©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Free Radicals Generated during the Glycation Reaction of Amino Acids by Methylglyoxal
A MODEL STUDY OF PROTEIN-CROSS-LINKED FREE RADICALS (*)

(Received for publication, July 13, 1995; and in revised form, September 27, 1995)

Hyung-Soon Yim (1) (2) Sa-Ouk Kang (2) Yung-Chil Hah (2) P. Boon Chock (1) Moon B. Yim (1)(§)

From the  (1)Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and the (2)Department of Microbiology, College of Natural Sciences and the Research Center for Molecular Microbiology, Seoul National University, Seoul 151-742, Korea

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The formation of alpha-dicarbonyl compounds seems to be an important step for cross-linking proteins in the glycation or Maillard reaction. To elucidate the mechanism for the cross-linking reaction, we studied the reaction between a three-carbon alpha-dicarbonyl compound, methylglyoxal, and amino acids. Our results showed that this reaction generated yellow fluorescent products as formed in some glycated proteins. In addition, three types of free radical species were also produced, and their structures were determined by EPR spectroscopy. These free radicals are 1) the cross-linked radical cation, 2) the methylglyoxal radical anion as the counterion, and 3) the superoxide radical anion produced only in the presence of oxygen. The generation of the cross-linked radical cations and the methylglyoxal radical anions does not require metal ions or oxygens. These results indicate that dicarbonyl compounds cross-link free amino groups of protein by forming Schiff bases, which donate electrons directly to dicarbonyl compounds to form the cross-linked radical cations and the methylglyoxal radical anions. Oxygen can accept an electron from the radical anion to generate a superoxide radical anion, which can initiate damaging chain reactions. Time course studies suggest that the cross-linked radical cation is a precursor of yellow fluorescent glycation end products.


INTRODUCTION

Free amino groups of proteins react slowly with reducing sugars such as glucose by the glycation or Maillard reaction to form poorly characterized brown fluorescent compounds(1) . This process is initiated by the condensation reaction of reducing sugars with free amino groups to form Schiff bases (Fig. 1A), which undergo rearrangement to form the relatively stable Amadori products(2, 3) . The Amadori products subsequently degrade into alpha-dicarbonyl compounds, deoxyglucosones(4, 5) . These compounds are more reactive than the parent sugars with respect to their ability to react with amino groups of proteins to form cross-links, stable end products called advanced Maillard products or advanced glycation end products (AGEs)(^1)(6) . AGEs are irreversibly formed and found to accumulate with aging, atherosclerosis, and diabetes mellitus, especially associated with long-lived proteins such as collagens(7, 8) , lens crystallines(9, 10) , and nerve proteins(11, 12) . It was suggested that the formation of AGEs not only modifies protein properties, but also induces biological damage in vivo(13, 14, 15, 16, 17, 18) . For example, AGEs deposited in the arterial wall could themselves generate free radicals capable of oxidizing vascular wall lipids and accelerate atherogenesis in hyperglycemic diabetic patients(17, 18) .


Figure 1: A, general scheme of the glycation or Maillard reaction; B, several advanced glycation end products or Maillard products: pentosidine (structure a), pyrrole derivatives (structure b), and pyrazine derivatives (structure c). CML, N-carboxymethyllysine.



The molecular structures of some AGEs have been identified as pentosidines (Fig. 1B, structure a)(19, 20, 21, 22) , pyrrole derivatives (structure b)(23) , pyrazine derivatives (structure c)(24, 25, 26, 27) , and N-carboxymethyllysine (Fig. 1A, CML)(28, 29, 30, 31, 32) . In the presence of molecular oxygen, the formation of these products from sugars is catalyzed by transition metal ions via glycoxidation, which oxidizes Amadori products to N-carboxymethyllysine (28, 29) , and the autoxidation of glucose, which produces superoxide radical anions (O(2)), H(2)O(2), and alpha-ketoaldehydes (33, 34, 35, 36, 37) . The major pathways of glycation reaction-mediated damage to macromolecules therefore involve both nonoxidative and oxidative processes. Their individual contributions to biological damage, however, are not well understood.

