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Methylglyoxal-induced modifications of hemoglobin: Structural and functional characteristics
Archives of Biochemistry and Biophysics 529 (2013) 99–104
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Methylglyoxal-induced modifications of hemoglobin: Structural and functional characteristics Tania Bose 1, Abhishek Bhattacherjee, Sauradipta Banerjee, Abhay Sankar Chakraborti ⇑ Department of Biophysics, Molecular Biology & Bioinformatics, University of Calcutta 92, Acharya Prafulla Chandra Road, Kolkata 700 009, India
a r t i c l e
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Article history: Received 2 August 2012 and in revised form 16 November 2012 Available online 8 December 2012 Keywords: Hemoglobin Methylglyoxal Diabetes mellitus Advanced glycation end products Hydroimidazolone Oxidative stress
a b s t r a c t Methylglyoxal (MG) reacts with proteins to form advanced glycation end products (AGEs). Although hemoglobin modification by MG is known, the modified protein is not yet characterized. We have studied the nature of AGE formed by MG on human hemoglobin (HbA0) and its effect on structure and function of the protein. After reaction of HbA0 with MG, the modified protein (MG-Hb) was separated and its properties were compared with those of the unmodified protein HbA0. As shown by MALDI-mass spectrometry, MG converted Arg-92a and Arg-104b to hydroimidazolones in MG-Hb. Compared to HbA0, MG-Hb exhibited decreased absorbance around 280 nm, reduced tryptophan fluorescence (excitation 285 nm) and increased a-helix content. However, MG modification did not change the quaternary structure of the heme protein. MG-Hb appeared to be more thermolabile than HbA0. The modified protein was found to be more effective than HbA0 in H2O2-mediated iron release and oxidative damages involving Fenton reaction. MG-Hb exhibited less peroxidase activity and more esterase activity than HbA0. MG-induced structural and functional changes of hemoglobin may enhance oxidative stress and associated complications, particularly in diabetes mellitus with increased level of MG. Ó 2012 Elsevier Inc. All rights reserved.
Introduction Post-translational modifications of proteins play important roles in controlling their functions. Protein modification by glucose (glycation) is a complex series of parallel and sequential reactions collectively called the Maillard reaction. The Schiff base formation, followed by Amadori rearrangement and advanced glycation end products (AGEs)2 formation are the key steps of protein glycation [1]. The nonenzymatic modification of proteins may be significant with increased level of blood glucose over prolonged periods of time in diabetes mellitus. Protein glycation reactions leading to AGEs are thought to be the root causes of different diabetic complications, including oxidative stress [2]. Both in vitro and in vivo studies from our laboratory have shown that glucose or fructose causes hemoglo⇑ Corresponding author. Fax: +91 33 2351 9755. E-mail address: [email protected] (A.S. Chakraborti). Present address: Stowers Institute for Medical research, Kansas City, MO 64110, USA. 2 Abbreviations used: AGEs, advanced glycation end products; CHCA, a-cyano hydroxy cinnamic acid; CID, collision-induced dissociation; DFO, desferrioxamine; DSC, differential scanning calorimetry; HbA0, human nonglycated hemoglobin; HbA1a1/HbA1a2/HbA1b/HbA1c, human glycated hemoglobins; MALDI-TOF, matrix assisted laser desorption ionization-time of flight; MG, methylglyoxal; MG-Hb, methylglyoxal-modified HbA0; MG-H1, Nd-(5-hydro-5 methyl-4-imidazolon-2-yl)ornithin; PAGE, polyacrylamide gel electrophoresis; PB, phosphate buffer (50 mM) pH 6.6; p-NPA, p-nitrophenyl acetate; ROS, reactive oxygen species. 1
0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2012.12.001
bin modification by Maillard reaction and promotes free radicalmediated oxidative reactions [3–7]. These findings are significant, because diabetes mellitus causes oxidative stress, which, in a vicious cycle, further aggravates the disease condition. The reactive a-oxoaldehydes namely, glyoxal, methylglyoxal and 3-deoxyglucosone are known to initiate Maillard-like reactions, and are more reactive than the parent hexose sugars with respect to their ability of protein modification and AGE formation [8,9]. Methylglyoxal (MG) level increases in different pathological conditions including diabetes mellitus [10,11], leading to formation of AGEs with different long-lived proteins, namely collagen [12], human serum albumin [9,12], a-crystallin [13], heat shock protein [14], insulin [15], etc. MG forms argpyrimidine, hydroimidazolones and tetrahydropyrimidine with arginine residues. On the other hand, carboxyethyllysine and MG-lysine dimer are the AGEs formed with lysine residues. Proteins susceptible to MG modification with related functional impairment are called the ‘‘dicarbonyl proteome’’ [16], which are predominantly modified on arginine residues with formation of dominant arginine adduct Nd-(5-hydro-5 methyl-4-imidazolon-2-yl)-ornithin (MG-H1). Kalapos has discussed MG-toxicity associated with free radical generation by its metabolism and modification of biological macromolecules [17]. Although hemoglobin glycation is well known and has been studied in details, there have been only two reports on its
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modification by MG. In a brief report, Chen et al. have shown that MG interacts with hemoglobin with modifications of arginine residues forming MG-H1 [18]. Gao and Wang have reported that the sites and extents of MG modification of arginine residues are correlated with solvent accessibility of these residues [19]. The findings are quite significant. However, MG-induced effects on the structure and function of the heme protein have not been reported. Considering the increased level of MG in diabetic condition as well as its very high reactivity (about 20,000 times more reactive than glucose), its interaction with hemoglobin should be studied in more detail. We have, therefore, undertaken the present study to separate MG-modified hemoglobin, to find the sites of modification by MG, to characterize the adduct formed, and to compare its structural and functional properties with those of normal protein for understanding any adverse effect associated with the modification.
Materials and methods Materials Methylglyoxal (MG), Sephadex G-100, Sephadex G-25, ferrozine, glyoxalase I (yeast), glyoxalase II (bovine liver), o-dianisidine, agarose, acrylamide, Coomasie R250, ethidium bromide, pnitrophenyl acetate (p-NPA), desferrioxamine (DFO), and a-cyano hydroxy cinnamic acid (CHCA) were purchased from Sigma Chemical Company, USA. Bio-Rex-70 resin (200–400 mesh) was obtained from Bio-Rad, India. All other reagents were AR grade and purchased locally. MG was distilled under low pressure and its concentration in stock solution was measured by end point enzymatic assay involving conversion to S-D-lactoylglutathione with glyoxalase I and hydrolysis catalyzed by glyoxalase II [20].
