Nickel-catalyzed N-terminal Oxidative Deamination in Peptides Containing Histidine at Position 2 Coupled with Sulfite Oxidation

Abstract

Peptides containing histidine at position 2 were observed to undergo spontaneous N-terminal oxidative deamination in aqueous solution in the presence of Ni(II), sulfite, and ambient oxygen. The reaction resulted in the formation of a free carbonyl on the N-terminal α-carbon (α-ketoamide) and was catalytic with respect to nickel. This oxidative deamination was confirmed by13C NMR, 1H NMR, mass spectrometry, and chemical tests. No evidence of modification of histidine was found. It was demonstrated that the nickel-dependent N-terminal oxidative deamination also occurred in His-2 peptides using potassium peroxymonosulfate (oxone) as an oxidant. When oxone was used, oxygen was not required for the deamination to proceed. The results suggest that both nickel-catalyzed reactions (sulfite and oxygen, and oxone) produce an imine intermediate that spontaneously hydrolyzes to form the free carbonyl. These findings may provide a physiologically relevant model for oxidative carbonyl formation in vivo, as well as a useful method for producing a site-specific carbonyl on peptides and proteins. Peptides containing histidine at position 2 were observed to undergo spontaneous N-terminal oxidative deamination in aqueous solution in the presence of Ni(II), sulfite, and ambient oxygen. The reaction resulted in the formation of a free carbonyl on the N-terminal α-carbon (α-ketoamide) and was catalytic with respect to nickel. This oxidative deamination was confirmed by13C NMR, 1H NMR, mass spectrometry, and chemical tests. No evidence of modification of histidine was found. It was demonstrated that the nickel-dependent N-terminal oxidative deamination also occurred in His-2 peptides using potassium peroxymonosulfate (oxone) as an oxidant. When oxone was used, oxygen was not required for the deamination to proceed. The results suggest that both nickel-catalyzed reactions (sulfite and oxygen, and oxone) produce an imine intermediate that spontaneously hydrolyzes to form the free carbonyl. These findings may provide a physiologically relevant model for oxidative carbonyl formation in vivo, as well as a useful method for producing a site-specific carbonyl on peptides and proteins. Excessive exposures to both nickel and sulfite are known to produce serious toxic and carcinogenic effects in animals and humans (1Athar M. Hasan S.K. Srivastava R.C. Biochem. Biophys. Res. Commun. 1987; 147: 1276-1281Crossref PubMed Scopus (85) Google Scholar, 2Neta P. Huie R.E. Environ. Health Perspect. 1985; 64: 209-217Crossref PubMed Scopus (242) Google Scholar, 3Reed G.A. Curtis J.F. Mottley C. Eling T.E. Mason R.P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7499-7502Crossref PubMed Scopus (65) Google Scholar, 4Zhuang Z. Huang X. Costa M. Toxicol. Appl. Pharmacol. 1994; 126: 319-325Crossref PubMed Scopus (28) Google Scholar). The generation of potentially harmful radicals from sulfite oxidation by transition metal catalysis has been well established (5Brandt C. van Eldik R. Chem. Rev. 1995; 95: 119-190Crossref Scopus (542) Google Scholar). Nickel salts are physiologically redox-inactive, and therefore nickel toxicity in vivo is inferred to involve activation by coordination with peptides and proteins. A variety of catalytic oxidative properties of Ni(II)/peptide complexes have been reported in the literature. These include decarboxylation of the Ni(II)-Gly-Gly-His complex in the presence of molecular oxygen (6Sakurai T. Nakahara A. Inorg. Chim. Acta. 1979; 34: L243-L244Crossref Scopus (29) Google Scholar), the ability of Ni(II)-histidyl peptide complexes to function as Fenton reaction catalysts (7Inoue S. Kawanishi S. Biochem. Biophys. Res. Commun. 1989; 159: 445-451Crossref PubMed Scopus (111) Google Scholar, 8Torreilles J. Guerin M.C. Slaoui-Hasnaoui A. Free Radical Res. Commun. 