- Email: [email protected]
Selective oxidation of Zn2+—insulin catalyzed by Cu2+
Selective Oxidation of Zn2þ—Insulin Catalyzed by Cu2þ ¨ NEICH VIKRAM SADINENI, CHRISTIAN SCHO Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047
Received 19 May 2006; revised 9 October 2006; accepted 27 October 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20834
ABSTRACT: The purpose of this study is to quantitate the sensitivity of Zn2þ-insulin to oxidation catalyzed by various redox active transition metals, Cu2þ, Fe2þ, Mn2þ, Ni2þ, Co2þ, Cr3þ. Human recombinant insulin (INS) was subjected to oxidation under various conditions in the presence and absence of Zn2þ and ascorbate. The extent of oxidation was monitored by RP-HPLC. Only Cu2þ, but none of the other metals or combination thereof, for example, Ni2þ/Co2þ, Co2þ/Cr3þ, and Ni2þ/Cr3þ, catalyzed INS oxidation, for example, to an extent of 45% when 20 mM INS/8.8 mM Zn2þ were exposed to 8 mM Cu2þ and 50 mM ascorbate for 90 min. The Cu2þ-catalyzed oxidation mainly targeted the B chain of INS, where the two histidine residues are located. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:1844–1847, 2007
Keywords:
formulation; stability; proteins; oxidation; mass spectrometry
INTRODUCTION Redox active transition metals are present in most buffers and are found to affect the processing and/or storage of biologic pharmaceuticals. In the presence of suitable reducing agents (prooxidants, example: ascorbate), these transition metals are able to activate O2 to reactive oxygen species (ROS, e.g., hydroxy radical, superoxide anion radical) which readily attack protein molecules. This proxidant/metal/O2 induced oxidation, called metal-catalyzed oxidation (MCO)1,2 poses a serious threat to the stability of recombinant proteins. Many amino acids are susceptible to MCO within a protein sequence, such as cysteine, methionine, tyrosine, lysine, proline, arginine, threonine, and histidine.3 Usually, MCO occurs site specifically, where amino acids ligating the redox active transition metal are targets for oxidation. Correspondence to: Christian Scho¨neich (Telephone: 785864-4880; Fax: 785-864-5736; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 1844–1847 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
1844
We have recently reported that insulin (INS) is specifically sensitive to oxidation catalyzed4 by Cu2þ even in the presence of high concentrations of Zn2þ. This process converts histidine to 2oxo-histidine, likely through a hydroxyl radicaldependent mechanism, as outlined in Scheme 1. In this scheme, the second electron transfer (reaction 2) is written as a direct reaction between the intermediary a-aminoalkyl type radical and Cu2þ because of the known propensity of a-amino alkyl radicals to reduce transition metals.5 However, a multistep mechanism, involving the reduction of oxygen, followed by electron transfer between superoxide and Cu2þ may work as well. The pronounced sensitivity of INS towards Cu2þ-dependent oxidation raised the question, whether other redox-active transition metals known to bind INS,6 or combinations thereof, such as Ni2þ/Co2þ, Co2þ/Cr3þ, and Ni2þ/Cr3þ, would catalyze oxidation as well, and to what extent. It is known that certain combinations of transition metals show a significantly higher catalytic activity compared to the individual metals alone; for example, Ni2þ and Co2þ are able to accelerate the autoxidation of Cu2þ/tetraglycine.7,8 This note
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 7, JULY 2007
SELECTIVE OXIDATION OF Zn2þ INSULIN
1845
Scheme 1. Hydroxyl radical OH. induced oxidation of histidine to 2-oxo-histidine.
will show that specifically Cu2þ but not Fe2þ, Ni2þ, Co2þ, Cr3þ, or Mn2þ catalyze the oxidation of INS even when Zn2þ of the original formulation was removed by dialysis.