The formation of alpha-dicarbonyl compounds seems to be an essential step for the cross-linking reaction, which leads to the formation of AGEs. To elucidate the mechanism for the cross-linking reaction (Fig. 1A, reaction 5), we studied the reaction between three-carbon alpha-dicarbonyl methylglyoxals and L-alanines. Our results showed that the yellow fluorescent products, formed in some glycated proteins, were generated by this reaction. In addition, we found that three types of free radical species were generated, and their structures were identified by EPR spectroscopy and other methods. These radicals are a cross-linked radical cation (the methylglyoxal dialkylimine radical cation or its protonated cation), the methylglyoxal radical anion, and the superoxide radical anion (which formed in the presence of oxygen molecules). The generation of the cross-linked radical cation and the methylglyoxal radical anion does not require metal ions or oxygens, suggesting that they are formed by a direct 1-electron transfer process.


EXPERIMENTAL PROCEDURES

Materials

Methylglyoxal, NaCNBH(3), EDTA, L-alanine, diethylenetriaminepentaacetic acid (DTPA), and catalase from bovine liver were purchased from Sigma. Cu,Zn-superoxide dismutase from bovine erythrocytes was obtained from Boehringer Mannheim, nitro blue tetrazolium (NBT) was from Calbiochem, and Chelex 100 resin (sodium form) was from Bio-Rad. Stable isotope-enriched alanines (N-, 1-C-, 2-C-, and 3-C-labeled), D(4)-alanines, and D(2)O were purchased from Cambridge Isotopes. Methylglyoxal was distilled twice before use. All solutions were treated with Chelex 100 to remove traces of transition metal ions.

Methods

EPR spectra were recorded on a Bruker ESP 300 spectrometer with a TM resonator operating at 9.79 GHz with 100-kHz magnetic field modulation. An aqueous flat cell (-150 µl) was used for the experiments. The reactions were started by injecting methylglyoxal into the samples initially containing 0.2 M alanine and 0.2 M methylglyoxal in 0.5 M carbonate buffer at pH 9.5 or in 0.1 M phosphate buffer at pH 7.5. The instrumental settings for spectral acquisition were, unless otherwise indicated, as follows: temperature, 25 °C; microwave power, 20 milliwatts; modulation amplitude, 1 G; conversion time, 10.24 ms; time constant, 82 ms; and sweep width, 100 G with 4096-point resolution.

The generation of O(2) in the aerobic reaction mixture was determined by NBT reduction and inhibition of its reduction by superoxide dismutase(18, 38, 39) . A 1-ml reaction mixture, which initially contained equal concentrations of methylglyoxal and alanine, was added at 30 s after initiation of the reaction to a 2-ml aliquot of 0.25 mM NBT in 100 mM carbonate buffer (pH 9.5). The reduction rate was determined as the increase in absorbance at 540 nm for 10 min at 30 °C. Electronic absorption and fluorescence spectra were obtained with a diode array spectrometer (Hewlett-Packard 8452A) and an LS-100 fluorescence spectrometer (Photon Technology International), respectively.


RESULTS

EPR Spectra of the Cross-linked Free Radical Cation

The first derivative EPR spectrum shown in Fig. 2A (upper spectrum) was obtained with the anaerobic reaction mixture of methylglyoxal and natural-abundance L-alanine in carbonate buffer at pH 9.5. A similar spectrum also appeared in phosphate buffer at pH 7.5 with a much slower rate and a weaker signal amplitude. For the assignment of hyperfine coupling constants (hfc) and structural identification of this radical, similar experiments were carried out in various isotope-enriched reaction mixtures. The upper spectrum in Fig. 2B, obtained from the reaction mixture prepared with L-[N]alanine, exhibits a different hyperfine splitting pattern compared with the spectrum in Fig. 2A. This alteration is entirely caused by changes of nitrogen nuclear spins, N (I = 1/2) in place of ^14N (I = 1), and their nuclear moments, µ(N)/µ(^14N) = 1.40. With this information, the experimental spectra in Fig. 2(A and B, lower spectra) were simulated. The simulation in the spectrum in Fig. 2A required two magnetically nonequivalent nitrogens (A


Figure 2: EPR spectra obtained from the reaction mixture containing methylglyoxal (0.2 M) and various isotope-enriched L-alanines (0.2 M) in carbonate buffer (0.5 M) at pH 9.5 (upper spectra) and simulated spectra (lower spectra). A, methylglyoxal (MG) and natural-abundance L-alanine; B, methylglyoxal and L-[N]alanine; C, methylglyoxal and L-[2-C]alanine. The hyperfine coupling constants used for the simulation are listed under ``Results.'' The spectrometer settings for the spectral acquisition are described under ``Experimental Procedures.''