1990; 11: 159-166Crossref PubMed Scopus (29) Google Scholar), and the ability of the Ni(II)-Gly-Gly-l-His complex to catalyze the oxidation and cleavage of DNA (9Chen K. Rokita S.E. Burrows C.J. J. Am. Chem. Soc. 1991; 113: 5884-5886Crossref Scopus (91) Google Scholar, 10Shullenberger D.F. Eason P.D. Long E.C. J. Am. Chem. Soc. 1993; 115: 11038-11039Crossref Scopus (52) Google Scholar) and to promote the formation of intermolecular protein cross-links (11Brown K.C. Yang S.-H. Kodadek T. Biochemistry. 1995; 34: 4733-4739Crossref PubMed Scopus (102) Google Scholar, 12Brown K.C. Yu Z. Burlingame A.L. Craik C.S. Biochemistry. 1998; 37: 4397-4406Crossref PubMed Scopus (53) Google Scholar). Muller et al. (13Muller J.G. Hickerson R.P. Perez R.J. Burrows C.J. J. Am. Chem. Soc. 1997; 119: 1501-1506Crossref Scopus (160) Google Scholar) and Lianget al. (14Liang Q. Ananias D.C. Long E.C. J. Am. Chem. Soc. 1998; 120: 248-257Crossref Scopus (78) Google Scholar) have recently reported that autooxidation of sulfite catalyzed by the Ni(II)-complexed Lys-Gly-His-amide tripeptide can oxidatively damage DNA. They postulate in situ formation of monoperoxysulfate, a strong oxidant, as an active intermediate in the damaging effect. We have recently reported on the identification of a unique Ni(II) binding site on hemoglobin that, in the presence of monoperoxysulfate, produces N-terminal oxidative deamination, as well as intramolecular cross-linking, both specific to the β-globins (15Levine J. Weickert M. Pagratis M. Etter J. Mathews A. Fattor T. Lippincott J. Apostol I. J. Biol. Chem. 1998; 273: 13037-13046Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). In the present study, we have employed small model peptides in order to verify the structural assignment of oxidative deamination, as well as to identify the minimal sequence requirements for reaction susceptibility. Additionally, we have tested a system that substitutes sulfite (SO32−) and oxygen (O2) for potassium peroxymonosulfate (oxone). 1The abbreviations oxonepotassium peroxymonosulfateAQC6-aminoquinolyl-N-hydoxysuccinimidyl carbamateDNP2,4-dinitrophenylhydrazineHPLChigh performance liquid chromatographyLC-MSHPLC interfaced directly to mass spectrometry Our findings show clearly that histidine at position 2 is a fundamental requirement for Ni(II)-catalyzed oxidative N-terminal deamination and that sulfite and ambient oxygen can readily substitute for the potent peroxyl oxidant oxone. Furthermore, by contrasting some of the differences between products produced by the sulfite/oxygen and the oxone reactions with Ni(II) peptides, we can exclude the formation of diffusible monoperoxysulfate (HSO5−) as a mediator in SO32−/O2-promoted deamination. potassium peroxymonosulfate 6-aminoquinolyl-N-hydoxysuccinimidyl carbamate 2,4-dinitrophenylhydrazine high performance liquid chromatography HPLC interfaced directly to mass spectrometry Sodium sulfite was purchased from Fluka Biochemika. Ammonium sulfite, potassium peroxymonosulfate (oxone), nickel chloride, butylated hydroxyanisole, tert-butyl alcohol, EDTA, mannitol, ethanol, sodium cyanoborohydride, sodium phosphate (as mono- and dibasic salts), and thiourea were from Sigma. The peptides bursin (KHG-amide), carnosine (β-alanyl-histidine), and homocarnosine (γ-amino butyryl-histidine) were purchased from Sigma. KGH-amide was synthesized by SynPep Corp. All other peptides were purchased from Bachem Bioscience Inc. and used without repurification. Unless otherwise noted, reaction mixtures contained 30 mm peptide and 5 mm nickel chloride, to which sodium sulfite was added from a 10× stock to a final concentration of 60 mm, all in 0.1m sodium phosphate, pH 8.2. 300-μl reactions were maintained at room temperature for 24 h under ambient air in 16 × 100-mm glass test tubes protected from light. Reactions were quenched by addition of 1: 10 (v/v) of 0.5 m EDTA, pH 8, and then diluted 1:10 into 2% formic acid in water and placed at 4 °C until analysis by reversed phase chromatography. Initial reaction mixtures contained 30 mm peptide and 30 mmnickel chloride in 0.1 m sodium phosphate, pH 8.2, to which oxone, also in 0.1 m sodium phosphate, pH 8.2, was added as a 10× stock to a final concentration of 30 mm. After approximately 5 min at room temperature, the oxone reactions were quenched by the addition of (at 1:10 (v/v)) 0.5 m EDTA, pH 8, and then