EXPERIMENTAL Materials Recombinant human INS, with a content of Zn2þ that is 0.5% of the weight of amorphous dry INS, corresponding to a molar ratio of 2:1 of INS: Zn2þ (hereafter represented as Zn:INS), was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA), and, unless otherwise stated, all other reagents were of highest grade commercially available. To ensure adequate solubility, stock solutions of INS were prepared in 0.1 N HCl. Concentrations of INS were determined spectrophotometrically (e275nm ¼ 5988 M1).9 All the stock solutions were prepared immediately prior to experimentation. Oxidation Conditions and Preparation of Insulin for Analysis
the concentrations and the order in which the respective reagents were added into the reaction mixture, with a 10-min interval between the addition of the redox active metal and ascorbic acid, to allow for binding of metal to Zn:INS. The reaction was monitored by RP-HPLC, taking samples at regular intervals after stopping the oxidation with EDTA (1 mM) to chelate the residual redox active metal. To ensure an adequate supply of oxygen, the reaction was carried out in 1.5 mL reaction vials. Experimental controls were performed in the absence of ascorbate. Zn:INS was then reduced and alkylated to
Table 1. Preparation of Oxidized Samples of Insulin Compound
Conclusion
Zn:INS or INS Buffer (pH 7.4) Redox active metal Ascorbic acid EDTA Guanidine HCl Dithiothreitol Iodoacetic acid b-mercaptoethanol
20 mM 20 mM 8/80 mM 50/100 mM 1 mM 6M 3 mM 6 mM 0.14 M
Reactions were performed in 20 mM sodium phosphate buffer, prepared from acid and base conjugates (pH 7.4) at room temperature and consisted of 500 mL solutions. Table 1 shows
Preparation of oxidized, reduced, and alkylated samples of Zn:INS and INS.
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 7, JULY 2007
1846
¨ NEICH SADINENI AND SCHO
Table 2. Oxidative Degradation of Zn:INS and INS under Various Conditions Outlined in Experimental Section Protein (20 mM)
Cu2þ
Zn:INS; INS Zn:INS; INS Zn:INS; INS Zn:INS; INS Zn:INS; INS
8 mM
Fe2þ
a
Ni2þ, Cr3þ, Co2þ, Mn2þ
Ascorbate
% Unoxidized (90 min)
8 mM 80 mM
50 mM 50/100 mM 50/100 mM 50/100 mM 50/100 mM
45%; 25% 100% 100% 100% 100%
8 mM 80 mM
a
This column represents the redox-active metals added individually into the reaction mixture.
quantify the oxidation levels of the individual A and B chains of INS. Dithiothreitol (DTT) at a final concentration of 3 mM was used to reduce the disulfide bonds after denaturation with 6 M guanidine hydrochloride. The free thiols were alkylated with iodoacetic acid (6 mM) for an hour in the dark and excess iodoacetic acid was reacted with 0.14 M b-mercaptoethanol for 10 min. For some experiments, Zn2þ was removed by dialysis before oxidation. In this case Zn:INS was dialyzed exhaustively in 20 mM sodium phosphate buffer (pH 7.4) with 5 mM EDTA for 12 h at 48C, with buffer changes every 4 h to obtain Zn2þ-free INS, confirmed using the PAR assay (PAR ¼ 4-(2Pyridylazo) resorcinol disodium salt).10 HPLC Analysis Reaction mixtures were fractionated by RP-HPLC on a Vydac C4 column (250 4.6 mm i.d.; Vydac; Hesperia, CA) utilizing a linear acetonitrile/ trifluoroacetic acid gradient with UV detection at 214 nm and a flow rate of 1 mL/min. The mobile phase ratio started with 100% mobile phase A (25% v/v acetonitrile/0.1% v/v trifluoroacetic acid) at 0 min and was increased linearly to 100% B (50% v/v acetonitrile/0.1% v/v trifluoroacetic acid)
within 12.5 min. The HPLC system consisted of two Shimadzu LC-10AS pumps and a Shimadzu SPD-10AV UV/Vis detector (Shimadzu; Columbia, MD). Data are reported as the ratio of the average peak area of the analyte at time ‘‘x’’ to the average peak area of analyte at time ‘‘0’’ (Equation 1). Fraction of insulin unoxidized ¼ peak areat¼x =peak areat¼0
ð1Þ
RESULTS AND DISCUSSION Oxidative Sensitivity of Insulin: Loss of B Chain The results obtained by exposure of 20 mm Zn:INS or INS to various concentrations of different transition metals are outlined in Table 2. Under the experimental conditions, low levels (8 mM) of Cu2þ caused significant oxidation of Zn:INS, evident through the extensive loss of chain B in the chromatogram after 90 min (Fig. 1). There was no quantifiable loss in the A chain of Zn:INS, confirming our previous result 4 that the oxidation is specific to His 5 and His.10 Experiments performed with far UV-CD spectroscopy also