Separation of nonglycated hemoglobin (HbA0) Human blood samples were collected from healthy volunteers (non-smokers) aged 20–25 years. Ethical principles formulated by the Institutional Ethics Committee were followed in carrying out the study. Hemoglobin (total) was isolated and purified from blood sample by using Sephadex G-100 column chromatography [21]. Hemoglobin sample in 50 mM phosphate buffer, pH 6.6 (PB) containing 0.15 M NaCl was applied to a cation exchange column containing BioRex-70 resin (20 1.5 cm) pre-equilibrated with PB. Different glycated hemoglobin species (HbA1a1, HbA1a2, HbA1b and HbA1c) and nonglycated hemoglobin (HbA0) were separated by using gradually increased concentration of NaCl in elution buffer (PB) following the method of Cohen and Wu [22] with some modifications described before [4]. HbA0 fraction in PB was collected after removal of NaCl by passing through Sephadex G-100 column, and its concentration was determined from Soret absorbance using an extinction coefficient (e415nm) of 125 mM 1 cm 1 (heme basis) [23].
Polyacrylamide gel electrophoresis (PAGE) MG-Hb and HbA0 were subjected to native PAGE (10%) for 3 h at constant voltage. Freshly prepared HbA0 was also included in the study. 20 ll of each sample (20 lM) was loaded. After electrophoresis, the gel was stained with Coomasie R250. Matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometric study After native PAGE of MG-Hb and HbA0, the bands were excised and subjected to reduction, alkylation and digestion with sequencing-grade trypsin in gel. After digestion at 37 °C for 16 h, the supernatants containing the peptide mixtures of MG-Hb and HbA0 were collected separately by centrifugation. The samples (0.5 ll each) were applied directly to the MALDI plate, mixed with 0.5 ll saturated CHCA solution (prepared in 50% acetonitrile and 0.1% trifluoroacetic acid) and allowed to crystallize. Mass spectra were recorded in a 4800 Proteomics Analyzer (MALDI-TOF/TOF mass spectrometer, Applied Biosystems). The location and chemical nature of modified amino acid residues were identified from MALDITOF data. MS/MS experiments were performed by using collision-induced dissociation (CID) technique for sequence information of the selected peptides of interest. Spectroscopic studies Different spectroscopic studies were done with isolated fractions MG-Hb and HbA0. Absorption spectra (250–650 nm) of the samples (12 lM each) were recorded in a UV–VIS spectrophotometer (Hitachi U2000) using 1 ml quartz cuvette of path length 1 cm. Fluorescence emission spectra (with excitation at 285 nm) of the protein fractions (6 lM) were monitored (300–400 nm) in a spectrofluorimeter (Hitachi F-3010) using 3 ml quartz cuvette of path length 1 cm. Circular dichroic (CD) measurements (200–250 nm, 250–350 nm and 400–600 nm) of MG-Hb and HbA0 fractions (3 lM each) were done in a spectropolarimeter (Jasco 600) using 1 mm path length cuvette. Differential scanning calorimetric (DSC) study DSC measurements were done in a Calorimetric Scientific Corporation instrument (N-DSCII). The protein fractions MG-Hb and HbA0 (750 ll, 40 lM) were degassed under vacuum at room temperature and loaded to the calorimeter. DSC scans were recorded from 25 °C to 85 °C at a heating rate of 2 °C/min. Estimation of free iron H2O2-induced iron release from separated fractions of HbA0 and MG-Hb was estimated, for which protein samples (250 ll and 40 lM) were incubated with varying concentrations of H2O2 (0, 0.25, 0.50, 0.75, and 1.0 mM) at 37 °C for 1 h. After protein precipitation with TCA, the supernatants were used to estimate free iron by ferrozine reaction [24], as described before [25]. Oxidative DNA degradation experiment
Separation of MG-modified HbA0 (MG-Hb) and unchanged HbA0 After in vitro reaction of HbA0 (100 lM) with MG (100 lM) at 25 °C for 3 days, MG-modified HbA0 fractions and unchanged HbA0 were separated by ion-exchange chromatography (BioRex70 resin, 20 1.5 cm) by stepwise increase of NaCl concentration (0, 0.05, 0.15 and 0.3 M) in PB, pH 6.6. MG-modified HbA0 (MGHb) and unchanged HbA0 were used in subsequent experiments.
For DNA degradation experiment, the reaction mixture (250 ll) containing plasmid (pGEM, 3 Kb) DNA (approximately 300 ng), HbA0 or MG-Hb (14 lM) and H2O2 (0.3%) was incubated at 37 °C for 1 h. DFO (1.5 mM) was added to the reaction mixture, as required. The reaction was stopped with 10% glycerol [26]. Different forms of DNA were separated by agarose (1%) gel electrophoresis and visualized by ethidium bromide staining.