The spectrum in Fig. 2C and the spectra in Fig. 3(B and C) were obtained with the reaction mixture containing L-[2-C]alanine, L-[1-C]alanine, and L-[3-C]alanine in place of natural-abundance alanine, respectively. They exhibited extra hyperfine interactions due to C (I = 1/2) nuclei: two 2-C (alpha-carbons) with A


Figure 3: EPR spectra obtained from the reaction mixture containing methylglyoxal (0.2 M) and various isotope-enriched L-alanines (0.2 M) in carbonate buffer (0.5 M) at pH 9.5 (upper spectra) and simulated spectra (lower spectra). A, methylglyoxal (MG) and natural-abundance L-alanine; B, methylglyoxal and L-[1-C]alanine; C, methylglyoxal and L-[3-C]alanine. The hyperfine coupling constants used for the simulation are listed under ``Results.'' The spectrometer settings for the spectral acquisition are described under ``Experimental Procedures.''



Although we have obtained a large number of hfc constants from this cross-linked radical, exact structural assignment for this radical is difficult because of asymmetry in hfc constants. Two equivocal structures are shown in Fig. 4(structures a-1 and a-2). In the case of structure a-1, the asymmetric nature of the spin distribution may be caused by the effect of the methyl group of methylglyoxal on the singly occupied molecular orbital, most likely the -orbital, which includes two N=C bonds. On the basis of experimental hfc constants, structure a-1` is assigned to the conformations of two alkyl groups with respect to the p-orbitals of the two nitrogen atoms. In this conformation, carboxyl, alpha-, and methyl carbons will have one small and one large C hfc constant in each carbon group because of the different dihedral angles to the p-orbitals of the nitrogen atoms (one carbon is close to = 90°, and the other is closer to 0°) and the cos^2 dependence of these hfc constants. The assignment of C hfc constants to individual carbons will be as follows: for the N-1 side, 8.52 G for 1-C, 4.10 G for 2-C, and 0.3 G for 3-C; and for the N-2 side, 0.3 G for 1-C, 0.2 G for 2-C, and 3.0 G for 3-C. This assignment gives a ratio of 3.7 for the total carbon spin densities between the N-1 and N-2 sides. This value is closest, among several possible assignments, to the value of 3.2, the ratio of spin densities on N-1 and N-2 atoms (A/A). The other possible structure of this radical is shown in Fig. 4(structure a-2). A protonation of a nitrogen in the cross-linked Schiff base will produce a triene-type compound, which may lose an electron to form the cross-linked radical cation. In this structure, the observed large C hfc constants will originate entirely from one alanine molecule in the cross-linked radical. In addition, we expect to detect two sets of methyl hydrogen hfc constants if the radical has this structure, in contrast to the experimental observation of only one set of methyl hydrogen hfc constants. It may be possible, however, that A^H(3) of one set is smaller than the line width, which may arise from the canceling effects of spin delocalization (hyperconjugation) and spin polarization in the spin transfer to the s-orbitals of the methyl hydrogens from the delocalized -center. Although we prefer structure a-1 as the structure of the cross-linked radical, structure a-2 cannot be ruled out at this time. It is certain, however, that the radical formed due to the cross-linking reaction contains two amino acids and one methylglyoxal.


Figure 4: Chemical structures of the cross-linked radical cation and the radical anion observed in this reaction and identified by EPR.