Figure 1. Degradation of B chain of Zn:INS in the presence of Cu2þ/ascorbic acid at 90 min. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 7, JULY 2007
DOI 10.1002/jps
SELECTIVE OXIDATION OF Zn2þ INSULIN
suggested that there was no change in secondary structure, where there is no visible difference between the spectra of Zn:INS in the presence and absence of metals. This suggests that the loss of B-chain is not due to conformational changes of Zn:INS in the presence of metals. Under similar conditions none of the other metals caused any significant loss of either chain A or B, even after 150 min reaction time. This result is rather surprising, especially for Fe2þ, which, is a well-documented catalyst of metal-induced protein oxidation, for example, b-amyloid peptides, a-synuclein, and prion protein during various pathological conditions such as Alzheimer’s disease,11,12 Parkinson’s disease13 and spongiform encephalopathies,14 respectively. There could be several reasons for the oxidative insensitivity of Zn:INS and INS towards these transition metals: (1) coordination of these metals with insulin could be such that the ROS generated do not react with the peptide; (2) the redox potentials of the respective metal:protein complexes are not favorable to initiate oxidation at these specific sites; (3) inadequate accessibility of the metal:protein complexes to ascorbate and oxygen and hence insignificant generation of ROS. The oxidative stability of Zn:INS was also studied with Cu2þ in the presence of peroxide instead of ascorbic acid and it was observed that there was no significant degradation of INS. The stability of Zn:INS was also studied in the presence of various metal combinations and ascorbic acid. Zn:INS was observed to be completely stable under these conditions.
CONCLUSION Our results suggest that among the common redox-active transition metals, Cu2þ is the only catalytically active metal able to induce INS oxidation.
REFERENCES 1. Stadtman ER. 1990. Metal ion-catalyzed oxidation of proteins: Biochemical mechanism and biological consequences. Free Radic Biol Med 9:315–325.
DOI 10.1002/jps
1847
2. Stadtman ER, Berlett BS. 1998. Reactive oxygenmediated protein oxidation in aging and disease. Drug Metab Rev 30:225–243. 3. Stadtman ER. 1993. Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu Rev Biochem 62:797–821. 4. Hovorka SW, Biesiada H, Williams TD, Huhmer A, Scho¨neich C. 2002. High sensitivity of Zn(II) insulin to metal-catalyzed oxidation: Detection of 2-oxo-histidine by tandem mass spectrometry. Pharm Res 19:530–537. 5. Hiller KO, Asmus KD. 1983. Formation and reduction reactions of a-amino radicals derived from methionine and its derivatives in aqueous solutions. J Phys Chem 87:3682–3688. 6. Kadima W. 1999. Role of metal ions in the T- to Rallosteric transition in the insulin hexamer. Biochemistry 38:13443–13452. 7. Alipazaga MV, Moreno RGM, Coichev N. 2004. Synergistic effect of Ni(II) and Co(II) ions on the sulfite induced autoxidation of Cu(II)/tetraglycine complex. Dalton T 2036–2040. 8. Green Brandon J, Tesfai Teweldemedhin M, Xie Y, Margerum Dale W. 2004. Oxidative selfdecomposition of the nickel(III) complex of glycylglycyl-L-histidylglycine. Inorg Chem 43:1463– 1471. 9. Fasman GD. 1989. Practical handbook of biochemistry and molecular biology. Boca Raton: CRC Press, Inc. 10. Miller RT, Martasek P, Raman CS, Masters BS. 1999. Zinc content of Escherichia coli-expressed constitutive isoforms of nitric-oxide synthase. Enzymatic activity and effect of pterin. J Biol Chem 274:14537–14540. 11. Castellani RJ, Honda K, Zhu X, Cash AD, Nunomura A, Perry G, Smith MA. 2004. Contribution of redox-active iron and copper to oxidative damage in Alzheimer disease. Ageing Res Rev 3: 319–326. 12. Mattson MP. 2004. Metal-catalyzed disruption of membrane protein and lipid signaling in the pathogenesis of neurodegenerative disorders. Ann NY Acad Sci 1012:37–50. 13. Cole NB, Murphy DD, Lebowitz J, Di Noto L, Levine RL, Nussbaum RL. 2005. Metal-catalyzed oxidation of a´-synuclein: Helping to define the relationship between oligomers, protofibrils and filaments. J Biol Chem 280:9678–9690. 14. Kim N-H, Choi J-K, Jeong B-H, Kim J-I, Kwon M-S, Carp RI, Kim Y-S. 2005. Effect of transition metals (Mn, Cu, Fe) and deoxycholic acid (DA) on the conversion of PrPC to PrPres. Faseb J 19:783–785.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 7, JULY 2007
