ARCHIVES
OF BIOCHEMISTRY
Vol. 283, No. 1, November
AND
BIOPHYSICS
15, pp. 20-26,199O
Site-Specific Oxidation of Angiotensin I by Copper(U) and L-Ascorbate: Conversion of Histidine Residues to Z-lmidazolones Koji Uchida’
and Shunro
Kawakishi
Laboratory of Chemistry of Plant Products, Department Nagoya l%;ersity, Nagoya 464-01, Japan -
of Food Science & Technology,
Received March 5,1990, and in revised form June 26,199O
The reaction of a histidine-containing peptide (angiotensin I) with copper(II)/ascorbate under physiological conditions has been studied chemically. In the presence of a catalytic amount of copper(I1) ion, ascorbate mediated the oxidative damage to the peptide via selective loss of the histidine residue. Furthermore, the reaction of copper(II)/ascorbate with the peptide gave two products (AGT-1 and AGT-2) selectively. From amino acid analysis of the modified peptides, it was found that either of the two histidine residues within the native peptide was modified. Amino-terminal sequence analysis indicated that AGT- 1 and AGT-2 were modified at the His’ and the His’, respectively. In addition, the data of FAB-MS and ‘H NMR suggested that the unknown residues (modified histidine) within AGT-1 and AGT-2 should have the 2-imidazolone structure. In order to confirm the 2-imidazolone residue in both modified peptides, they were hydrolyzed and analyzed by reversephase HPLC. The result demonstrated that the acid hydrolysis of modified peptides gave a product which was identical to authentic 2-imidazolone residue. Consequently, it was confirmed that the reaction of Cu(II)/ ascorbate occurs specifically at the C-2 position of the imidazole ring of the histidine residue within a peptide. 0 1990 Academic Press, Inc.
Ascorbate is relatively stable in pure water, while in the presence of a catalytic amount of metal ion, it rapidly oxidizes to dehydroascorbate through an electron transfer from ascorbate to metal (l-4). The rate of reaction is known to depend on pH, catalyst, oxygen pressure, temperature, etc. Specifically, the acceleration of ascorbate autoxidation by copper(I1) ion is well known and is 1 To whom correspondence
should be addressed.
accompanied by the one-electron reduction of molecular oxygen to yield some free radical species such as superoxide (0;) and the hydroxyl radical (‘OH) (l-3). These partially reduced oxygen species have been implicated by many authors to be important causative agents of oxygen toxicity in cancer, aging, and other human diseases. These intermediates may be responsible for the action of various biological and synthetic materials as enzymes, antibiotics, carcinogens, and reducing materials under physiological conditions. Among the oxygen-derived intermediates, ‘OH is an extremely reactive species that oxidizes cellular constituents or added agents via direct addition (e.g., ring-hydroxylation), hydrogen atom abstraction, and electron transfer. Accordingly, the cytotoxicity of ascorbate in the presence of metal ions has been interpreted in terms of the generation of oxygenderived free radicals (5). It has been reported that, in the presence of a micromolar concentration of copper(I1) ion, ascorbate enhances the oxidation of various biological materials such as polysaccharides (6), proteins (5,7-lo), and DNA (ll13). In particular, it is of specific interest that active species generated by Cu(II)/ascorbate cause a site-specific modification of proteins in which copper ions are bound to proteins. The chemical nature of the modified protein is poorly understood; however, specific loss of the histidine residue, the most characteristic change in the primary structure of protein, has been demonstrated (10). Further, we have investigated the reaction of histidine residues in proteins and peptides with copper(II)/ascorbate (10, 14) and established a novel monooxygenation reaction of the imidazole ring of the histidine derivative (15-17). Such a site-specific reaction has not yet been characterized in either peptides or proteins; hence, we have attempted to detect this oxidized form of histidine residues using a peptide, angiotensin I (Asp-Arg-ValTyr-Ile-His-Pro-Phe-His-Leu), as the substrate. In