To find whether the Schiff base is the precursor of this radical, the base was reduced with NaCNBH(3), which is known to reduce Schiff bases selectively and to inhibit the subsequent reactions. When NaCNBH(3) (1.0 M) was added to the reaction mixture, the EPR signal of the cross-linked radical and the yellow color were not detected. The effect of NaCNBH(3) may indicate that methylglyoxal dialkylimine, O(2)C(CH(3))- HCN=C(CH(3))HC=NCH(CH(3))CO(2), is the intermediate for the formation of this cross-linked radical.

Observation of the Enediol Radical Anion of Methylglyoxal

Spectra B and C in Fig. 5were obtained from the reaction mixture containing natural-abundance methylglyoxal and isotope-enriched 2,3,3,3-D(4)-alanine in carbonate buffer prepared in D(2)O. These spectra were compared with spectrum A, which is the identical spectrum shown in Fig. 2A. Spectrum B, recorded at 5 min, exhibited an additional set of resonance lines (marked by asterisks), indicating the presence of another radical species. These resonance lines were not resolved in spectrum A because the signal amplitude of the cross-linked radical was much higher in spectrum A. This species is relatively more stable than the cross-linked radical as shown in spectrum C, which was obtained at 15 min. The simulation of this five-line spectrum (spectrum D) required three magnetically equivalent hydrogens with A^H(3) = 8.73 G and one hydrogen with A^H = 9.81 G and g = 2.0043. These EPR parameters are identical to those of the cis-form of the methylglyoxal radical anion (see Fig. 4, structure b) reported previously(40, 41) . This result suggests that the cross-linked radical observed in this reaction is a cation formed with the methylglyoxal radical anion being the radical counterion.


Figure 5: EPR spectra obtained from the reaction mixture containing methylglyoxal (0.2 M) and various isotope-enriched L-alanines (0.2 M) in carbonate buffer (0.5 M) at pH 9.5. Spectrum A, spectrum of methylglyoxal and natural-abundance L-alanine recorded at 5 min after starting the reaction. Spectrum B, spectrum of methylglyoxal and 2,3,3,3-D(4)-alanine in carbonate buffer solution prepared in D(2)O. The spectrum was recorded at 5 min after initiating the reaction. The resonance lines marked by asterisks belong to another species as explained under ``Results.'' Spectrum C, spectrum recorded at 15 min with the same sample used for spectrum B. Spectrum D, simulated spectrum obtained by using the hyperfine coupling constants listed under ``Results.''



Effects of Molecular Oxygens and Metal Ions

Fig. 6A shows the effects of molecular oxygen on the amplitude of the EPR signal of the cross-linked radical cation. The rate of radical cation formation was faster and reached a higher amplitude under anaerobic conditions than under aerobic conditions. Similar results were also obtained for the formation of methylglyoxal radical anions (data not shown). The decay rate of the cation was also faster under anaerobic conditions. These results indicate that oxygen molecules are not required for the generation of the radical cations and anions. However, one cannot rule out that molecular oxygen may be involved in subsequent reactions. The effect of metal ions on the reaction was also studied. The time courses (Fig. 6B) showed that the addition of either FeCl(2) or a chelating agent (DTPA) did not exert any significant effects on the formation of the cross-linked radical cations. It demonstrates that transition metal ions also are not required for the formation of the radical cations.


Figure 6: Effects of oxygen (A) and transition metal ions (B) on the amplitude of the EPR signal of the cross-linked radical cation. All samples contain 0.2 M methylglyoxal and 0.2 ML-alanine in 0.5 M carbonate buffer at pH 9.5. Additional treatments were as follows: A, aerobic conditions (circle) and anaerobic conditions (bullet); B, no addition (circle), 0.1 mM FeCl(2) (bullet), and 1 mM DTPA (). Reactions were started by injecting methylglyoxal. arb. unit, arbitrary unit.



These results together indicate that the cross-linked radical cation and the methylglyoxal radical anion are generated from the direct electron transfer between methylglyoxal (MG) and a Schiff base, probably methylglyoxal dialkylimine (MGDI) or its protonated species, as shown in .

On-line formulae not verified for accuracy

Previous investigations of the reactions of methylglyoxal with protein or methylamine also suggested that a condensation product, which was not well characterized, served as the electron donor and that methylglyoxal acted as an electron acceptor (41, 42, 43) .