Received 19 May 2006; revised 9 October 2006; accepted 27 October 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20834
ABSTRACT: The purpose of this study is to quantitate the sensitivity of Zn2þ-insulin to oxidation catalyzed by various redox active transition metals, Cu2þ, Fe2þ, Mn2þ, Ni2þ, Co2þ, Cr3þ. Human recombinant insulin (INS) was subjected to oxidation under various conditions in the presence and absence of Zn2þ and ascorbate. The extent of oxidation was monitored by RP-HPLC. Only Cu2þ, but none of the other metals or combination thereof, for example, Ni2þ/Co2þ, Co2þ/Cr3þ, and Ni2þ/Cr3þ, catalyzed INS oxidation, for example, to an extent of 45% when 20 mM INS/8.8 mM Zn2þ were exposed to 8 mM Cu2þ and 50 mM ascorbate for 90 min. The Cu2þ-catalyzed oxidation mainly targeted the B chain of INS, where the two histidine residues are located. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:1844–1847, 2007
Keywords:
formulation; stability; proteins; oxidation; mass spectrometry
INTRODUCTION Redox active transition metals are present in most buffers and are found to affect the processing and/or storage of biologic pharmaceuticals. In the presence of suitable reducing agents (prooxidants, example: ascorbate), these transition metals are able to activate O2 to reactive oxygen species (ROS, e.g., hydroxy radical, superoxide anion radical) which readily attack protein molecules. This proxidant/metal/O2 induced oxidation, called metal-catalyzed oxidation (MCO)1,2 poses a serious threat to the stability of recombinant proteins. Many amino acids are susceptible to MCO within a protein sequence, such as cysteine, methionine, tyrosine, lysine, proline, arginine, threonine, and histidine.3 Usually, MCO occurs site specifically, where amino acids ligating the redox active transition metal are targets for oxidation. Correspondence to: Christian Scho¨neich (Telephone: 785864-4880; Fax: 785-864-5736; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 1844–1847 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
1844
We have recently reported that insulin (INS) is specifically sensitive to oxidation catalyzed4 by Cu2þ even in the presence of high concentrations of Zn2þ. This process converts histidine to 2oxo-histidine, likely through a hydroxyl radicaldependent mechanism, as outlined in Scheme 1. In this scheme, the second electron transfer (reaction 2) is written as a direct reaction between the intermediary a-aminoalkyl type radical and Cu2þ because of the known propensity of a-amino alkyl radicals to reduce transition metals.5 However, a multistep mechanism, involving the reduction of oxygen, followed by electron transfer between superoxide and Cu2þ may work as well. The pronounced sensitivity of INS towards Cu2þ-dependent oxidation raised the question, whether other redox-active transition metals known to bind INS,6 or combinations thereof, such as Ni2þ/Co2þ, Co2þ/Cr3þ, and Ni2þ/Cr3þ, would catalyze oxidation as well, and to what extent. It is known that certain combinations of transition metals show a significantly higher catalytic activity compared to the individual metals alone; for example, Ni2þ and Co2þ are able to accelerate the autoxidation of Cu2þ/tetraglycine.7,8 This note
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 7, JULY 2007
SELECTIVE OXIDATION OF Zn2þ INSULIN
1845
Scheme 1. Hydroxyl radical OH. induced oxidation of histidine to 2-oxo-histidine.
will show that specifically Cu2þ but not Fe2þ, Ni2þ, Co2þ, Cr3þ, or Mn2þ catalyze the oxidation of INS even when Zn2þ of the original formulation was removed by dialysis.
