Reversible Intramolecular Hydrogen Transfer between Protein

J. Phys. Chem. B 2008, 112, 15921–15932

15921

Reversible Intramolecular Hydrogen Transfer between Protein Cysteine Thiyl Radicals and r C-H Bonds in Insulin: Control of Selectivity by Secondary Structure Olivier Mozziconacci,† Todd D. Williams,‡ Bruce A. Kerwin,§ and Christian Scho¨neich*,† Department of Pharmaceutical Chemistry, 2095 Constant AVenue, UniVersity of Kansas, Lawrence, Kansas 66047; Mass Spectrometry Laboratory, UniVersity of Kansas, Lawrence, Kansas 66045; and the Department of Process and Product DeVelopment, Amgen Inc., Seattle, Washington 98119 ReceiVed: July 26, 2008; ReVised Manuscript ReceiVed: October 2, 2008

The selective oxidative modification of proteins can have significant consequences for structure and function. Here, we show that protein cysteine thiyl radicals (CysS•) can reversibly abstract hydrogen atoms from the RC-H bonds of selected amino acids in a protein (insulin). CysS• were generated photolytically through homolysis of cystine and through photoionization of an aromatic residue, followed by one-electron reduction of cystine. Intramolecular hydrogen transfer was monitored through the covalent incorporation of deuterium into specific amino residues. Of 51 insulin amino residues, only six incorporated significant levels of deuterium: Leu(B6), Gly(B8), Ser(B9), Val(B18), Gly(B20), and Cys(A20). All these amino acids are located at the beginning/end or outside of R-helices and β-sheets, in accordance with theory, which predicts that specifically the RC-H bonds of amino acids in these secondary structures have higher homolytic C-H bond dissociation energies compared to the RC-H bonds of amino acids in extended conformations. Through such hydrogen transfer mechanisms, thiyl radicals are able to catalyze the oxidation of amino acids in a protein through oxidants, which would not necessary directly react with these amino acids. This feature has important consequences for protein stability under conditions of oxidative stress and/or protein production in pharmaceutical biotechnology. 1. Introduction Oxidative damage to proteins plays a major role in biological aging and various pathologies.1-5 Under conditions of oxidative stress, reactive oxygen and nitrogen species can modify key redox-sensitive amino acids, potentially leading to altered biological activities and/or structures of the target proteins. Cysteine (Cys) constitutes one of the most reactive amino acids, especially in its deprotonated thiolate form, where, depending on the protein, the Cys pKa varies between ca. 5 and 9.6,7 In fact, the Cys residue has frequently been referred to as a “sink” for oxidizing species.8 However, specifically for the one-electron oxidation product of Cys, the Cys thiyl radical (CysS•), this paradigm must be re-evaluated as our recent experiments with a model peptide suggest a considerable intramolecular reactivity of CysS• with the C-H bonds of spatially close amino acid residues.9 The design of these experiments, important for the present paper, is outlined in Scheme 1, where CysS• radicals are generated through photolytic cleavage of a disulfide bond.10,11 Through the reversible intramolecular hydrogen transfer with adjacent Gly residues, CysS• and Gly exist in equilibrium 1a with the thiol and an RC• radical. In D2O, the thiol undergoes spontaneous H/D exchange so that the reverse hydrogen transfer from the deuterated thiol to RC• leads to the formation of an R C-D bond, which can be monitored by mass spectrometry analysis. Here, we have for the first time evaluated the potential for hydrogen abstraction by thiyl radicals in a full protein. On the basis of theoretical calculations by Rauk and co-workers,12,13 we expected that CysS• radicals would preferentially attack Gly residues located outside R-helices, which was experimentally * Corresponding author: Fax (785) 864-5736; e-mail [email protected]. † Pharmaceutical Chemistry Department, University of Kansas. ‡ Mass Spectrometry Laboratory, University of Kansas. § Amgen Inc.

SCHEME 1: Reaction Scheme for Covalent H/D Exchangea

a

The intramolecular H atom transfer occurs between the thiyl radical of the cysteine residue and RC-H. The mechanism was confirmed by the incorporation of a deuterium atom into the final product.

confirmed in this paper. Nevertheless, some reactivity of CysS• with additional amino acids outside the R-helical structure was observed, underlining that Gly residues are not the only targets for CysS• radicals. Our experiments have important implications for biology and biotechnology. First, several enzymes utilize CysS• radicals for catalytic turnover, for example, the ribonucleotide reductases,14 pyruvate formate lyase,15,16 and benzyl succinate synthase.17 Maintenance of functional integrity of these enzymes would require conformational restrictions, which do not allow their CysS• radicals to react with RC-H bonds of surrounding amino acid. The reversible reaction of CysS• with the RC-H bond of any amino acid except Gly could lead to L/D isomerization. Second, CysS• radicals have been proposed as intermediates in the S-nitrosation of proteins by NO/O2.18 Again, such a mechanism would require conformational restrictions, which

10.1021/jp8066519 CCC: $40.75  2008 American Chemical Society Published on Web 11/17/2008

15922 J. Phys. Chem. B, Vol. 112, No. 49, 2008

Mozziconacci et al.