diluted 1:10 into 2% formic acid in water and placed at 4 °C until analysis by reversed phase chromatography. With time, optimized conditions for the oxone reactions were established. Where noted as optimized oxone conditions, the peptides, NiCl2, and oxone stocks were made up in 0.2 msodium bicarbonate, pH 8.2. Additionally, under optimized conditions, the oxone was added in five equal increments 2 min apart, rather than as a single bolus. Peptides were subjected to gas phase hydrolysis at 165 °C for 1 h in the presence of HCl containing 1% phenol, using a Savant AP100AminoPrep Hydrolyzer. Amino acids were analyzed using precolumn derivatization with AQC as described previously (16Apostol I. Levine J. Lippincott J. Leach J. Hess E. Glascock C.B. Weickert M.J. Blackmore R. J. Biol. Chem. 1997; 272: 28980-28988Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Derivatizations were performed as described previously (15Levine J. Weickert M. Pagratis M. Etter J. Mathews A. Fattor T. Lippincott J. Apostol I. J. Biol. Chem. 1998; 273: 13037-13046Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Electrospray mass spectrometry was performed using a Finnigan Mat LCQTM ion trap mass spectrometer interfaced with an HP1090M HPLC. The LCQ™ spectrometer was calibrated as per the manufacturer's recommendations, which provide for a specified accuracy of 0.01% for m/z masses of 100–2000 Da. Peptides were separated at room temperature using a Zorbax 300SB C18 column with a gradient of 1–40% acetonitrile in 0.1% trifluoroacetic acid/water (v/v) in 20 min at 1 ml/min, following an initial 4-min isocratic (1%) hold. 1H and 13C NMR spectra were collected on a Varian VXR-300S instrument. Peptides were dissolved in either deuterium oxide or deutero-Me2SO. Chemical shifts are reported relative to DSS for deuterium oxide samples or tetramethylsilane for deutero-Me2SO samples. 1H chemical shift versus pH studies were performed using a D2O-phosphate system. No corrections of pH measurement for D2O content were made. Reaction of the tripeptide Ala-His-Ala with Ni(II) and oxone under initial standard conditions resulted in >60% modification of the peptide. The modified peptide eluted as a broad, asymmetric peak several minutes later than the parent peptide peak when analyzed by reversed phase C18 HPLC (Fig.1, A and B). The modified peptide displayed significantly increased absorption at 245 nm. Also, the modified peptide displayed a mass of 296.1 Da when analyzed by in-line mass spectrometry. This represents a discrete loss of 1 Da when compared with the parent peptide (296.1 versus297.1 Da for the parent peptide). MS2 fragmentation mapped the 1-Da loss to the A2, B2, and B3fragment ions A ions were not in either The ion which can to was to loss of the from the tripeptide J. Am. Soc. 1990; 1: Scopus Google Scholar). were confirmed by A of the histidine ion was observed m/z which that the histidine was These findings all that the 1-Da mass loss was on the the N-terminal and the histidine fragmentation parent peptide and peptide at and min as in modified peptide from oxone reaction without Ni(II) of ions to parent The modified peptide was to protein that a chemical modification occurred at the This was by a of with AQC which was used to for the presence of or Amino acid analysis of the modified peptide approximately of the of relative to histidine that was observed for the parent peptide. in to the parent peptide the modified peptide peak with a peptide with and mass of Da This represents a mass of which is of a and therefore of the presence of a free carbonyl on the modified peptide. The presence of a free the loss of free and the 1-Da mass all that the modified peptide was oxidatively at the in an peptide 1H and 13C NMR of the modified peptide peak were used to verify structural The 1H of the oxidatively peptide displayed a shift of the N-terminal from to with a from a to a with formation of a at the α-carbon of The of the histidine was observed to significantly relative to the parent peptide. an that the of pH versus 1H chemical shift of the histidine demonstrated in a of the following evidence that the histidine was not