EXPERIMENTAL Materials Recombinant human INS, with a content of Zn2þ that is 0.5% of the weight of amorphous dry INS, corresponding to a molar ratio of 2:1 of INS: Zn2þ (hereafter represented as Zn:INS), was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA), and, unless otherwise stated, all other reagents were of highest grade commercially available. To ensure adequate solubility, stock solutions of INS were prepared in 0.1 N HCl. Concentrations of INS were determined spectrophotometrically (e275nm ¼ 5988 M1).9 All the stock solutions were prepared immediately prior to experimentation. Oxidation Conditions and Preparation of Insulin for Analysis
the concentrations and the order in which the respective reagents were added into the reaction mixture, with a 10-min interval between the addition of the redox active metal and ascorbic acid, to allow for binding of metal to Zn:INS. The reaction was monitored by RP-HPLC, taking samples at regular intervals after stopping the oxidation with EDTA (1 mM) to chelate the residual redox active metal. To ensure an adequate supply of oxygen, the reaction was carried out in 1.5 mL reaction vials. Experimental controls were performed in the absence of ascorbate. Zn:INS was then reduced and alkylated to
Table 1. Preparation of Oxidized Samples of Insulin Compound
Conclusion
Zn:INS or INS Buffer (pH 7.4) Redox active metal Ascorbic acid EDTA Guanidine HCl Dithiothreitol Iodoacetic acid b-mercaptoethanol
20 mM 20 mM 8/80 mM 50/100 mM 1 mM 6M 3 mM 6 mM 0.14 M
Reactions were performed in 20 mM sodium phosphate buffer, prepared from acid and base conjugates (pH 7.4) at room temperature and consisted of 500 mL solutions. Table 1 shows
Preparation of oxidized, reduced, and alkylated samples of Zn:INS and INS.
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 7, JULY 2007
1846
¨ NEICH SADINENI AND SCHO
Table 2. Oxidative Degradation of Zn:INS and INS under Various Conditions Outlined in Experimental Section Protein (20 mM)
Cu2þ
Zn:INS; INS Zn:INS; INS Zn:INS; INS Zn:INS; INS Zn:INS; INS
8 mM
Fe2þ
a
Ni2þ, Cr3þ, Co2þ, Mn2þ
Ascorbate
% Unoxidized (90 min)
8 mM 80 mM
50 mM 50/100 mM 50/100 mM 50/100 mM 50/100 mM
45%; 25% 100% 100% 100% 100%
8 mM 80 mM
a
This column represents the redox-active metals added individually into the reaction mixture.
quantify the oxidation levels of the individual A and B chains of INS. Dithiothreitol (DTT) at a final concentration of 3 mM was used to reduce the disulfide bonds after denaturation with 6 M guanidine hydrochloride. The free thiols were alkylated with iodoacetic acid (6 mM) for an hour in the dark and excess iodoacetic acid was reacted with 0.14 M b-mercaptoethanol for 10 min. For some experiments, Zn2þ was removed by dialysis before oxidation. In this case Zn:INS was dialyzed exhaustively in 20 mM sodium phosphate buffer (pH 7.4) with 5 mM EDTA for 12 h at 48C, with buffer changes every 4 h to obtain Zn2þ-free INS, confirmed using the PAR assay (PAR ¼ 4-(2Pyridylazo) resorcinol disodium salt).10 HPLC Analysis Reaction mixtures were fractionated by RP-HPLC on a Vydac C4 column (250 4.6 mm i.d.; Vydac; Hesperia, CA) utilizing a linear acetonitrile/ trifluoroacetic acid gradient with UV detection at 214 nm and a flow rate of 1 mL/min. The mobile phase ratio started with 100% mobile phase A (25% v/v acetonitrile/0.1% v/v trifluoroacetic acid) at 0 min and was increased linearly to 100% B (50% v/v acetonitrile/0.1% v/v trifluoroacetic acid)
within 12.5 min. The HPLC system consisted of two Shimadzu LC-10AS pumps and a Shimadzu SPD-10AV UV/Vis detector (Shimadzu; Columbia, MD). Data are reported as the ratio of the average peak area of the analyte at time ‘‘x’’ to the average peak area of analyte at time ‘‘0’’ (Equation 1). Fraction of insulin unoxidized ¼ peak areat¼x =peak areat¼0
ð1Þ
RESULTS AND DISCUSSION Oxidative Sensitivity of Insulin: Loss of B Chain The results obtained by exposure of 20 mm Zn:INS or INS to various concentrations of different transition metals are outlined in Table 2. Under the experimental conditions, low levels (8 mM) of Cu2þ caused significant oxidation of Zn:INS, evident through the extensive loss of chain B in the chromatogram after 90 min (Fig. 1). There was no quantifiable loss in the A chain of Zn:INS, confirming our previous result 4 that the oxidation is specific to His 5 and His.10 Experiments performed with far UV-CD spectroscopy also
Figure 1. Degradation of B chain of Zn:INS in the presence of Cu2þ/ascorbic acid at 90 min. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 7, JULY 2007
DOI 10.1002/jps
SELECTIVE OXIDATION OF Zn2þ INSULIN
suggested that there was no change in secondary structure, where there is no visible difference between the spectra of Zn:INS in the presence and absence of metals. This suggests that the loss of B-chain is not due to conformational changes of Zn:INS in the presence of metals. Under similar conditions none of the other metals caused any significant loss of either chain A or B, even after 150 min reaction time. This result is rather surprising, especially for Fe2þ, which, is a well-documented catalyst of metal-induced protein oxidation, for example, b-amyloid peptides, a-synuclein, and prion protein during various pathological conditions such as Alzheimer’s disease,11,12 Parkinson’s disease13 and spongiform encephalopathies,14 respectively. There could be several reasons for the oxidative insensitivity of Zn:INS and INS towards these transition metals: (1) coordination of these metals with insulin could be such that the ROS generated do not react with the peptide; (2) the redox potentials of the respective metal:protein complexes are not favorable to initiate oxidation at these specific sites; (3) inadequate accessibility of the metal:protein complexes to ascorbate and oxygen and hence insignificant generation of ROS. The oxidative stability of Zn:INS was also studied with Cu2þ in the presence of peroxide instead of ascorbic acid and it was observed that there was no significant degradation of INS. The stability of Zn:INS was also studied in the presence of various metal combinations and ascorbic acid. Zn:INS was observed to be completely stable under these conditions.