SCHEME 2: Tagging of Thiol Residues Formed after Photolytic or Chemical Cleavage of the Disulfide Bondsa

a Reactions 2-7: photolytic cleavage and derivatization of the thiol residue with N-ethylmaleamide (NEM). Reactions 8-9: reduction by dithiotreitol (DTT) and derivatization of the thiol residues with diethyl maleate (DEM).

would not allow the protein CysS• radical to react with surrounding RC-H bonds. Third, during production and storage, biotechnology products such as protein pharmaceuticals, diagnostic, and therapeutic antibodies are exposed to light. These conditions can generate CysS• radicals via direct homolysis of disulfide bonds11 or through photoionization of Trp,19 followed by one-electron reduction of disulfide bonds. The ensuing reaction of CysS• with RC-H bonds may potentially lead to various protein oxidation products (including fragments and aggregates), of which potentially antigenic products such as aggregates are a major concern to the biotechnology industry. The mechanistic studies described in this paper will be useful for the design of similar experiments with antibodies and/or biologically important proteins in order to evaluate the role of CysS• radicals in irreversible protein degradation. Indeed, theoretical calculations20 predict that specifically within random peptide conformations (but not R-helix or β-sheet) the RC-H bond dissociation energy (BDE) of most amino acids is lower than the BDE of the S-H bond, resulting in a thermodynamic preference for hydrogen atom abstraction from the RC-H bond of amino acids by CysS•. Earlier, we determined second-order rate constants on the order of 103-105 M-1 s-1 for the bimolecular hydrogen abstraction by thiyl radicals from the R C-H bonds of model peptides containing Val, Ala, and Gly and from the side chains of Phe, Met, Ser, and Thr.21,22 More recently, we measured high rate constants of k ) 104-105 s-1 for the intramolecular hydrogen abstraction by CysS• from Ala and Gly, respectively, in short model heptapeptides, demonstrating that such reactions may occur under biological conditions, i.e., in the presence of O2 and endogenous antioxidants.23,24 2. Experimental Section 2.1. Materials. N-Ethylmaleimide (>99%, NEM) was supplied by Fluka (Saint Louis, MO); DL-dithiothreitol (>99%, DTT), diethyl maleate (97%, DEM), reduced glutathione

(>99%), and methylene chloride (>98%) were supplied by Sigma-Aldrich (Saint Louis, MO). ThioGlo1 was supplied by Calbiochem (La Jolla, CA). These products were used without further purification. Three stock solutions containing NEM (solution A), DTT (solution B), and DEM (solution C) were prepared at a concentration of 0.1 M in ammonium bicarbonate buffer (pH 7.8, 50 mM). Endoproteinase Glu-C and carboxypeptidase (CPY) (sequencing grade) were supplied by Roche (Indianapolis, IN) and used to prepare a digestion buffer consisting of 500 µg/mL enzyme in ammonium bicarbonate buffer (pH 7.8, 50 mM). Human insulin, zinc-free (Chart 1) was supplied by Roche Applied Science (Indianapolis, IN) at a purity level of >95% and used without further purification. 2.2. Reactions. 2.2.1. UV Irradiation. Insulin was dissolved in 1 mL (or 10 mL) of H2O (Milli-Q) or in deuterium oxide (99.9%, Cambridge Isotope Laboratories, Inc., Andover, MA) at a final concentration of 500 µM (or 50 µM), and aliquots of 400 µL (or 1 mL) were placed in quartz tubes open to air (or saturated with Ar). The samples were irradiated at room temperature (25 °C) for 0.5-30 min by means of four UV lamps (RMA-500, Southern New England, Branford, CT) at 253.7 nm in a Rayonet (Southern New England, Branford, CT) photochemical reactor. 2.2.2. Enzymatic Digestion. Two classes of enzymes were used to digest insulin. Protease S. aureus V8 (EndoproteinaseGlu-C) specifically cleaves peptide bonds on the carboxyterminal side of either aspartic or glutamic acid. In the presence of ammonium ions, the enzyme specificity is limited to glutamic acid.25 Thus, the use of Glu-C in ammonium bicarbonate buffer, to digest insulin, releases sufficiently short peptides to simplify their mass spectrometry analysis. Carboxypeptidase Y has a broad amino acid specificity and is able to release every amino acid from the carboxy terminal of peptides, though glycine and aspartic acid are released kinetically slower than other amino acid residues.26

Photolysis of Insulin TABLE 1: B-Chain Products of Insulin Generated after Photoirradiation in Air-Saturated Aqueous Solution