modified in the reaction 13C NMR also the presence of an histidine acid and a carbonyl at all in with oxidative deamination NMR NMR of parent in NMR of in NMR of in NMR of and in NMR of and in in a NMR NMR of in in a Sodium of the modified peptide peak of the in a that eluted in the reversed phase gradient as a of both mass of 2 Da 1 These were as from of the carbonyl. MS2 fragmentation 2 as well as 1H NMR analysis of confirmed AQC derivatization of the deamination reaction analyzed by evidence of free in the The was with the of peptide oxidatively that deamination was with the of free The oxone reaction on the tripeptide in the of added Ni(II) was It was that when Ni(II) was of the under otherwise conditions, of the peptide was The modified an increased at and eluted the reversed phase HPLC gradient as a peak later than either the parent peptide or the oxidatively 1 analysis of peak a mass of Da the parent peptide mass versus Da for the This modified peak was to to that a chemical modification occurred at the In to the oxidatively peptide modified peptide peak was with the of free carbonyl MS2 ion trap fragmentation mapped the to the A2, B2, and ions 2 we that modification was the of chemical oxidation of the to an J. and Scholar). 1H NMR analysis confirmed The N-terminal was from to and as a observed was the of a at which is with an Muller et al. (13Muller J.G. Hickerson R.P. Perez R.J. Burrows C.J. J. Am. Chem. Soc. 1997; 119: 1501-1506Crossref Scopus (160) Google Scholar) have situ formation of in the presence of ambient oxygen, Ni(II), and the tripeptide Ni(II) complexes of sequence have been reported to catalytic properties from that of histidine at position 2 (7Inoue S. Kawanishi S. Biochem. Biophys. Res. Commun. 1989; 159: 445-451Crossref PubMed Scopus (111) Google Scholar), we to test for of peroxymonosulfate formation from sulfite autooxidation on a peptide with histidine at position The tripeptide Ala-His-Ala was with sodium sulfite in the presence of nickel under the standard conditions described This was observed to produce modification of >60% of the peptide. The modified displayed the time, 1-Da mass and MS2 fragmentation by analysis as the oxidatively produced using oxone. chemical and AQC as described for the modified of using Ni(II) and oxone NMR confirmed that an oxidatively peptide was produced using Ni(II), sulfite, and ambient oxygen. The sulfite reaction with in the of added nickel was It was that in to the oxone formation was In when nickel was from the reaction of the tripeptide with sulfite, modification of was We have previously that histidine at position 2 is an of an oxidatively Ni(II) binding site on the of hemoglobin (15Levine J. Weickert M. Pagratis M. Etter J. Mathews A. Fattor T. Lippincott J. Apostol I. J. Biol. Chem. 1998; 273: 13037-13046Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). as a with the we the of the tripeptide Reaction of the tripeptide with Ni(II) and oxone under standard conditions also resulted in >60% modification of the peptide. This modified peptide eluted as a peak several minutes later than the parent peptide peak when analyzed by reversed phase C18 HPLC A The modified peptide displayed increased absorption at and a discrete mass of Da when analyzed by compared with the parent peptide versus Da for the parent This was to to MS2 fragmentation also that the modification was on the N-terminal on we that modification was an on the N-terminal When nickel was from the oxone reaction with the an was observed 5 formation to Reaction of the tripeptide with Ni(II) and sodium sulfite under standard (SO32−) conditions produced modification of This that in the of the is by that peptide in the presence of Ni(II), and ambient oxygen. at position 2 therefore to required for both to oxidative deamination, as well as the ability to a from sulfite and oxygen. sequence effects and requirements for to oxidative deamination, we reactions of Ni(II) and a of small peptides with either or oxone as the oxidant. We the reactions for evidence of modification of the parent peptide. The results of are in acid sequence effects on oxidative to intramolecular to