20 All
0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
SITE-SPECIFIC
OXIDATION
the course of this study, we found that Cu(II)/ascorbate caused selective damage to the histidine residues in the peptide accompanied by the formation of two products in good yields. In the present study, we have characterized the products chemically and succeeded in the detection of the 2-imidazolone residue within the modified peptides. MATERIALS
AND
METHODS
Angiotensin I acetate salt was obtained from Sigma. LMaterials. Ascorbate, CuSO,. 5Hs0, heptafluorobutyric acid, and ethylenediaminetetraacetic acid disodium salt (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. Trifluoroacetic acid was purchased from Tokyo Kasei Kogyo Co., Ltd. Other reagents were of the highest grades commercially available. The reaction Reaction of angiotensin I with copper(II)/ascorbate. was carried out at room temperature. The solution (2 ml) contained 0.5 mg angiotensin I, 5 mM ascorbate, and 0.05 mM CuSO, in 0.1 M sodium phosphate buffer (pH 7.4). The reaction was initiated by the addition of ascorbate and stopped by the addition of EDTA solution (0.05 mM). The peptide was determined by reverse-phase HPLC on a Develosil ODS-5 column (4.6 X 250 mm). The reaction mixture was applied to a column equilibrated in a solution of 0.1% trifluoroacetic acid. The peptide was eluted with a linear gradient of methanol (2.5%/ min) at a flow rate of 0.8 ml/min, the elution being monitored by absorbance at 210 nm. Areas of the chromatographic peaks of each material were calculated using a Shimadzu Chromatopac Integrator, Model CR3A. Amino acid analysis was performed with Amino acid composition. a JEOL JLC-300 amino acid analyzer equipped with a JEOL LC30DK20 data analyzing system. Sodium citrate buffers (pH 3.15, 3.20, 4.20, and 9.70) and 0.3 N NaOH were all products of Eiken Chemicals Co., Ltd. The samples for amino acid analysis were prepared as follows: the reaction mixture at each time was freeze-dried and then hydrolyzed with 6 N HCl at a concentration of 1 mg peptide/ml in U~CUO at 110°C for 20 h. The hydrolysates were concentrated, dissolved in aqueous HCl (pH 2.2), and then submitted for amino acid analysis. Similarly, the modified peptides (50 fig), AGT-1 and AGT-2, were hydrolyzed with 6 N HCl (50 ~1) in cacao at 110°C for 20 h and then submitted for amino acid analysis. Amino acids were separated on a LCR-6 column (6 X 100 mm) packed with fine particles of strong acidic cation exchanger, reacted with ninhydrin, and detected by visible absorbance at 440 and 570 nm. Amino-terminal sequence analysis. Amino-terminal sequence analysis was performed on an Applied Biosystems Model 477A gasphase protein sequencer equipped with an Applied Biosystems Model 120A phenylthiohydantoin analyzer for the on-line detection of PTH* derivatives. All chemicals and reverse-phase HPLC column used for this analysis were Applied Biosystems Products. Angiotensin I (2.0 fig in 30 ~1 aqueous HCl) and the isolated peptide samples (1.6 wg AGT-1 in 30 ~1 methanol and 2.0 /Ig AGT-2 in 30 pl methanol) were loaded onto a trifluoroacetic acid-treated fiber filter. Prior to sample application, the filter was coated with Polybrene and subjected to three cycles of Edman degradation. Anilinothiazolinone derivatives were automatically converted to PTH derivatives and injected into the on-line analyzer for identification using reverse-phase HPLC. PTH-amino acids were eluted with a stepwise gradient of 5% tetrahydrofuran in HZ0 and acetonitrile at a flow rate of 210 pl/min, programming with the Applied Biosystems gradient system. PTH-amino acids were monitored by absorbance at 270 nm. Data reduction and quantification
* Abbreviations used: FAB-MS, fast atom bombardment-mass trometry; PTH, phenylthiohydantoin.
21
OF ANGIOTENSIN were performed using a Nelson 760 interface, a Hewlett-Packard computer, and Applied Biosystems developed software.