2.2.3. Thiol DeriWatization Reactions and Digestion. An aliquot of 50 µL was taken from the irradiated solutions (H2O or D2O) of insulin and added to 700 µL of 50 mM ammonium bicarbonate buffer and 50 µL of solution A in order to derivatize thiol residues generated through UV irradiation with NEM (1: 16 dilution). After 1 h, 50 µL of solution B (DTT) was added to reduce the remaining disulfide bonds. After further 30 min at room temperature, 150 µL of solution C (DEM) was added to derivatize the thiol residues formed through DTT reduction of the disulfide bonds. That is, all thiols labeled with NEM represent photoirradiation products whereas thiols labeled with DEM are released through the reduction of disulfides. To digest the samples, 20 µL of the stock solutions containing either endoproteinase Glu-C or carboxypeptidase CPY was added, and the solutions were incubated at 37 °C for 3 h (Glu-C) or 1 h (CPY). 2.2.4. Quantification of Thiol Residues. The fluorogenic thiol reagent ThioGlo1 was used for the quantification of free thiol.27 A spectrofluorimeter (RF 5000U, Shimadzu) was set at λex ) 379 nm and λem ) 513 nm and was calibrated by different known concentrations of reduced glutathione in the range of 0-2 µM. The derivatization with ThioGlo1 (20 µM) was carried out in 1 mL solution of ammonium bicarbonate buffer (50 mM, pH 7.3) containing 10 µL of the irradiated sample. The fluorescence intensity was measured after 1 h of incubation with ThioGlo1. 2.3. HPLC-MS Analysis. The samples were injected onto a narrow-bore column (MicroTech Scientific, 5 cm × 1 mm

J. Phys. Chem. B, Vol. 112, No. 49, 2008 15923 Zorbax C18, 3.5 µm) and eluted with two successive linear gradients delivered at the rate of 12 µL min-1 by an AcquityUltra-Performance liquid chromatography system (Waters Corp., Milford, MA). Mobile phases consisted of water/ acetonitrile/formic acid at a ratio of 99%, 1%, 0.08% (v:v:v) for solvent A and a ratio of 0%, 99%, 0.06% (v:v:v) for solvent B. The following linear gradients were set: 1% of solvent B for 1 min, 10-30% of solvent B within 8 min, and 30-70% of solvent B within 3 min. 2.4. Nano-Electrospray Ionization Time-of-Flight (ESI TOF) Analysis. ESI spectra were acquired on a Q-TOF-2 (Micromass Ltd., Manchester, U.K.) hybrid mass spectrometer operated in the MS1 mode and acquiring data with the timeof-flight analyzer. The instrument was operated for maximum resolution with all lenses optimized on the [M + 2H]2+ ion from the cyclic peptide gramicidin S. The cone voltage was 30 eV, Ar was admitted to the collision cell at a pressure that attenuates the beam to about 20%, and the cell was operated at 5 eV (maximum transmission). Spectra were acquired at 11 364 Hz pusher frequency covering the mass range 100-2000 amu and accumulating data for 5 s per cycle. Time to mass calibration was made with CsI cluster ions acquired under the same conditions. 2.5. MS/MS Analysis. Collision-induced dissociation (CID) spectra were acquired by setting the MS1 quadrupole to transmit a precursor mass window of (1.5 amu centered on the most abundant isotopomer. Ar was the collision gas admitted at a density that attenuates the beam to 20%; this corresponds to 16 psi on the supply regulator or 5.3 × 10-5 mbar on a penning gauge near the collision cell. MS/MS spectra were acquired with two different collision energies (25 and 40 eV). 2.6. Covalent H/D Exchange and Isotopic Correction. The deuterium composition of peptide ions and their fragments was determined from the differences between the average mass of a covalently deuterated peptide and the average mass of the corresponding fully protonated peptide. The average masses were calculated from centroided isotopic distributions. The distribution of deuterium incorporation was obtained after isotopic correction by subtracting the isotope abundance distribution in the product formed during UV irradiation in H2O from the isotope abundance distribution of the same product generated in D2O. This variation of the isotopic distribution between the experiments performed in D2O and H2O will be given along this paper by the variation of the percent base peak intensity (%∆BPI). 3. Results 3.1. Photolytic Processes. In insulin, the chromophores absorbing photons with λ ) 253.7 nm are the three disulfide bonds and the aromatic residues Tyr, Phe, and His. There are two independent pathways for thiyl radical generation: The direct photolysis of the disulfide bonds predominantly leads to homolytic cleavage and thiyl radicals (Scheme 2, reaction 1),10,11 which ultimately disproportionate generating thioaldehyde and thiol (Scheme 2, reaction 2).9,11,28 In addition, photoionization of aromatic amino acids leads to radical cations and hydrated electrons (reaction 3), where in the case of Tyr the radical cations deprotonate to yield tyrosyl radicals (reaction 4).29 The hydrated electrons will ultimately reduce protein disulfide bonds to thiyl radical and thiol (reaction 5).30 The relative contribution of both pathways can be quantitated by the addition of appropriate scavengers for hydrated electrons. Specifically, CH2Cl2 will react with eaq- with k6 ) 6.0 × 109 M-1 s-1.31 Thus, when CH2Cl2 is added to insulin-containing solutions at a ratio of [CH2Cl2]/[insulin] > 4.0, we expect to prevent thiyl


Mozziconacci et al.

Figure 1. HPLC-MS analysis of the reduced B-chain formed after UV irradiation of insulin (500 µM) in aqueous solution (bottom) and in deuterium oxide solution (top).

radical generation via reaction 6. In addition, a fraction of hydrated electrons will also be scavenged through reaction with O2 in O2-containing solutions (reaction 7, k7 ) 1.9 × 1010 M-1 s-1).32 Therefore, in the following product studies several representative reaction products (e.g., thiols) were quantified specifically in the absence or presence of either O2 or CH2Cl2.