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Assay of peroxidase and esterase activities Peroxidase activities of hemoglobin fractions were assayed according to the method of Everse et al. [27]. The reaction mixture (1 ml) contained 50 mM citrate buffer, pH 5.4, 0.5 lM HbA0 or MGHb and 0.002% o-dianisidine. The reaction was initiated by adding 17.6 mM H2O2. The absorbance at 450 nm was followed at 25 °C for 2 min. For estimation of esterase activities of HbA0 and MG-Hb following the method of Elbaum and Nagel [28], hydrolysis of p-NPA (1.5 mM) was monitored in a reaction mixture (1 ml) containing 3.5 lM protein sample in 50 mM Phosphate buffer, pH 7.4 and the change in absorbance at 400 nm was recorded at 25 °C for 2 min. Results In vitro reaction of HbA0 with MG and separation of the modified proteins Following in vitro reaction of HbA0 with MG, the modified and unchanged HbA0 were separated by ion exchange chromatography. A representative elution profile obtained with absorbances of the eluted samples at 415 nm is shown in Fig. 1. Four fractions (I, II, III and IV) were separated. HbA0 solution, when applied to the column under similar condition, was found to elute at the same position as that of fraction IV, indicating fraction IV as the unchanged HbA0. Other three fractions (I, II and III) might be MG-modified HbA0. Fraction I (denoted as MG-Hb) and fraction IV (unchanged HbA0) were collected. When applied to a calibrated Sephadex G100 column pre-equilibrated with PB, both fractions were eluted at the same position corresponding to tetrameric hemoglobin (M.W 66,800), indicating no change in the quaternary structure of MG-Hb. These two fractions were used for their characterization. Nondenaturing gel electrophoresis of fractions I and IV indicated different band positions (inset of Fig. 1). Fraction I exhibited greater electrophoretic mobility than fraction IV. On the other hand, fraction IV appeared to be the unchanged protein, as its band position was similar to that of HbA0. Identification of the chemical nature and molecular location of AGEs in MG-Hb In comparison with HbA0, new peptides appeared in the mass spectrum of MG-Hb (Supplemental Fig. S1a and b). For example, the peptide with m/z value of 1141.67 Da matched exactly with
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the peptide 91–99 of a chain of HbA0 with theoretical m/z value 1087.71 Da plus an addition of 54 Da, a mass increase characteristic of arginine modification to MG-H1. Arg-92a was thus a target of modification. Similarly, the peptide with m/z value of 2880.87 Da might correspond to the peptide 96–120 of b chain of HbA0 with theoretical m/z value 2826.51 Da plus an addition of 54 Da, indicative of the modification of Arg-104b to MG-H1. De novo analysis of the CID mass spectrum of the peptide with m/z value 1141.67 Da matched with the sequence of peptide 91–99 of a chain as LR⁄VDPVNFK, with R⁄ representing Arg-92a modified to MG-H1 (Supplemental Fig. S1c). The modified peptide with m/z value 2880.87 Da was also fragmented by CID to obtain sequence information and locate arginine modification. The peptide sequence 96–120 of b chain appeared to be LHVDPENFR⁄EVLIQLFTGHPETLEK, with Arg-104 modified to MG-H1 (CID mass spectrum not shown). The results are summarized in Table 1. Spectroscopic studies Absorption spectra of HbA0 and MG-Hb were recorded in the region 250–650 nm (Fig. 2a). The spectrum of MG-Hb was found to be almost similar with that of HbA0, with a slight decrease in the absorbance of MG-Hb spectrum around 280 nm (Fig. 2a inset). When excited at 285 nm, both HbA0 and MG-Hb showed similar profile in the emission spectra (300–400 nm) (Fig. 2b). However, the arbitrary fluorescence emission of MG-Hb was much less than that of HbA0. Comparison with other protein concentrations even having absorbance as low as 0.05 at 285 nm showed decreased protein fluorescence of MG-Hb than HbA0, suggesting a correlation of MG-induced protein modification with observed tryptophan fluorescence. MG-induced conformational change of hemoglobin was studied by CD spectral analysis. Fig. 2c exhibits spectra of HbA0 and MG-Hb at far UV region (200–250 nm). Compared to HbA0, MG-Hb showed an increase in negative ellipticity in the region 210–225 nm. Molar ellipticity [h] values were obtained using the relation [29]: [h] = [MrW]/10.l.c, where c (g/ml) is the concentration of the protein, h (mdeg), obtained directly from the dichrograph chart, is the observed rotation, l (cm) is the path length and MrW is the mean residual molecular weight 110 of the amino acid. The a -helical contents of HbA0 and MG-Hb were estimated according to the relation [30]: Fraction of a -helix = ([h]222 + 2340)/ 30300, where [h]222 is the ellipticity at 222 nm. The a -helical contents of HbA0 and MG-Hb were found to be approximately 75% and 82%, respectively. Both HbA0 and MG-Hb exhibited more or less identical spectra in near UV region (250–350 nm) and visible region (400– 600 nm) (not shown). Thermal denaturation study For thermal denaturation study, HbA0 and MG-Hb samples were subjected to DSC analysis. A representative experiment is shown in Fig. 3. The thermograms (heat flow vs. temperature) exhibited 73 °C as the melting temperature (Tm) for HbA0 and 62 °C for MG-Hb, depicting higher thermolabile nature of the modified protein.
Fig. 1. The elution profiles of MG-modified HbA0 and unchanged HbA0 fractions separated by ion-exchange chromatography (–). After in vitro reaction of HbA0 (100 lM) with MG (100 lM) at 25 °C for 3 days, unchanged and modified fractions were separated in Bio-Rex-70 column by stepwise increase of NaCl concentrations in PB. NaCl concentrations 0, 0.05, 0.15 and 0.3 M were used to separate fractions I, II, III and IV, respectively. Absorbances of eluted fractions at 415 nm were plotted against fraction numbers. The elution profile of control HbA0 subjected to similar conditions of ion-exchange chromatography (d–d). Inset: Nondenaturing PAGE (10%) of freshly prepared HbA0 (lane 1) and fractions I (lane 2) and IV (lane 3) isolated after in vitro reaction of HbA0 with MG.
Iron release experiment and iron-mediated oxidative reactions Iron release from HbA0 and MG-Hb was studied by incubating the protein fractions with different concentrations of H2O2 at 37 °C for 1 h and the amount of free iron was estimated by ferrozine reaction, as shown in Fig. 4a. H2O2 induced iron release from both HbA0 and MG-Hb fractions. However, more iron was released from MG-Hb than from HbA0.
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Table 1 Assignment of modified amino acid residues. Observed mass (Da) 1141.67 2880.87
Theoretical mass (Da) 1087.71 2826.51
Peptide sequence ⁄
LR VDPVNFK (91–99) LHVDPENFR⁄EVLIQLFTGHPETLEK (96–120)
Mass increase (Da)
AGE formed
Modified residue (R⁄)
54 54
MG-H1 MG-H1
Arg-92a Arg-104b
Fig. 3. Differential scanning calorimeter thermograms (heat flow vs. temperature) of HbA0 and MG-Hb. 40 lM protein fractions (750 ll) were used for the DSC scans.
both H2O2 and protein samples (lanes 3 and 6), as evident from the prominent form II DNA in the lanes. MG-Hb and H2O2 together (lane 3) exerted more effect than HbA0 and H2O2 combination (lane 6). Besides form II, form III DNA, which arises due to further breakdown of DNA, appeared in lane 3. DFO prevented both H2O2/MGHb and H2O2/HbA0–mediated DNA breakdown considerably, as shown in lanes 4 and 7, respectively.