9816
Authentic 2-imidPreparation of authentic 2-imidazolone residue. azolone residue was prepared from the acid hydrolysis of N-protected (benzoyl group) 2-imidazolone compound which was obtained through the reaction of N-benzoylhistidine with copper(II)/ascorbate (15,16). First, the N-benzoyl-2-imidazolone residue was prepared from the reaction of 50 mM N-benzoylhistidine with 50 mM L-ascorbate and 0.5 mM CuSO, in 300 ml of 0.1 M sodium phosphate buffer (pH 7.4) at room temperature. Oxygen gas was bubbled into the mixture for 48 h. Isolation of the product was performed with reverse-phase HPLC on a Lop ODS-5 column (20 X 250 mm). The reaction mixture was concentrated and applied to the column equilibrated in a solution of 25% methanol in 0.1% trifluoroacetic acid. Products were eluted at a flow rate of 6 ml/min, the elution being monitored by absorbance at 230 nm. Finally, we obtained 35.7 mg of authentic N-benzoyl-2-imidazolone residue. The chemical structure of N-benzoyl-2-imidazolone residue has been convincingly established by FAB-MS, ‘H NMR, i3C NMR (15,16) and tandem mass spectrometry.s Subsequently, authentic N-benzoyl-2-imidazolone residue was hydrolyzed with 6 N HCl in uucuo at 110°C for 20 h. HPLC analysis of the hydrolyzed sample revealed the formation of single product from N-benzoyl-2-imidazolone residue. The product was purified with reverse-phase HPLC on a Develosil ODS-5 column (4.6 X 250 mm). The sample was applied to a column equilibrated in a solution of 5% methanol in 0.1% heptafluorobutyric acid and eluted at a flow rate of 0.8 ml/ min, the elution being monitored by absorbance at 210 nm. Chemical characterization of the product was performed by ‘H NMR, i3C NMR, and FAB-MS. Spectral data of this residue were as follows: ‘H NMR (DMSO-de) 6 2.96 (2H, m, CH2), 4.12 (lH, m, CH
OF BIOCHEMISTRY
Vol. 283, No. 1, November
AND
BIOPHYSICS
15, pp. 20-26,199O
Site-Specific Oxidation of Angiotensin I by Copper(U) and L-Ascorbate: Conversion of Histidine Residues to Z-lmidazolones Koji Uchida’
and Shunro
Kawakishi
Laboratory of Chemistry of Plant Products, Department Nagoya l%;ersity, Nagoya 464-01, Japan -
of Food Science & Technology,
Received March 5,1990, and in revised form June 26,199O
The reaction of a histidine-containing peptide (angiotensin I) with copper(II)/ascorbate under physiological conditions has been studied chemically. In the presence of a catalytic amount of copper(I1) ion, ascorbate mediated the oxidative damage to the peptide via selective loss of the histidine residue. Furthermore, the reaction of copper(II)/ascorbate with the peptide gave two products (AGT-1 and AGT-2) selectively. From amino acid analysis of the modified peptides, it was found that either of the two histidine residues within the native peptide was modified. Amino-terminal sequence analysis indicated that AGT- 1 and AGT-2 were modified at the His’ and the His’, respectively. In addition, the data of FAB-MS and ‘H NMR suggested that the unknown residues (modified histidine) within AGT-1 and AGT-2 should have the 2-imidazolone structure. In order to confirm the 2-imidazolone residue in both modified peptides, they were hydrolyzed and analyzed by reversephase HPLC. The result demonstrated that the acid hydrolysis of modified peptides gave a product which was identical to authentic 2-imidazolone residue. Consequently, it was confirmed that the reaction of Cu(II)/ ascorbate occurs specifically at the C-2 position of the imidazole ring of the histidine residue within a peptide. 0 1990 Academic Press, Inc.