CHART 1: Representation of the Primary Structure of Insulin and the Digestion Sites for Endoproteinase Glu-C and the Localization of the Sites of Covalent H/D Exchange (Gray Background)a

hν

TyrOH 98 TyrOH•+ + eaq-

(3)

hν

TyrOH•+ 98 H+ + TyrO•

(4)

H+

eaq- + RSSR 98 RS · + RSH

(5)

eaq- + CH2Cl2 f •CH2Cl + Cl-

(6)

eaq- + O2 f O2•-

(7)

3.2. Analysis of the Photoproducts. 3.2.1. QualitatiWe Analysis of the Products. The HPLC-MS analysis of photoirradiated aqueous solutions containing insulin revealed photoproducts mainly originating from the B-chain. The disproportionation (Scheme 2, reaction 2) following homolytic cleavage of the disulfides Cys(B7)-Cys(A7) and Cys(B19)-Cys(A20) generates either reduced Cys(B7) and Cys(B19) (Table 1 and Figure 1, bottom panel MH4+: m/z ) 857.6) or their respective thioaldehydes, which subsequently transform into aldehyde9 (Table 1 and Figure 1, MH4+: m/z ) 857.6). Thus, the photoproducts of the B-chain are a combination of reduced Cys and cysteine-derived aldehyde (Table 1 and Figure 1, MH4+: m/z ) 849.4 and m/z ) 853.7). We note that in the presence of

a

The positions of R-helices are highlighted by dashed frames.

O2 the free thiol residues of the B-chain are also converted into sulfinic acid (Table 1 and Figure 1, MH4+: m/z ) 861.9 and m/z ) 865.7), likely via addition of O2 to an initial thiyl radical. The MS/MS data of the main product (m/z ) 857.6) are presented in the Supporting Information (Figure S1). In addition, we detected a cross-link between Cys and Tyr, potentially a radical-radical recombination product between tyrosyl (TyrO•) and CysS• radicals (Figure 2, MH2+: m/z ) 751.8). 3.2.2. Product Formation in H2O and D2O. Aqueous and deuterium oxide solutions containing insulin (500 µM) were photoirradiated for up to 10 min and analyzed by HPLC-MS (Figure 1). In H2O, the photoproduct of the B-chain is characterized by its quadruply charged ions with m/z ) 857.6 while photoirradiation in D2O results in a product with m/z 859.4. From the difference m/z(D2O) - m/z(H2O) ) 1.8, we conclude that a maximum of 6-7 deuteriums atoms are incorporated covalently into the cleaved B-chain. No significant deuterium incorporation was detected for nonirradiated insulin (control). The different individual products resulting from the photoirradiation shown in Table 1 (thiol, thioaldehyde, aldehyde, sulfinic acid) incorporate significant amounts of deuterium (see below). 3.2.3. Quantification of the Thiol Residues. Free thiols were quantified representatively to evaluate the relative contribution

Photolysis of Insulin

J. Phys. Chem. B, Vol. 112, No. 49, 2008 15925

Figure 2. MS/MS spectrum of the photoproduct m/z ) 751.8 (doubly charged). The NYCN-NEM derivative is cross-linked with the fragment ALYLVCGE through residues Tyr(A19) and Cys(B19).


Figure 3. (A) Formation of free thiol residues after 10 min of UV irradiation of insulin (50 µM) in Ar-saturated aqueous solution in the presence of different concentrations of CH2Cl2. (B) Time course of the formation of the free thiol residues during UV irradiation of insulin (50 µM) in air-saturated aqueous solution.

of reactions 1 and 5 to thiyl radical formation. Insulin (50 µM) was irradiated in both Ar- and air-saturated aqueous solution. Ar-Saturated Solution. Prior to photoirradiation, the insulin solutions (1 mL) were placed in quartz tubes and saturated with Ar for 5 min. Final concentrations of 0-10 mM CH2Cl2 were added to the Ar-saturated samples after Ar saturation. The solutions were photoirradiated for up to 10 min and diluted 1:100 in the presence of 20 µM of ThioGlo1. Figure 3A demonstrates that increasing concentrations of CH2Cl2 lower the yield of thiol from 60 µM to a plateau value of 40 µM for 200 µM CH2Cl2, beyond which no further decrease is observed. At the plateau, all hydrated electrons are scavenged by CH2Cl2 (reaction 6), inferring that under these conditions all thiols are formed through the direct homolytic cleavage of the disulfide bonds (reactions 1 and 2). Thus, in Ar-saturated solution in the absence of CH2Cl2 67% of the thiols are formed through direct homolysis of the disulfide bonds and 33% through hydrated electrons. The latter implies that some cross-links between thiyl radicals and tyrosyl radicals may form, consistent with the product displayed in Figure 2. Air-Saturated Solution. The insulin solution (1 mL) was placed in a quartz tube open to air. Aliquots (10 µL) of the photoirradiated samples were taken after different times of irradiation and diluted 1:100 before the addition of a final concentration of ThioGlo1 of 20 µM. After 10 min of irradiation, the concentration of thiol residues reached a plateau (35 µM, Figure 3B) which is approximately equivalent to the concentration of thiols measured after scavenging all hydrated electrons in Ar-saturated solution (40 µM, Figure 1A). The fraction of hydrated electrons able to reduce disulfide bonds in air-saturated solution is estimated to be less than 4%, calculated as follows: the concentration of O2 in air-saturated solution is 2.2 × 10-4 M (P ) 1 atm, 25 °C), and the concentration of the disulfide