CONCLUSION Our results suggest that among the common redox-active transition metals, Cu2þ is the only catalytically active metal able to induce INS oxidation.
REFERENCES 1. Stadtman ER. 1990. Metal ion-catalyzed oxidation of proteins: Biochemical mechanism and biological consequences. Free Radic Biol Med 9:315–325.
DOI 10.1002/jps
1847
2. Stadtman ER, Berlett BS. 1998. Reactive oxygenmediated protein oxidation in aging and disease. Drug Metab Rev 30:225–243. 3. Stadtman ER. 1993. Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu Rev Biochem 62:797–821. 4. Hovorka SW, Biesiada H, Williams TD, Huhmer A, Scho¨neich C. 2002. High sensitivity of Zn(II) insulin to metal-catalyzed oxidation: Detection of 2-oxo-histidine by tandem mass spectrometry. Pharm Res 19:530–537. 5. Hiller KO, Asmus KD. 1983. Formation and reduction reactions of a-amino radicals derived from methionine and its derivatives in aqueous solutions. J Phys Chem 87:3682–3688. 6. Kadima W. 1999. Role of metal ions in the T- to Rallosteric transition in the insulin hexamer. Biochemistry 38:13443–13452. 7. Alipazaga MV, Moreno RGM, Coichev N. 2004. Synergistic effect of Ni(II) and Co(II) ions on the sulfite induced autoxidation of Cu(II)/tetraglycine complex. Dalton T 2036–2040. 8. Green Brandon J, Tesfai Teweldemedhin M, Xie Y, Margerum Dale W. 2004. Oxidative selfdecomposition of the nickel(III) complex of glycylglycyl-L-histidylglycine. Inorg Chem 43:1463– 1471. 9. Fasman GD. 1989. Practical handbook of biochemistry and molecular biology. Boca Raton: CRC Press, Inc. 10. Miller RT, Martasek P, Raman CS, Masters BS. 1999. Zinc content of Escherichia coli-expressed constitutive isoforms of nitric-oxide synthase. Enzymatic activity and effect of pterin. J Biol Chem 274:14537–14540. 11. Castellani RJ, Honda K, Zhu X, Cash AD, Nunomura A, Perry G, Smith MA. 2004. Contribution of redox-active iron and copper to oxidative damage in Alzheimer disease. Ageing Res Rev 3: 319–326. 12. Mattson MP. 2004. Metal-catalyzed disruption of membrane protein and lipid signaling in the pathogenesis of neurodegenerative disorders. Ann NY Acad Sci 1012:37–50. 13. Cole NB, Murphy DD, Lebowitz J, Di Noto L, Levine RL, Nussbaum RL. 2005. Metal-catalyzed oxidation of a´-synuclein: Helping to define the relationship between oligomers, protofibrils and filaments. J Biol Chem 280:9678–9690. 14. Kim N-H, Choi J-K, Jeong B-H, Kim J-I, Kwon M-S, Carp RI, Kim Y-S. 2005. Effect of transition metals (Mn, Cu, Fe) and deoxycholic acid (DA) on the conversion of PrPC to PrPres. Faseb J 19:783–785.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 7, JULY 2007