Generation of Superoxide Radical Anions

Several reports have shown that glycated proteins generate superoxide radical anions, which initiate lipid peroxidation(13, 14, 15, 16, 17, 18) . This finding was thought to be a possible mechanism for accelerated atherogenesis in diabetes(17, 18) . To examine whether O(2) was also generated in this model reaction, we performed NBT reduction experiments by adding varying concentrations of the reaction mixture. Fig. 7A shows that the reduction rates of NBT measured at 540 nm were increased with the addition of an increasing concentration of reactants in aerobic solutions. This reduction was inhibited by the addition of Cu,Zn-superoxide dismutase in the reaction mixture (Fig. 7B). Catalase did not inhibit, but rather somewhat enhanced the reduction of NBT (data not shown), probably by preventing reoxidation of reduced NBT by removing H(2)O(2). When the glycation products obtained under anaerobic conditions were used for titration, the reduction of NBT began as soon as the solution in the cuvette was exposed to air.


Figure 7: Generation of the superoxide radical anion in the methylglyoxal and alanine reaction. A, reduction rates of NBT were measured by increasing concentrations of reaction products. The reaction products (1 ml) were added to 2 ml of 0.25 mM NBT in 100 mM carbonate buffer at pH 9.5. The absorbance changes were monitored at 540 nm for 10 min at 30 °C. B, the effect of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) on the reduction rate of NBT is shown. The concentrations of methylglyoxal and alanine were both 40 mM.



These results demonstrate that although the initiation of the cross-linking reaction does not require molecular oxygen, the reaction products generate superoxide radical anions in the presence of oxygen. The methylglyoxal radical anion is most likely responsible for O(2) generation by its electron transfer reaction to oxygen via .

On-line formulae not verified for accuracy

This result is consistent with the observation that the EPR signal amplitude of the methylglyoxal anion was higher under anaerobic conditions than under aerobic conditions.

Color Formation Versus Cross-linked Radical Cation Formation

Fig. 8A shows electronic absorption spectra as a function of time (3-min intervals) obtained from the reaction mixture containing 40 mM alanine and 40 mM methylglyoxal in 0.5 M carbonate buffer at pH 9.5. The reaction was initiated by the addition of methylglyoxal. The absorbance at 285 and 334 nm increased with time (marked by arrows). The time course of the absorbance at 334 nm (Fig. 8B, curve a) was compared with that of the EPR signal, which monitored the cross-linked radical cation (curve b). The EPR signal indicated that the cross-linked radical cations were formed rapidly and subsequently converted to other species or decayed away. However, the color formation monitored by the absorbance at 334 nm was relatively slow and increased continuously. These results suggest that the cross-linked radical cation is the precursor of the yellow end products. These products also exhibit fluorescence around 385 nm when excited at 334 nm (data not shown).


Figure 8: Optical absorbance and cross-linked radical cation formation. A: the reaction mixtures contained 40 mM alanine and 40 mM methylglyoxal in 0.5 M carbonate buffer at pH 9.5. The spectra were recorded at 3-min intervals. The arrows indicate the direction of absorbance change with time. B: curve a, the absorbances at 334 nm were obtained with diluents of 0.2 M methylglyoxal and 0.2 M alanine in 0.5 M carbonate buffer at pH 9.5. A 10-µl portion of reaction mixture was diluted with 1 ml of 3 M HCl. Curve b, the signal amplitude of EPR spectra is plotted. The EPR sample contained 0.2 M methylglyoxal and 0.2 M alanine in 0.5 M carbonate buffer at pH 9.5. arb. unit, arbitrary unit.