Mozziconacci et al. bond ([RSSR]) is taken as the concentration of the protein. The fraction of thiols formed through reaction 5 in the absence of O2 is 0.33 (see Ar-saturated section). Therefore, based on the rate constants for reactions 5 and 7 (k5 ) 1.1 × 1010 M-1 s-1 and k7 ) 1.9 × 1010 M-1 s-1),32,33 the equation [1 - k7[O2]/ {(k7[O2] + k5[RSSR])}] × 0.33 gives an approximation of the yield of thiol residues generated through reaction 5 in airsaturated solution. 3.3. Localization of Deuterium Incorporation. In order to localize the amino acid residues involved in covalent H/D exchange (Scheme 1), the reduced Cys residues generated under photoirradiation (Scheme 2, reaction 2) were derivatized with NEM (Scheme 2, reaction 7). To simplify chromatographical separation and the identification of the products, the disulfide bonds remaining after irradiation were reduced with DTT and the generated Cys residues were derivatized with DEM (Scheme 2, reactions 8 and 9). HPLC-MS analysis of photoirradiated insulin, treated according to the derivatization protocol and digested with endoproteinase Glu-C, highlighted eight products (Figure 4A-E). The MS/MS data of the digested and derivatized products are presented in the Supporting Information (Figures S2-S7). 3.3.1. Deuterium Incorporation into the A-Chain. Sequence GIVE (m/z 417.2). This N-terminal fragment from the A-chain, which eluted after 2.36 min, does not undergo any significant deuterium incorporation. Indeed, the variation of the base peak intensity (%∆BPI) at m/z 418.2 does not exceed 5% (Figure 4A), which can be considered as measurement uncertainty. MS/ MS sequencing of the fragment confirmed that none of the amino acids incorporated deuterium (Table 2). This fragment serves as an additional control that our data are not contaminated by “noncovalent” H/D exchange of ionizable residues. Sequence NYCN. Located at the C-terminus of the A-chain, this fragment contains Cys(A20) involved in the interchain disulfide bond Cys(A20)-Cys(B19). Two different fragments with m/z 638.25 and 685.28 eluted after 2.58 and 4.21 min, containing CysA20 labeled with NEM and DEM, respectively (Figure 4B). Based on our labeling sequence with NEM and DEM, the Cys(A20)-NEM labeled peptide represents a photolytically generated thiol whereas the Cys(A20)-DEM labeled peptide represents the original disulfide (or a disulfide formed through recombination of thiyl radicals generated initially through homolytic cleavage of the Cys(A20)-Cys(B19) disulfide bridge). After isotopic correction and comparison of the data obtained during insulin photolysis in H2O (full line) and D2O (dashed line), only the fragment derivatized with NEM (m/z 638.2) shows significant deuterium incorporation with %∆BPI(m/z ) 639.2) ) 20%, whereas the same fragment derivatized with DEM displays only a very low %∆BPI(m/z ) 685.2) ) 2% (Figure 4B). The %∆BPI of the individual b and y ions of the product with m/z 638.25 obtained during MS/MS analysis reveal that deuterium incorporation in NYCN predominantly occurs at Cys(A20) (Table 2). Importantly, we detected the Tyr(A19) immonium ion, which displayed no incorporation of deuterium. Sequence QCCTSICSLYQLE. This internal fragment of the A-chain could not be directly detected by MS/MS analysis, likely due to some incomplete cleavage. However, in the absence of reduction and derivatization with NEM and DEM, a peptide product with m/z 1123.2 (Figure 5) that contains the sequence QCCTSICSLYQLE was identified by MS/MS (Figure S7). Besides an intact disulfide between Cys(A7) and Cys(B7), the original disulfide bond between Cys(A6) and Cys(A11) is cleaved, consistent with photolytic cleavage at this site. After



Figure 4. HPLC-MS analysis of fragments of the reduced and derivatized A and B chains obtained through endoproteinase Glu-C. NEM and DEM were used to derivatize the cysteine residues. The MS spectra of the fragments generated in H2O (full lines) and D2O (dashed lines) solutions were compared after isotopic correction in order to determine the deuterium incorporation into individual fragments. The variation of the percent base peak intensity measured between H2O and D2O samples was as follows: (A) ∆(418.2 - 417.2) ) 5%, (B) ∆(686.2 - 685.2) ) 2%, ∆(639.2 - 638.2) ) 20%, (C) ∆(805.4 - 804.4) ) 20%, ∆(828.9 - 827.9) ) 7%, (D) ∆(1040.6 - 1039.6) ) 8%, ∆(993.5 - 992.5) ) 32%, (E) ∆(559.8 - 558.8) ) 5%.