Enzyme activities Hemoglobin possesses enzyme-like activities. It interacts with H2O2 to yield a potent oxidant ferrylhemoglobin, which is capable
Fig. 2. (a) The representative absorption spectra of 12 lM HbA0 ( ) and MG-Hb () in the region 250–650 nm. Inset: The same spectra of HbA0 ( ) and MG-Hb () in 250–300 nm region with enlarged scale. (b) The representative fluorescence emission spectra with excitation at 285 nm of 6 lM HbA0 ( ) and MG-Hb ( ) in the wavelength region 300–400 nm. (c) The representative CD spectra of 3 lM HbA0 ( ) and MG-Hb ( ) in far UV region (200–250 nm).
DNA (plasmid) degradation by HbA0 and MG-Hb was studied in the absence or presence of H2O2. After incubation, the samples were subjected to gel electrophoresis (Fig. 4b). When plasmid DNA was incubated with MG-Hb (lane 2) or HbA0 (lane 5), DNA degradation occurred to some extent in comparison with that of only DNA (lane 1). However, degradation was more pronounced in the presence of
Fig. 4. (a) H2O2-induced iron release from HbA0 (j) and MG-Hb (d). Hemoglobin samples (40 lM and 250 ll) were incubated at 37 °C for 1 h with varying concentrations of H2O2. After protein precipitation with TCA, the supernatant was used to estimate free iron by ferrozine reaction. The results (expressed as lg iron released) are mean ± SEM of three individual sets of experiments. (b) H2O2mediated plasmid DNA degradation by HbA0 and MG-Hb. Reaction systems containing DNA and different components, as indicated, were incubated at 37 °C for 1 h, after which 10% glycerol was added and the samples were subjected to agarose (1%) gel electrophoresis and ethidium bromide staining. This is a representative experiment from three different experiments. Lanes: 1. DNA, 2. DNA + MG-Hb, 3. DNA + MG-Hb + H2O2, 4. DNA + MG-Hb + H2O2 + DFO, 5. DNA + HbA0, 6. DNA + HbA0 + H2O2, 7. DNA + HbA0 + H2O2 + DFO.
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of oxidizing a wide variety of electron donors resembling peroxidase-like activities [27]. Peroxidase activities of the hemoglobin fractions were assayed using o-dianisidine. In comparison with HbA0, MG-Hb exhibited less peroxidase activity as shown in Fig. 5a. Hemoglobin also exhibits esterase-like activity [28]. The rate of p-NPA hydrolysis by hemoglobin samples was followed to estimate their esterase activities. As shown in Fig. 5b, MG-Hb exhibited more p-NPA hydrolysis over time than HbA0, suggesting the potentiation effect of MG-induced modification on the esterase activity of the heme protein. The rate of p-NPA hydrolysis by HbA0 and MG-Hb were found to be pH dependent (Fig. 5c). Compared to HbA0, MG-Hb-mediated esterase activity became more pronounced with gradual increase in pH.
Discussion Our finding together with the existing reports [18,19] suggests that MG reacts with hemoglobin. Mass spectrometric studies reveal that Arg-92 in a -chains and Arg-104 in b -chains of HbA0 are modified by MG to MG-H1 adducts (Supplemental Fig. S1 and Table 1). Chen et al. [18] have shown that incubation of 100 lM hemoglobin with 500 lM MG at 37°C for 24 h results in modifications of Arg-31 in the a -chains and Arg-30, 40 and 104 residues in the b -chains of the protein, with the latter two being the most reactive sites. Gao and Wang [19] have reported that Arg-92 and 141 residues in the a -chains and Arg-40 and 104 residues in the b -chains are hotspot sites of modification by MG in the reaction mixtures containing hemoglobin and different concentrations of MG (10 lM–200 mM) at 37 °C for 24 h. Our study differs from the existing reports in several respects. Instead of total hemoglobin, we have used HbA0 (100 lM) for reaction with MG (100 lM) under different condition (25 °C for 3 days). Moreover, the modified protein (MG-Hb) has been separated and used for its characterization. However, Arg-104 b appears to be the common site of modification by MG in all these studies. Compared to other arginines, Arg-104 b is the most surface accessible residue of hemoglobin [19], and is the most preferable target for modification by MG. Compared to HbA0, MG-Hb exhibits increased electrophoretic mobility possibly due to the loss of positive charge (Fig. 1 inset), decreased absorbance around 280 nm (Fig. 2a), reduced fluorescence emission with excitation at 285 nm (Fig. 2b) and increased a -helix content (Fig. 2c), indicating more compact structure of the modified protein. The thermal stability of HbA0 decreases due to interaction with MG (Fig. 3). MG-modification of a -crystallin has also been reported to suggest structural changes by partial unfolding, decreased tryptophan fluorescence intensity, reduced thermal stability [13] and increased ellipticity [31]. On the other hand, studies from our laboratory indicate that compared to normal hemoglobin and myoglobin, glucose or fructose-modified heme proteins exhibit reduced thermal stability, but increased fluorescence emission and reduced level of a -helix contents representing increased volume of the proteins [4,5,32]. Thus although hexoses and MG induce modifications through Maillard reactions, the observed effects on the molecules are different. Iron in hemoglobin is completely domesticated and tamed within protoporphyrin cage and further surrounded by globin boundary. Under certain conditions, iron is liberated from heme moiety and loosely bound to another moiety [24]. Iron release exhibits a positive correlation with the extent of in vitro reaction of MG with HbA0 (data not shown). H2O2 is known to induce iron release from hemoglobin [33,34]. However, it causes more iron release from MG-Hb than from HbA0 (Fig. 4a). Release of trace metals such as iron and copper in biological systems may be significant, because they may be a source of reactive oxygen
Fig. 5. (a) Peroxidase-like activities of HbA0 (j) and MG-Hb (d). The reaction mixture (1 ml) contained 50 mM citrate buffer, pH 5.4, 0.5 lM HbA0 or MG-Hb and 0.002% o-dianisidine. The reaction was initiated by addition of 17.6 mM H2O2. The absorbance at 450 nm was monitored. The results are mean ± SEM of four individual experiments. (b) Esterase activities of HbA0 (j) and MG-Hb (d). Hydrolysis of p-NPA (1.5 mM) was monitored at 400 nm in a reaction mixture containing 3.5 lM protein sample in 50 mM phosphate buffer, pH 7.4. Hydrolysis of p-NPA by buffer alone was also monitored (N). The results are mean ± SEM of four individual experiments. (c). Effect of pH on the rate of p-NPA hydrolysis by HbA0 (j) and MG-Hb (d). Hemoglobin sample used was 3.5 lM. The results are mean ± SEM of three sets of experiments after correction for buffer hydrolysis.