DISCUSSION

The late stage of the glycation reaction between deoxyglucosones and free amino groups of proteins shown in Fig. 1A (step 5) was studied using a model system of methylglyoxal and L-alanine. The results are consistent with the reaction scheme shown in Fig. 9. We detected three free radical species: the cross-linked methylglyoxal dialkylimine radical cation ( Fig. 9(structure a) and 4 (structure a-1)) or its protonated species (Fig. 4, structure a-2), the enediol radical anion of methylglyoxal (Fig. 9, structure b), and the superoxide radical anion (Fig. 9, structure c). The addition of NaCNBH(3) inhibited the formation of the cross-linked radical and the yellow color. This result indicates that the formation of the Schiff bases is an essential step (Fig. 9, steps i and ii) for cross-linking. Furthermore, transition metal ions and oxygen were not required for the generation of the cross-linked radical cations or the methylglyoxal radical anions (Fig. 6, A and B). These results together suggest that a direct 1-electron transfer between a Schiff base methylglyoxal dialkylimine (or its protonated form) and methylglyoxal is responsible for the generation of the cross-linked radical cation and the radical counteranion of methylglyoxal (Fig. 9, step iii). Under aerobic conditions, molecular oxygen can then accept an electron from the methylglyoxal anion to generate the superoxide radical anion (Fig. 9, step iv). The time course studies (Fig. 8) indicate that the cross-linked radical cations are precursors of the yellow end products.


Figure 9: Partial reaction scheme between methylglyoxal (MG) and amino acids (AA).



Previous investigations have shown that N,N`-dialkylpyrazine radical cations are formed by the addition of ascorbate in the reaction mixture containing glucose and amine or amino acid in water at 80 °C(24, 25, 26, 27) . The intermediate for the free radical was glyoxal dialkylimine, which was formed immediately after glycosylamine formation, prior to Amadori rearrangement. It was proposed that the formation of the dialkylpyrazine radical cation from the dialkylimine was via the reverse aldol condensation reaction, which requires acid hydrolysis followed by reduction(26, 27) . In the absence of reducing agents, two-carbon and three-carbon fragments in the form of glyoxal and methylglyoxal were formed from glucose, depending on where the fragmentation occurred(26) . It appears, on the basis of our results, that methylglyoxal, once formed, is able to act as a reducing agent as well as a cross-linker under mild conditions. In addition, the structure of the cross-linked radical cation is different from that obtained with glyoxal.

Glycation is believed to be modulated by oxidative stress. Wolff and co-workers (34, 35, 36, 37) demonstrated that reducing sugars can undergo oxidation in the presence of oxygen and transition metal ions, which generates H(2)O(2), oxygen radicals, and alpha-ketoaldehydes. This reaction leads to protein browning, conformational changes, and fragmentation. In addition, the formation of N-carboxymethyllysine (Fig. 1, step 4) and pentosidine also requires transition metal ions and oxygen(22, 28, 29) . Therefore, AGEs in vivo are products of the combined processes of glycation and oxidative modification. Our results also suggest that the formation of alpha-ketoaldehydes or deoxyglucosones is a critical step that leads to protein cross-linking, formation of radical cation sites on the cross-linked proteins, and generation of radical counteranions. O(2) and H(2)O(2) generated from radical counteranions can initiate free radical chain reactions including lipid peroxidation. The cross-linked radical cations, which have an extensively delocalized unpaired electron, are quite stable. These radical sites in cross-linked proteins may be more persistent and could be a reactive site for putative reducing (A) and oxidizing (B) molecules, which produce free radicals for a long duration. One of many possible reaction schemes could be as shown in .

On-line formulae not verified for accuracy

This kind of reaction by long-lived glycated protein may contribute to the increased peroxidation of lipids when glycated protein was added in vitro and may also contribute to accelerating oxidative modification of vascular wall lipid in diabetes and atherosclerosis.


FOOTNOTES

*
This work was supported in part by the International Collaborative Research Program of NHLBI, National Institutes of Health and by the Korea Science and Engineering Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Lab. of Biochemistry, NHLBI, NIH, Bldg. 3, Rm. 202, Bethesda, MD 20892-0340. Tel.: 301-496-9494; Fax: 301-496-0599.

(^1)
The abbreviations used are: AGEs, advanced glycation end products; DTPA, diethylenetriaminepentaacetic acid; NBT, nitro blue tetrazolium; G, gauss; hfc, hyperfine coupling.


ACKNOWLEDGEMENTS

We (M. B. Y. and P. B. C.) thank Dr. Earl R. Stadtman for introducing us to the glycation reaction.


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