Mozziconacci et al.

TABLE 2: Variation of the Percent Base Peak Intensities (%∆BPI) Calculated after Isotopic Correction for the Molecular Ions and Their MS/MS Fragmentsa

Figure 5. HPLC-MS analysis of a partially digested fragment of insulin after UV irradiation. The MS spectra of the fragment generated in H2O (full lines) and D2O (dashed lines) solutions were compared after isotopic correction. No significant incorporation could be observed within this fragment. The two cysteine residues (C*) are a couple of thiol and thioaldehyde.

a The cysteine residues are derivatized with NEM. The CID mass spectra of precursor ion scans were carried out by means of a Q-TOF mass spectrometer.

isotopic correction, the calculation of the %∆BPI for each m/z between 1122.9 and 1124.9 was negative. Moreover, the MS spectrum obtained from the experiment performed in D2O is not moved toward higher masses compared to the MS spectrum obtained from the experiment performed in H2O. Hence, we conclude the sequence QCCTSICSLYQLE does not undergo significant deuterium incorporation subsequent to the photolytic cleavage of the disulfide bond between Cys(A6) and Cys(A11). 3.3.2. Deuterium Incorporation into the B-Chain. Sequence FVNQHLCGSHLVE. After derivatization with NEM and DEM, two peptides were chromatographically separated with retention times of 4.36 and 5.75 min, which display m/z 804.4 and 827.9 of doubly charged ions, respectively. Analogous to the data presented in Figure 4B, the NEM-labeled sequence represents a thiol generated through photodissociation of a disulfide followed by disproportionation (reactions 1 and 2). In contrast, the DEM-labeled thiol is generated through DTT reduction of the original disulfide between Cys(B7) and Cys(A7) (Chart 1). The %∆BPI measured for m/z 828.45 (incorporation of one deuterium) and 828.94 (incorporation of two deuterium) does not exceed 5% (Figure 4C), indicating no covalent H/D exchange. Instead, for both ions with m/z 805.44 (and 804.94) the %∆BPI amounts to 20%, indicating significant covalent H/D exchange in the peptide sequence, which evolves from the photolytically generated CysS• radical. Besides, we note that the intensity of the main peak (804.44) decreases in D2O (Figure

4C, dashed line) compared to H2O (Figure 4C, full line). The %∆BPI of the individual b and y ions obtained through an MS/ MS experiment displays significant deuterium incorporation between residues Leu(B6) and Ser(B9). Sequence ALYLVCGE. Two products with m/z 1039.5, eluting after 6.76 min, and m/z 992.5, eluting after 8.9 min, were detected for ALYLVCGE, where Cys was derivatized with DEM or NEM, respectively. A significant deuterium incorporation could be observed only in the NEM derivative (%∆BPI(m/z ) 993.55) ) 32%), which represents the photogenerated thiol. Instead, the DEM derivative displays a %∆BPI(m/z ) 1040.58) ) 8% (Figure 4D). The MS/MS fragments of the product with m/z 992.54 reveal a continuous increase of the %∆BPI within the b and y fragments from b2 to b6 and y3 to y5. The maximal %∆BPI (32%) is reached for the fragments b6 and y5. The combined information from the b and y fragments, and the internal fragments VC and VCG, where %∆BPI are 22% and 31%, respectively, leads to the conclusion that deuterium incorporation occurs predominantly between residues Val(B18) and Gly(B20) (Table 2). Sequence RGFFYTPKT. This doubly charged fragment with m/z 558.8 is located at the C-terminus of the B-chain. No significant deuterium incorporation was observed after isotopic correction and comparison of experiments performed in H2O and D2O (%∆BPI(m/z ) 559.84) ) 5%) (Figure 4E). The absence of deuterium incorporation in this fragment was confirmed by an independent set of experiments, using carboxypeptidase instead of Glu-C endoproteinase (data not shown). 3.3.3. Variation of Temperature. In addition to these experiments at room temperature, we performed photolytic H/D incorporation of insulin in H2O and D2O at 50-55 °C. Essentially, the patterns of deuterium incorporation were identical to those observed at room temperature. 4. Discussion Our results indicate several selective target areas for hydrogen abstraction by CysS• in insulin. These are highlighted by a gray



SCHEME 3: Covalent H/D Exchange during the Derivatization of Thiol Residues with NEM or DEMa

CHART 2: Schematic Representation of Hydrogen Bond Patterns for Insulin Adapted from Zoete et al.35 a

a Experimentally, this covalent exchange is estimated to account for not more than 8% of deuterium incorporation based on the 1:16 dilution of D2O samples into H2O.

a The double arrows indicate hydrogen bonds. The positions of R-helices are highlighted by dashed frames.