species (ROS). The accumulation of ROS in cells leads to various forms of oxidative modifications of proteins (carbonylation or nitro-modifications), lipids (hydroperoxide lipid derivatives) and DNA (adducts and breaks) leading to loss of their molecular functions [35]. OH radicals are produced by Fenton reaction: Fe2+ + H2O2 = Fe3+ + OH + OH [33]. More iron released from MG-Hb than HbA0 leading to increased formation of OH radicals by Fenton reaction may explain the increased breakdown of DNA by the modified protein (Fig. 4b). Inhibition of the reaction by DFO indicates that this is an iron-dependent reaction. In the presence of H2O2, both HbA0 and MG-Hb induce lipid peroxidation and deoxyribose degradation, and MG-Hb appears to be more effective than HbA0 in these reactions (data not shown). MG-H1 modifications in several proteins have been associated with physiological
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abnormalities like oxidative stress, dyslipidemia, mitochondrial dysfunction, cell detachment, and apoptosis [16,17]. However, there has been no study on the effect of MG-H1 modification of hemoglobin. Our finding suggests that MG-induced iron release from hemoglobin is another source of ROS generation in the development of MG toxicity, and is in agreement with our earlier reports on glucose and fructose toxicities of heme proteins [3–5,36]. Hemoglobin samples isolated from streptozotocin-induced diabetic rats also exhibit enhanced iron-dependent oxidative reactions of the cell constituents [6,7]. MG, being a much stronger reactive agent than its parent hexoses, may have significant contribution to the observed pathological effect in diabetic condition. However, in contradiction with existing reports on harmful effects of AGEs, we have shown in a recent study that fructose-induced arginine modification (arg-pyrimidine) in myoglobin exhibits antioxidative activity and is involved in the conversion of met (Fe3+) to oxy (Fe2+) form of the protein [37]. AGE-mediated effects should, therefore, be further explored. The decreased peroxidase activity exhibited by MG-Hb (Fig. 5a) may be associated with a modulation mechanism linked to structural change of the modified protein. The change may cause reduced ferryl formation or may interfere with entry of the secondary substrate molecule o-dianisidine to the heme pocket leading to decreased peroxidase activity. Compared to HbA0, a reduced peroxidase activity of HbA1c has been reported by using o-dianisidine [3] and 5-aminosalicylic acid [38]. On the other hand, glycated myoglobin exhibits more peroxidase activity than normal myoglobin, due to enhanced ferryl formation in the modified protein [36]. Increased esterase activity of MG-Hb (Fig. 5b) is in agreement with that of glycated or fructated heme proteins [39,5]. Human hemoglobin has 38 histidyl residues of which His2b is very crucial for the catalytic activity [40]. His2b is in close proximity to the bound MG as found in molecular modeling study (not shown), and may be influenced by the modified arginine residue MG-H1, leading to a potentiating effect on the catalytic activity of hemoglobin. The difference between esterase activities of HbA0 and MG-Hb at physiological pH appears to be small. However, considering high concentration of hemoglobin within erythrocytes, total esterase activity may be considerably high, particularly in uncontrolled diabetes with increased level of MG. The increased esterolytic activity is associated with vascular alterations, including impaired structure and function of erythrocytes in diabetes [41] . The pH dependence of the esterase activity (Fig. 5c) indicates that the catalysis is related to deprotonation of the appropriate side chains with increase in pH, and is also consistent with the participation of histidine residues in this process. In a related study, Ahmed et al. [9] have shown that at high pH (pH 9.4) MG-treated human serum albumin exhibits higher esterase activity than the native protein. Conclusion MG induces modification of arginine residues forming MG-H1 with consequent change in the structural and functional properties of hemoglobin, including enhanced free iron-mediated oxidative reactions. Considering the increased level of MG in diabetes mellitus as well as its high reactivity, MG-induced modifications of hemoglobin may be associated with the oxidative stress and pathological complications of the metabolic disorder. Acknowledgments T.B and A.B received research fellowships [Grant Nos. F.3-20/ 2002 (SR-II) and F.4-1/2006 (BSR)/5-16/2007(BSR), respectively]
