, and our results

are in agreement with this hypothesis.Based on the

first-order rate constants measured in the model peptide

compounds with oligoprolines as spacers and using these

results as a ruler for the proteins, the value of 120 s)1found

for HEWL and TEWL suggests a separation distance

between donor and acceptor of 1.4 nm [7,8,10] and thus a

similar pattern of oxidation.Indeed, the similarity of NOE

data, hydrogen-exchange rates, chemical shifts and coupling

constants of the two proteins are indicative that structures

of HEWL and TEWL are essentially identical [37].It should

be noted that the presence of Tyr at position 3 did not affect

the rate constant suggesting that this residue does not

participate in the intramolecular process.Nevertheless,

dimerization of Tyr23 could come also from Trp62 and/or

Trp63.However Trp108 and Trp111 are at a closer distance

and since they remain oxidized, we conclude that no

intramolecular electron transfer was initiated from these

residues.It seems that the argument of distance is not

sufficiently explanatory

In conclusion, the long-range intramolecular electron

transfer process appears to be much more complicated than

was previously thought.Some tryptophan residues do not

lead to LRET and no explanation comes from examination

of the distances.Some tyrosine residues (such as Tyr20 and

Tyr3 in TEWL) are not involved in the process and similarly

the argument of distances does not provide explanations.In

addition, an unexpected fragmentation site is observed: the

Asn103–Gly104 sequence.More careful investigations of

LRET are needed, including analysis of the final

com-pounds, to reach an understanding of the process

Acknowledgements

M.F thanks the Curie Institute, the Yvette Mayent award and Dr

D.Lavalette, director of the INSERM unit 350 for his hospitality.We

are indebted to the Curie Institute and especially to Dr V.Favaudon for

the use and maintenance of the linear accelerator and for fruitful

discussions.

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