background in Chart 1. In the following, we will discuss the primary processes of thiyl radical formation as well as the selectivity of hydrogen transfer based on the available structural information for insulin.34 Two assumptions are made for the mechanistic analysis. First, we will use the published NMR structures of insulin (free of zinc) and assume that the overall structure of insulin does not change significantly over the time range of the reversible hydrogen transfer reactions. Second, we expect no more than one photolytic disulfide cleavage per insulin molecule over the time scale of the reversible hydrogen transfer reactions. Formation and Reaction of Thiyl Radicals. In insulin, the photolytic generation of thiyl radicals occurs via two distinct pathways: direct photodissociation of the disulfide bond (reaction 1) and photoionization of aromatic residues, followed by oneelectron reduction of the disulfide bond (reactions 3-5). The direct photolysis of the disulfide bond accounts for 67% of disulfide cleavage in Ar-saturated solution and photoionization of an aromatic residue (likely Tyr) followed by disulfide reduction accounts for 33% of disulfide cleavage. Disproportionation represents an important bimolecular reaction of the thiyl radicals evidenced by the formation of thiol and thioaldehyde (Scheme 2, reaction 2). Such products were also observed during photodissociation of small disulfide-containing peptides in solution9 and disulfides in the gas phase.11 The nature of the detected reaction products (Table 1, Figure 5) indicates that all insulin disulfide bonds are susceptible to photolytic cleavage. The distinct intermediacy of thiyl radicals in this process is indicated by the occurrence of intramolecular hydrogen transfer reactions, evidenced by covalent H/D exchange according to the general mechanism represented in Scheme 1. In D2O, the thiyl radical exists in equilibrium (1a) with an RC• radical, and the generated thiol converts into deuterated thiol before D atom transfer to the RC• radical occurs (reaction 1c). Our HPLC-MS results reveal the incorporation of at least six deuterons into the peptide backbone of the insulin B-chain (Figure 1) and one deuteron into the peptide backbone of the A-chain, indicating multiple target sites for H/D exchange. Before a discussion of the quantitative localization of the sites of H/D exchange, we need to briefly comment on the uncertainty of the experimental measurement. This is important to differentiate the covalent H/D exchange due to the formation of thiyl radicals from any H/D exchange which may occur during the derivatization of thiols with NEM or DEM in the presence of residual D2O (Scheme 3). Derivatization of Thiol Residues and Uncertainties of the %∆BPI Measurement. Only the thiols resulting from photolytic generation of cysteine thiyl radicals were derivatized with

NEM (Scheme 2, reaction 3), whereas thiol residues formed after reduction of the disulfide bonds by DTT were derivatized with DEM (Scheme 2, reaction 5). Thus, the observation of covalent deuterium atom incorporation was expected only for thiols derivatized with NEM because the formation of the cysteine thiyl radical must precede the covalent H/D exchange, as described in Scheme 1. Nevertheless, some low levels of deuterium were observed for thiol residues derivatized with DEM. This can easily be explained by the solvent mixture of 16:1 H2O/D2O (v/v) as a result of dilution of the original D2O sample. Thus, the derivatization product could incorporate a deuterium atom according to Scheme 3. As this pathway does not dependent on intramolecular H atom transfer, we chose to use the greatest variation of %∆BPI observed during DEM derivatization of insulin without photolysis as threshold of the %∆BPI measurement. The largest %∆BPI ) 8% in the absence of photolysis was obtained during derivatization of the fragment ALYLVCGE with DEM (Figure 4D). As a consequence, we used %∆BPI ) 8% as a threshold where %∆BPI < 8% was defined as no photolytic H/D exchange and %∆BPI > 8% was defined as indicating covalent H/D exchange through the mechanism shown in Scheme 1. Quantitative Discussion of Deuterium Incorporation into Specific Amino Acids Based on MS/MS Analysis (Table 2). In the fragment NYCN, the %∆BPI measured in the fragments b3, y2 and the internal fragment ion Y confirmed that deuterium incorporation (%∆BPI ) 15%) is localized on Cys(A20). The value of this %∆BPI is 2-fold higher than the decision threshold (8%) allowing to assign deuterium incorporation on Cys(A20). In the fragment ALYLVCGE, the combined information on %∆BPI measured for the internal fragment ions YLVC (29%) and VC (22%) leads to the conclusion that %∆BPI for the fragment ion YL < 7%, confirmed by the %∆BPI measured for the b4 fragment (6%) and a direct measurement of the %∆BPI of the internal fragment YL (4%). Using the fragment b5 and the internal fragment ion VCG with %∆BPI equal of 10% and 31%, respectively, we conclude that deuterium incorporation is equally spread (about 10%) on the three amino acids, Val(B18), Cys(B19), and Gly(B20). Cys(B19) is, however, derivatized with NEM and, therefore, is rather close to our threshold of 8%. Hence, covalent deuterium incorporation through thiyl radical reaction appears to target predominantly Val(B18) and Gly(B20). In the fragment FVNQHLCGSHLVE, the MS/MS fragments b6 and b8 show mainly deuterium incorporation into the section LCGSHLVE. The value of the %∆BPI (22%) in the internal fragment ion NQHLCGSH covers the overall %∆BPI (20%) measured in the molecular ion. Hence, the deuterium incorporation can be limited to the section LCGSH. The similarity of


Mozziconacci et al.