from the University Grants Commission, New Delhi. S.B received a research fellowship [No. 09/028(0802/2010-EMR-1] from the Council of Scientific and Industrial Research, New Delhi. The study was supported by financial assistances from the Department of Science and Technology, New Delhi [Grant No. DST/SR/FST/LSI-286/ 2006(c)] and the University Grants Commission, New Delhi [Grant No. UGC (DSA) F.4-1/2009 (SAP-II)]. We are thankful to Prof. U. Chaudhuri, Ex-Professor, Department of Biophysics, Molecular Biology and Bioinformatics, Calcutta University and Dr. D. Mukherjee, Saha Institute of Nuclear Physics, Kolkata for helpful discussions.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2012.12.001. References [1] I. Giardino, D. Edelstein, M. Brownlee, J. Clin. Invest. 94 (1994) 110–117. [2] A. Negre-Salvayre, R. Salvayre, N. Augé, R. Pamplona, M. Portero-Otín, Antioxid. Redox. Signal 11 (2009) 3071–3109. [3] M. Kar, A.S. Chakraborti, Curr. Sci. 80 (2001) 770–773. [4] S. Sen, M. Kar, A. Roy, A.S. Chakraborti, Biophys. Chem. 113 (2005) 289–298. [5] T. Bose, A.S. Chakraborti, Biochim. Biophys. Acta 1780 (2008) 800–808. [6] M. Roy, S. Sen, A.S. Chakraborti, Life Sci. 82 (2008) 1102–1111. [7] S. Sen, M. Roy, A.S. Chakraborti, J. Phar. Pharmacol. 63 (2011) 287–296. [8] M.P. Kalapos, Toxicol. Lett. 110 (1999) 145–175. [9] N. Ahmed, D. Dobler, M. Dean, P.J. Thornalley, J. Biol. Chem. 280 (2005) 5724– 5732. [10] A.C. McLellan, P.J. Thornally, J. Benn, P.H. Sonksen, Clin. Sci. 87 (1994) 21–29. [11] R. Ramasamy, S.F. Yan, A.M. Schmidt, Cell 124 (2006) 258–260. [12] F.A. Shamsi, A. Partal, C. Sady, M.A. Glomb, R.H. Nagaraj, J. Biol. Chem. 273 (1998) 6928–6936. [13] M.S. Kumar, P.Y. Reddy, P.A. Kumar, T. Surolia, G.B. Reddy, Biochem. J. 379 (2004) 273–282. [14] C.G. Schalkwijk, J. van Bezu, R.C. van der Schors, K. Uchida, C.D. Stehouwer, V.W. van Hinsbergh, FEBS Lett. 580 (2006) 1565–1570. [15] X. Jia, D.J.H. Olson, A.R.S. Ross, L. Wu, Faseb J. 20 (2006) 1555–1557. [16] N. Rabbani, P.J. Thornalley, Amino acids 42 (2012) 1133–1142. [17] M.P. Kalapos, Chem. Biol. Interact. 171 (2008) 251–271. [18] Y. Chen, N. Ahmed, P.J. Thornalley, Ann. N. Y. Acad. Sci. 1043 (2005) 905. [19] Y. Gao, Y. Wang, Biochemistry 45 (2006) 15654–15660. [20] A.C. McLellan, S.A. Phillips, P.J. Thornalley, Anal. Biochem. 206 (1992) 17–23. [21] J. Bhattacharyya, M. Bhattacharyya, A.S. Chakraborti, U. Chaudhuri, R.K. Poddar, Int. J. Biol. Macromol. 23 (1998) 11–18. [22] M.P. Cohen, V. Wu, Methods Enzymol. 231 (1994) 65–67. [23] J. Bhattacharyya, M. Bhattacharyya, A.S. Chakraborti, U. Chaudhuri, R.K. Poddar, Biochem. Pharmacol. 47 (1994) 2049–2053. [24] S.S. Panter, Methods Enzymol. 231 (1994) 502–514. [25] M. Kar, A.S. Chakraborti, Ind. J. Exptl. Biol. 37 (1999) 190–192. [26] W.J. Dixon, J.J. Hayes, J.R. Levin, M.F. Weidner, B.A. Dombroski, T.D. Tullius, Methods Enzymol. 208 (1991) 380–413. [27] J. Everse, M.C. Johnson, M.A. Marini, Methods Enzymol. 231 (1994) 547–561. [28] D. Elbaum, R.L. Nagel, J. Biol. Chem. 256 (1981) 2280–2283. [29] G. Geraci, L.J. Parkhurst, Methods Enzymol. 76 (1981) 262–275. [30] Y.H. Chen, J.T. Yan, H.M. Martinez, Biochemistry 11 (1972) 4120–4131. [31] R. Nagaraj, T. Oya-Ito, P.S. Padayatti, R. Kumar, S. Mehta, K. West, B. Levison, J. Sun, W. Crabb, A.K. Padival, Biochemistry 42 (2003) 10746–10755. [32] A. Roy, R. Sil, A.S. Chakraborti, Mol. Cell. Biochem. 338 (2010) 105–114. [33] J.M.C. Gutteridge, FEBS Lett. 201 (1986) 291–295. [34] B. Halliwell, G.M.C. Gutteridge, Methods Enzymol. 186 (1990) 1–88. [35] M. Giorgio, M. Trinei, E. Migliaccio, P.G. Pelicci, Nat. Rev. Mol. Cell Biol. 8 (2007) 722–728. [36] A. Roy, S. Sen, A.S. Chakraborti, Free Radic. Res. 38 (2004) 139–146. [37] A. Bhattacherjee, A.S. Chakraborti, Int. J. Biol. Macromol. 48 (2011) 202–209. [38] U.Y. Khoo, D.J. Newman, W.K. Miller, C.P. Price, Eur. J. Clin. Chem. Clin. Biochem. 32 (1994) 435–440. [39] S. Sen, T. Bose, A. Roy, A.S. Chakraborti, Mol. Cell. Biochem. 301 (2007) 251– 257. [40] D. Elbaum, B. Weidenmann, R.L. Nagel, J. Biol. Chem. 27 (1982) 8454–8458. [41] A. Symeonidis, G. Althanassiou, A. Psiroyannis, V. Kyriazopoulou, A. KapataisZoumb, Y. Missirlis, N. Zoumbos, Clin. Lab. Haematol. 23 (2001) 103–109.
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Methylglyoxal-induced modifications of hemoglobin: Structural and functional characteristics Tania Bose 1, Abhishek Bhattacherjee, Sauradipta Banerjee, Abhay Sankar Chakraborti ⇑ Department of Biophysics, Molecular Biology & Bioinformatics, University of Calcutta 92, Acharya Prafulla Chandra Road, Kolkata 700 009, India
a r t i c l e
i n f o
Article history: Received 2 August 2012 and in revised form 16 November 2012 Available online 8 December 2012 Keywords: Hemoglobin Methylglyoxal Diabetes mellitus Advanced glycation end products Hydroimidazolone Oxidative stress
a b s t r a c t Methylglyoxal (MG) reacts with proteins to form advanced glycation end products (AGEs). Although hemoglobin modification by MG is known, the modified protein is not yet characterized. We have studied the nature of AGE formed by MG on human hemoglobin (HbA0) and its effect on structure and function of the protein. After reaction of HbA0 with MG, the modified protein (MG-Hb) was separated and its properties were compared with those of the unmodified protein HbA0. As shown by MALDI-mass spectrometry, MG converted Arg-92a and Arg-104b to hydroimidazolones in MG-Hb. Compared to HbA0, MG-Hb exhibited decreased absorbance around 280 nm, reduced tryptophan fluorescence (excitation 285 nm) and increased a-helix content. However, MG modification did not change the quaternary structure of the heme protein. MG-Hb appeared to be more thermolabile than HbA0. The modified protein was found to be more effective than HbA0 in H2O2-mediated iron release and oxidative damages involving Fenton reaction. MG-Hb exhibited less peroxidase activity and more esterase activity than HbA0. MG-induced structural and functional changes of hemoglobin may enhance oxidative stress and associated complications, particularly in diabetes mellitus with increased level of MG. Ó 2012 Elsevier Inc. All rights reserved.