Ascorbate is relatively stable in pure water, while in the presence of a catalytic amount of metal ion, it rapidly oxidizes to dehydroascorbate through an electron transfer from ascorbate to metal (l-4). The rate of reaction is known to depend on pH, catalyst, oxygen pressure, temperature, etc. Specifically, the acceleration of ascorbate autoxidation by copper(I1) ion is well known and is 1 To whom correspondence
should be addressed.
accompanied by the one-electron reduction of molecular oxygen to yield some free radical species such as superoxide (0;) and the hydroxyl radical (‘OH) (l-3). These partially reduced oxygen species have been implicated by many authors to be important causative agents of oxygen toxicity in cancer, aging, and other human diseases. These intermediates may be responsible for the action of various biological and synthetic materials as enzymes, antibiotics, carcinogens, and reducing materials under physiological conditions. Among the oxygen-derived intermediates, ‘OH is an extremely reactive species that oxidizes cellular constituents or added agents via direct addition (e.g., ring-hydroxylation), hydrogen atom abstraction, and electron transfer. Accordingly, the cytotoxicity of ascorbate in the presence of metal ions has been interpreted in terms of the generation of oxygenderived free radicals (5). It has been reported that, in the presence of a micromolar concentration of copper(I1) ion, ascorbate enhances the oxidation of various biological materials such as polysaccharides (6), proteins (5,7-lo), and DNA (ll13). In particular, it is of specific interest that active species generated by Cu(II)/ascorbate cause a site-specific modification of proteins in which copper ions are bound to proteins. The chemical nature of the modified protein is poorly understood; however, specific loss of the histidine residue, the most characteristic change in the primary structure of protein, has been demonstrated (10). Further, we have investigated the reaction of histidine residues in proteins and peptides with copper(II)/ascorbate (10, 14) and established a novel monooxygenation reaction of the imidazole ring of the histidine derivative (15-17). Such a site-specific reaction has not yet been characterized in either peptides or proteins; hence, we have attempted to detect this oxidized form of histidine residues using a peptide, angiotensin I (Asp-Arg-ValTyr-Ile-His-Pro-Phe-His-Leu), as the substrate. In
20 All
0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
SITE-SPECIFIC
OXIDATION
the course of this study, we found that Cu(II)/ascorbate caused selective damage to the histidine residues in the peptide accompanied by the formation of two products in good yields. In the present study, we have characterized the products chemically and succeeded in the detection of the 2-imidazolone residue within the modified peptides. MATERIALS
AND
METHODS
Angiotensin I acetate salt was obtained from Sigma. LMaterials. Ascorbate, CuSO,. 5Hs0, heptafluorobutyric acid, and ethylenediaminetetraacetic acid disodium salt (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. Trifluoroacetic acid was purchased from Tokyo Kasei Kogyo Co., Ltd. Other reagents were of the highest grades commercially available. The reaction Reaction of angiotensin I with copper(II)/ascorbate. was carried out at room temperature. The solution (2 ml) contained 0.5 mg angiotensin I, 5 mM ascorbate, and 0.05 mM CuSO, in 0.1 M sodium phosphate buffer (pH 7.4). The reaction was initiated by the addition of ascorbate and stopped by the addition of EDTA solution (0.05 mM). The peptide was determined by reverse-phase HPLC on a Develosil ODS-5 column (4.6 X 250 mm). The reaction mixture was applied to a column equilibrated in a solution of 0.1% trifluoroacetic acid. The peptide was eluted with a linear gradient of methanol (2.5%/ min) at a flow rate of 0.8 ml/min, the elution being monitored by absorbance at 210 nm. Areas of the chromatographic peaks of each material were calculated using a Shimadzu Chromatopac Integrator, Model CR3A. Amino acid analysis was performed with