intramolecular of of oxidative oxidative N-terminal and oxidative oxidative deamination, of oxidative in a all of the peptides with histidine at position 2 demonstrated to oxidative deamination under both of conditions, of the peptides histidine at position 2 demonstrated oxidative deamination under either It noted that in the of both and a of the from reaction at 30 was to in the form of of a mass of Da in the of peptide). These were not observed when the reaction was performed at a peptide concentration of mm, that formation was of the by 1H NMR to a of of of not These were not observed when oxone was used as the oxidant. the carnosine and homocarnosine (γ-amino butyryl-histidine) were to to deamination. These results suggest that are to of deamination. peptides formation in the reaction with oxone. Peptides with as the also produced N-terminal a is not to as to the oxidative of as other No in either reaction with was and in both a modified peptide that was demonstrated a loss of 2 Da versus Da for the parent peptide). The modified peptide was with that the was This with formation of a imine that was spontaneous Peptides and KGH-amide with a histidine in position were also conditions, peptides have been by Muller et al. (13Muller J.G. Hickerson R.P. Perez R.J. Burrows C.J. J. Am. Chem. Soc. 1997; 119: 1501-1506Crossref Scopus (160) Google Scholar) and K.C. Yu Z. Burlingame A.L. Craik C.S. Biochemistry. 1998; 37: 4397-4406Crossref PubMed Scopus (53) Google Scholar, Q. Ananias D.C. Long E.C. J. Am. Chem. Soc. 1998; 120: 248-257Crossref Scopus (78) Google Scholar, M. Burrows C. Rokita S.E. Chem. Res. Toxicol. 1997; PubMed Scopus Google Scholar) to produce radicals of DNA damage and protein No deamination of either the or the KGH-amide peptides were in reactions with The KGH-amide peptide was modified in the reaction to an a mass of with the of by an This was not In the reactions with oxone, decarboxylation of and other peptide of both peptides were of which to include deamination. the tripeptide to readily with both and the to spontaneously form an intramolecular with the of the This was confirmed by and reaction with conditions, evidence of deamination was found. Peptides with and as the N-terminal also to present to to oxidative deamination. The of of using sulfite is to formation as a for oxidative in the of was relative to with either oxidant C. Biophys. Acta. 1993; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). It is also that other chemical and properties of the of the the reaction The of deamination of the is radicals have been reported in G.A. J. 1993; PubMed Scopus Google Scholar, S. R. M.J. Biochem. J. 1997; PubMed Scopus Google Scholar). evidence of a known of oxidative of in protein cross-linking, was K.C. Yu Z. Burlingame A.L. Craik C.S. Biochemistry. 1998; 37: 4397-4406Crossref PubMed Scopus (53) Google Scholar, G.A. J. 1993; PubMed Scopus Google Scholar), and evidence of was by or to by the method of and J. Am. Chem. Soc. Scopus (65) Google Scholar). with an increased of sulfite, we of a of the which a mass of Da of standard conditions, using of sulfite the reaction resulted in a of MS2 and protein were all with decarboxylation by the of the a We that a a of potentially by the M.J. Biochem. Biophys. PubMed Scopus Google Scholar). products were with other in containing with at 1 order of The was not observed when Ni(II) was from the The reaction with produced a small of oxidative deamination. Additionally, a of other occurred formation of a In all oxone produced a of in peptides histidine at position This with a to for evidence of the generation of a diffusible peroxymonosulfate from reactions with This tested by the tripeptide in reactions with peptides and for These conditions resulted in deamination of the peptides, as was with oxone No of formation was on the tripeptide when was with the standard can inferred that the oxidative deamination reaction not of diffusible in the of all His-2 peptides, situ formation of a which is either from or in a reaction to is A nickel of the reaction was using the under standard The results that in the of oxidative deamination a Ni(II) to peptide