Introduction Post-translational modifications of proteins play important roles in controlling their functions. Protein modification by glucose (glycation) is a complex series of parallel and sequential reactions collectively called the Maillard reaction. The Schiff base formation, followed by Amadori rearrangement and advanced glycation end products (AGEs)2 formation are the key steps of protein glycation [1]. The nonenzymatic modification of proteins may be significant with increased level of blood glucose over prolonged periods of time in diabetes mellitus. Protein glycation reactions leading to AGEs are thought to be the root causes of different diabetic complications, including oxidative stress [2]. Both in vitro and in vivo studies from our laboratory have shown that glucose or fructose causes hemoglo⇑ Corresponding author. Fax: +91 33 2351 9755. E-mail address: [email protected] (A.S. Chakraborti). Present address: Stowers Institute for Medical research, Kansas City, MO 64110, USA. 2 Abbreviations used: AGEs, advanced glycation end products; CHCA, a-cyano hydroxy cinnamic acid; CID, collision-induced dissociation; DFO, desferrioxamine; DSC, differential scanning calorimetry; HbA0, human nonglycated hemoglobin; HbA1a1/HbA1a2/HbA1b/HbA1c, human glycated hemoglobins; MALDI-TOF, matrix assisted laser desorption ionization-time of flight; MG, methylglyoxal; MG-Hb, methylglyoxal-modified HbA0; MG-H1, Nd-(5-hydro-5 methyl-4-imidazolon-2-yl)ornithin; PAGE, polyacrylamide gel electrophoresis; PB, phosphate buffer (50 mM) pH 6.6; p-NPA, p-nitrophenyl acetate; ROS, reactive oxygen species. 1
0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2012.12.001
bin modification by Maillard reaction and promotes free radicalmediated oxidative reactions [3–7]. These findings are significant, because diabetes mellitus causes oxidative stress, which, in a vicious cycle, further aggravates the disease condition. The reactive a-oxoaldehydes namely, glyoxal, methylglyoxal and 3-deoxyglucosone are known to initiate Maillard-like reactions, and are more reactive than the parent hexose sugars with respect to their ability of protein modification and AGE formation [8,9]. Methylglyoxal (MG) level increases in different pathological conditions including diabetes mellitus [10,11], leading to formation of AGEs with different long-lived proteins, namely collagen [12], human serum albumin [9,12], a-crystallin [13], heat shock protein [14], insulin [15], etc. MG forms argpyrimidine, hydroimidazolones and tetrahydropyrimidine with arginine residues. On the other hand, carboxyethyllysine and MG-lysine dimer are the AGEs formed with lysine residues. Proteins susceptible to MG modification with related functional impairment are called the ‘‘dicarbonyl proteome’’ [16], which are predominantly modified on arginine residues with formation of dominant arginine adduct Nd-(5-hydro-5 methyl-4-imidazolon-2-yl)-ornithin (MG-H1). Kalapos has discussed MG-toxicity associated with free radical generation by its metabolism and modification of biological macromolecules [17]. Although hemoglobin glycation is well known and has been studied in details, there have been only two reports on its
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modification by MG. In a brief report, Chen et al. have shown that MG interacts with hemoglobin with modifications of arginine residues forming MG-H1 [18]. Gao and Wang have reported that the sites and extents of MG modification of arginine residues are correlated with solvent accessibility of these residues [19]. The findings are quite significant. However, MG-induced effects on the structure and function of the heme protein have not been reported. Considering the increased level of MG in diabetic condition as well as its very high reactivity (about 20,000 times more reactive than glucose), its interaction with hemoglobin should be studied in more detail. We have, therefore, undertaken the present study to separate MG-modified hemoglobin, to find the sites of modification by MG, to characterize the adduct formed, and to compare its structural and functional properties with those of normal protein for understanding any adverse effect associated with the modification.
Materials and methods Materials Methylglyoxal (MG), Sephadex G-100, Sephadex G-25, ferrozine, glyoxalase I (yeast), glyoxalase II (bovine liver), o-dianisidine, agarose, acrylamide, Coomasie R250, ethidium bromide, pnitrophenyl acetate (p-NPA), desferrioxamine (DFO), and a-cyano hydroxy cinnamic acid (CHCA) were purchased from Sigma Chemical Company, USA. Bio-Rex-70 resin (200–400 mesh) was obtained from Bio-Rad, India. All other reagents were AR grade and purchased locally. MG was distilled under low pressure and its concentration in stock solution was measured by end point enzymatic assay involving conversion to S-D-lactoylglutathione with glyoxalase I and hydrolysis catalyzed by glyoxalase II [20].
Separation of nonglycated hemoglobin (HbA0) Human blood samples were collected from healthy volunteers (non-smokers) aged 20–25 years. Ethical principles formulated by the Institutional Ethics Committee were followed in carrying out the study. Hemoglobin (total) was isolated and purified from blood sample by using Sephadex G-100 column chromatography [21]. Hemoglobin sample in 50 mM phosphate buffer, pH 6.6 (PB) containing 0.15 M NaCl was applied to a cation exchange column containing BioRex-70 resin (20 1.5 cm) pre-equilibrated with PB. Different glycated hemoglobin species (HbA1a1, HbA1a2, HbA1b and HbA1c) and nonglycated hemoglobin (HbA0) were separated by using gradually increased concentration of NaCl in elution buffer (PB) following the method of Cohen and Wu [22] with some modifications described before [4]. HbA0 fraction in PB was collected after removal of NaCl by passing through Sephadex G-100 column, and its concentration was determined from Soret absorbance using an extinction coefficient (e415nm) of 125 mM 1 cm 1 (heme basis) [23].