Amino acid composition. a JEOL JLC-300 amino acid analyzer equipped with a JEOL LC30DK20 data analyzing system. Sodium citrate buffers (pH 3.15, 3.20, 4.20, and 9.70) and 0.3 N NaOH were all products of Eiken Chemicals Co., Ltd. The samples for amino acid analysis were prepared as follows: the reaction mixture at each time was freeze-dried and then hydrolyzed with 6 N HCl at a concentration of 1 mg peptide/ml in U~CUO at 110°C for 20 h. The hydrolysates were concentrated, dissolved in aqueous HCl (pH 2.2), and then submitted for amino acid analysis. Similarly, the modified peptides (50 fig), AGT-1 and AGT-2, were hydrolyzed with 6 N HCl (50 ~1) in cacao at 110°C for 20 h and then submitted for amino acid analysis. Amino acids were separated on a LCR-6 column (6 X 100 mm) packed with fine particles of strong acidic cation exchanger, reacted with ninhydrin, and detected by visible absorbance at 440 and 570 nm. Amino-terminal sequence analysis. Amino-terminal sequence analysis was performed on an Applied Biosystems Model 477A gasphase protein sequencer equipped with an Applied Biosystems Model 120A phenylthiohydantoin analyzer for the on-line detection of PTH* derivatives. All chemicals and reverse-phase HPLC column used for this analysis were Applied Biosystems Products. Angiotensin I (2.0 fig in 30 ~1 aqueous HCl) and the isolated peptide samples (1.6 wg AGT-1 in 30 ~1 methanol and 2.0 /Ig AGT-2 in 30 pl methanol) were loaded onto a trifluoroacetic acid-treated fiber filter. Prior to sample application, the filter was coated with Polybrene and subjected to three cycles of Edman degradation. Anilinothiazolinone derivatives were automatically converted to PTH derivatives and injected into the on-line analyzer for identification using reverse-phase HPLC. PTH-amino acids were eluted with a stepwise gradient of 5% tetrahydrofuran in HZ0 and acetonitrile at a flow rate of 210 pl/min, programming with the Applied Biosystems gradient system. PTH-amino acids were monitored by absorbance at 270 nm. Data reduction and quantification
* Abbreviations used: FAB-MS, fast atom bombardment-mass trometry; PTH, phenylthiohydantoin.
21
OF ANGIOTENSIN were performed using a Nelson 760 interface, a Hewlett-Packard computer, and Applied Biosystems developed software.
9816
Authentic 2-imidPreparation of authentic 2-imidazolone residue. azolone residue was prepared from the acid hydrolysis of N-protected (benzoyl group) 2-imidazolone compound which was obtained through the reaction of N-benzoylhistidine with copper(II)/ascorbate (15,16). First, the N-benzoyl-2-imidazolone residue was prepared from the reaction of 50 mM N-benzoylhistidine with 50 mM L-ascorbate and 0.5 mM CuSO, in 300 ml of 0.1 M sodium phosphate buffer (pH 7.4) at room temperature. Oxygen gas was bubbled into the mixture for 48 h. Isolation of the product was performed with reverse-phase HPLC on a Lop ODS-5 column (20 X 250 mm). The reaction mixture was concentrated and applied to the column equilibrated in a solution of 25% methanol in 0.1% trifluoroacetic acid. Products were eluted at a flow rate of 6 ml/min, the elution being monitored by absorbance at 230 nm. Finally, we obtained 35.7 mg of authentic N-benzoyl-2-imidazolone residue. The chemical structure of N-benzoyl-2-imidazolone residue has been convincingly established by FAB-MS, ‘H NMR, i3C NMR (15,16) and tandem mass spectrometry.s Subsequently, authentic N-benzoyl-2-imidazolone residue was hydrolyzed with 6 N HCl in uucuo at 110°C for 20 h. HPLC analysis of the hydrolyzed sample revealed the formation of single product from N-benzoyl-2-imidazolone residue. The product was purified with reverse-phase HPLC on a Develosil ODS-5 column (4.6 X 250 mm). The sample was applied to a column equilibrated in a solution of 5% methanol in 0.1% heptafluorobutyric acid and eluted at a flow rate of 0.8 ml/ min, the elution being monitored by absorbance at 210 nm. Chemical characterization of the product was performed by ‘H NMR, i3C NMR, and FAB-MS. Spectral data of this residue were as follows: ‘H NMR (DMSO-de) 6 2.96 (2H, m, CH2), 4.12 (lH, m, CH