of of the parent peptide was 4 of peptide per 1 of a catalytic of a 1:10 of and 4 of sulfite, the catalytic of Ni(II) increased to the Ni(II)/peptide not have a on the of deamination a of was as an for the standard deamination reaction with A Ni(II) using the was with oxone. In to the under initial conditions, oxidative deamination required when oxone was under optimized conditions for the oxone the oxone was added to the reaction in five equal rather than as a single bolus. In deamination was using approximately of that of Ni(II) can in the oxone reaction as well of oxone with a standard sulfite both with the at are in It can from that the sulfite reaction results in significantly products compared with oxone. It that when nickel was formation was a potentially We therefore the and with Ni(II) and oxone under standard No evidence of oxidative deamination was peptide This that formation may a and relevant reaction when Ni(II) is reaction with oxone. were to the of oxidative deamination of the the of either or oxone as can the oxidative deamination using oxone as the oxidant was (at several minutes of the reaction using and ambient oxygen displayed a initial of deamination, which to the min of No in deamination was observed h to 1 When reaction was under an at approximately 2 in the of peptide modification was This that oxygen is not under the standard conditions with when oxygen was from the sulfite reaction peptide modification was of oxygen from the oxone reaction not oxidative deamination. studies were to the of the addition of on the of oxidative modification in the reactions using either sulfite or oxone. These known and The results of as of of deamination under the standard conditions are in of oxidative deamination of by mm mm mm mm mm mm mm mm butylated in a butylated The of observed in the presence of ethanol, alcohol, or the of diffusible or radicals the oxidative deamination in either reaction P. Huie R.E. Environ. Health Perspect. 1985; 64: 209-217Crossref PubMed Scopus (242) Google Scholar, C. Mason R.P. Biochem. Biophys. PubMed Scopus Google Scholar). The of to either reaction that is that oxidative deamination in either formation of diffusible by a as in a for oxidative protein carbonyl formation by Rev. Biochem. 1993; PubMed Scopus Google Scholar). test was to was by the reaction conditions, as has been reported for sulfite of several R. J. 1995; PubMed Scopus Google Scholar). The of oxidative deamination by the metal suggest that of nickel from the peptide is the of by which the The for by thiourea is the effects of and reactions using versus using to suggest that may by the coordination of the nickel complexes J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). effects also to suggest that the complex that produces an from may from the in the reaction with (oxone). studies under standard conditions using oxone as the oxidant that approximately was to deamination of the It was also observed that resulted in of the peptide than the in with under standard conditions that significantly than 1 of to peptide was required to deamination. With of sulfite, than of the peptide was oxidatively with than of Reactions with the system using oxone or for effects of reaction the results of which are in the oxone reaction pH both reactions displayed a high relative of oxidative deamination the physiologically relevant pH of This to the that model system for oxidative peptide modification may relevant to of nickel and sulfite in vivo (1Athar M. Hasan S.K. Srivastava R.C. Biochem. Biophys. Res. Commun. 1987; 147: 1276-1281Crossref PubMed Scopus (85) Google Scholar, 3Reed G.A. Curtis J.F. Mottley C. Eling T.E. Mason R.P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7499-7502Crossref PubMed Scopus (65) Google Scholar). We have that the reaction of peptides containing histidine at position 2 with Ni(II) and oxone produces oxidative deamination of the peptides with a of other of the and we evidence of modification of the histidine to 1 of oxone, which other When Ni(II) was from the formation on the N-terminal was produced In peptides histidine at position formation was of Ni(II) was present or