SCHEME 4: Reaction Scheme for Covalent H/D Exchange at Ser(B9)

the %∆BPI for the fragments y6 and y7 indicates that incorporation of deuterium into Cys(B7) is minimal. The internal fragment ion CGSHL displays %∆BPI equal of 14%, and together with the %∆BPI of the fragments b9 and b10, we can conclude that Gly(B8) and Ser(B9) are the most probable sites for the covalent H/D exchange. Finally, we can assign a %∆BPI of 8% to Leu(B6) and 14% to Gly(B8) and Ser(B9). Correlation between the Sites of Covalent H/D Exchange and Secondary Structure. The experimental values of %∆BPI measured for each peptide fragment of insulin (Table 2) demonstrate a preferred incorporation of deuterium on the immediate neighbors of Cys and in one case Cys itself (Cys(A20)). Within insulin, incorporation of at least one deuteron (in italics) is observed in the sequences [Leu(B6)Cys(B7)-Gly(B8)-Ser(B9)] and [Val(B18)-Cys(B19)-Gly(B20)] and into [Cys(A20)]. All these sequences/amino acids are located at the beginning or the end of R-helices, which are highlighted with dashed frames in Chart 1. No covalent H/D exchange is observed for amino acids deeply buried within R-helices, for example for Cys(A6), which nevertheless clearly forms a thiyl radical as it is cleaved from Cys(A11) (see Figure 5 and Chart 1). This observation is consistent with calculations,12 which document that the RC-H bonds of amino acid residues located in R-helices have higher bond dissociation energies (BDE) compared to those in extended conformations. No H/D

exchange is also observed for the sequence Gly(B21)-Thr(B30), which forms an antiparallel β-sheet stabilized through insulin quaternary structure.35 As a consequence, RC-H bonds of these residues are not accessible to oxidants, except for Gly(B23), which is the only residue which contains a second RC-H bond. Nevertheless, our data show that Gly(B23) does not undergo H/D exchange. This could be explained by conformational restrictions of insulin, which do not allow for efficient interaction of any insulin CysS• radical with Gly(B23). We note that for H atom transfer from RC-H to CysS• the ideal geometry of the transition state would have to adopt an angle of 95° for H · · · S-R and about 180° for RC-H · · · S.20 These requirements are only achievable in more flexible random coil regions of proteins. The absence of deuterium incorporation into Gly(B23) suggests that the degrees of freedom of the protein limit the possibility for any CysS• to interact with Gly(B23). Similar geometrical constraints would rationalize that no H/D exchange is observed in Gly(A1). Protein conformation and the BDEs of R C-H bonds are strongly affected by H bonds.12,36 We, therefore, analyzed the selectivity of deuterium incorporation with respect to the localization of hydrogen bonds in insulin, using a recent molecular modeling study by Zoete et al.35 as reference. The latter was performed on porcine insulin, but human and porcine insulin are highly homologous except for a single residue, Thr(B30), in human insulin, which is replaced

Photolysis of Insulin by Ala(B30) in porcine insulin. Interestingly, it appears that hydrogen bonds between amino acid residues may affect the efficiency of covalent H/D exchange, most likely through restriction in conformational flexibility. For example, the fragment displaying the most efficient deuterium incorporation (ALYLVCGE, %∆BPI ) 32%) contains Gly(B20), which is not involved in hydrogen bonding (Chart 2). In the two other sequences/residues, which show smaller deuterium incorporation compared to the fragment ALYLVCGE, [Leu(B6), Cys(B7), Gly(B8)], and [Cys(A20)], all amino acids except Ser(B9) are involved in hydrogen bonds. Specifically, Ser(B9) contains not only a reactive RC-H bond but also an activated βC-H bond, where H/D exchange could occur via a mechanism depicted in Scheme 4. In fact, the possibility of H/D exchange with the β C-H bond was detected during earlier experiments.21 Other examples where hydrogen bonding could prevent covalent H/D exchange can be discussed based on the hydrogen-bond network presented in Chart 2. For example, Asn(A21) is adjacent to Cys(A20), located outside of any R-helix, and Gly(B23) is the only one amino acid residue within the antiparallel β-sheet susceptible to H atom abstraction and in close proximity to Cys(B19). Both residues (Asn(A21) and Gly(B23)) would be candidates for covalent H/D exchange. However, MD simulations exhibit hydrogen bonds between Asn(A21) and Gly(B23). Similarly, Ile(A10), Cys(A11), and Ser(A12) would be candidates for H/D exchange, but no significant incorporation of deuterium was observed. Again, MD simulations show that Ile(A10) and Ser(A12) are involved in hydrogen bonds with Ser(A9) and Gln(A15), respectively. In order to increase the dynamic flexibility of insulin, we carried out photoirradiation experiments in H2O and D2O at 50-55 °C, where the protein is not denatured.37 The %∆BPI within each fragment of insulin was more or less identical to the data presented for 25 °C in Table 2. Based on an activation free energy for H atom transfer between CysS• and the RC-H bond in the order of 60-70 kJ mol-1,38 and an estimated activation energy for radical-radical recombination reactions (between CysS• and RC•) of

Reversible Intramolecular Hydrogen Transfer between Protein

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