Polyacrylamide gel electrophoresis (PAGE) MG-Hb and HbA0 were subjected to native PAGE (10%) for 3 h at constant voltage. Freshly prepared HbA0 was also included in the study. 20 ll of each sample (20 lM) was loaded. After electrophoresis, the gel was stained with Coomasie R250. Matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometric study After native PAGE of MG-Hb and HbA0, the bands were excised and subjected to reduction, alkylation and digestion with sequencing-grade trypsin in gel. After digestion at 37 °C for 16 h, the supernatants containing the peptide mixtures of MG-Hb and HbA0 were collected separately by centrifugation. The samples (0.5 ll each) were applied directly to the MALDI plate, mixed with 0.5 ll saturated CHCA solution (prepared in 50% acetonitrile and 0.1% trifluoroacetic acid) and allowed to crystallize. Mass spectra were recorded in a 4800 Proteomics Analyzer (MALDI-TOF/TOF mass spectrometer, Applied Biosystems). The location and chemical nature of modified amino acid residues were identified from MALDITOF data. MS/MS experiments were performed by using collision-induced dissociation (CID) technique for sequence information of the selected peptides of interest. Spectroscopic studies Different spectroscopic studies were done with isolated fractions MG-Hb and HbA0. Absorption spectra (250–650 nm) of the samples (12 lM each) were recorded in a UV–VIS spectrophotometer (Hitachi U2000) using 1 ml quartz cuvette of path length 1 cm. Fluorescence emission spectra (with excitation at 285 nm) of the protein fractions (6 lM) were monitored (300–400 nm) in a spectrofluorimeter (Hitachi F-3010) using 3 ml quartz cuvette of path length 1 cm. Circular dichroic (CD) measurements (200–250 nm, 250–350 nm and 400–600 nm) of MG-Hb and HbA0 fractions (3 lM each) were done in a spectropolarimeter (Jasco 600) using 1 mm path length cuvette. Differential scanning calorimetric (DSC) study DSC measurements were done in a Calorimetric Scientific Corporation instrument (N-DSCII). The protein fractions MG-Hb and HbA0 (750 ll, 40 lM) were degassed under vacuum at room temperature and loaded to the calorimeter. DSC scans were recorded from 25 °C to 85 °C at a heating rate of 2 °C/min. Estimation of free iron H2O2-induced iron release from separated fractions of HbA0 and MG-Hb was estimated, for which protein samples (250 ll and 40 lM) were incubated with varying concentrations of H2O2 (0, 0.25, 0.50, 0.75, and 1.0 mM) at 37 °C for 1 h. After protein precipitation with TCA, the supernatants were used to estimate free iron by ferrozine reaction [24], as described before [25]. Oxidative DNA degradation experiment
Separation of MG-modified HbA0 (MG-Hb) and unchanged HbA0 After in vitro reaction of HbA0 (100 lM) with MG (100 lM) at 25 °C for 3 days, MG-modified HbA0 fractions and unchanged HbA0 were separated by ion-exchange chromatography (BioRex70 resin, 20 1.5 cm) by stepwise increase of NaCl concentration (0, 0.05, 0.15 and 0.3 M) in PB, pH 6.6. MG-modified HbA0 (MGHb) and unchanged HbA0 were used in subsequent experiments.
For DNA degradation experiment, the reaction mixture (250 ll) containing plasmid (pGEM, 3 Kb) DNA (approximately 300 ng), HbA0 or MG-Hb (14 lM) and H2O2 (0.3%) was incubated at 37 °C for 1 h. DFO (1.5 mM) was added to the reaction mixture, as required. The reaction was stopped with 10% glycerol [26]. Different forms of DNA were separated by agarose (1%) gel electrophoresis and visualized by ethidium bromide staining.
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Assay of peroxidase and esterase activities Peroxidase activities of hemoglobin fractions were assayed according to the method of Everse et al. [27]. The reaction mixture (1 ml) contained 50 mM citrate buffer, pH 5.4, 0.5 lM HbA0 or MGHb and 0.002% o-dianisidine. The reaction was initiated by adding 17.6 mM H2O2. The absorbance at 450 nm was followed at 25 °C for 2 min. For estimation of esterase activities of HbA0 and MG-Hb following the method of Elbaum and Nagel [28], hydrolysis of p-NPA (1.5 mM) was monitored in a reaction mixture (1 ml) containing 3.5 lM protein sample in 50 mM Phosphate buffer, pH 7.4 and the change in absorbance at 400 nm was recorded at 25 °C for 2 min. Results In vitro reaction of HbA0 with MG and separation of the modified proteins Following in vitro reaction of HbA0 with MG, the modified and unchanged HbA0 were separated by ion exchange chromatography. A representative elution profile obtained with absorbances of the eluted samples at 415 nm is shown in Fig. 1. Four fractions (I, II, III and IV) were separated. HbA0 solution, when applied to the column under similar condition, was found to elute at the same position as that of fraction IV, indicating fraction IV as the unchanged HbA0. Other three fractions (I, II and III) might be MG-modified HbA0. Fraction I (denoted as MG-Hb) and fraction IV (unchanged HbA0) were collected. When applied to a calibrated Sephadex G100 column pre-equilibrated with PB, both fractions were eluted at the same position corresponding to tetrameric hemoglobin (M.W 66,800), indicating no change in the quaternary structure of MG-Hb. These two fractions were used for their characterization. Nondenaturing gel electrophoresis of fractions I and IV indicated different band positions (inset of Fig. 1). Fraction I exhibited greater electrophoretic mobility than fraction IV. On the other hand, fraction IV appeared to be the unchanged protein, as its band position was similar to that of HbA0. Identification of the chemical nature and molecular location of AGEs in MG-Hb In comparison with HbA0, new peptides appeared in the mass spectrum of MG-Hb (Supplemental Fig. S1a and b). For example, the peptide with m/z value of 1141.67 Da matched exactly with
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the peptide 91–99 of a chain of HbA0 with theoretical m/z value 1087.71 Da plus an addition of 54 Da, a mass increase characteristic of arginine modification to MG-H1. Arg-92a was thus a target of modification. Similarly, the peptide with m/z value of 2880.87 Da might correspond to the peptide 96–120 of b chain of HbA0 with theoretical m/z value 2826.51 Da plus an addition of 54 Da, indicative of the modification of Arg-104b to MG-H1. De novo analysis of the CID mass spectrum of the peptide with m/z value 1141.67 Da matched with the sequence of peptide 91–99 of a chain as LR⁄VDPVNFK, with R⁄ representing Arg-92a modified to MG-H1 (Supplemental Fig. S1c). The modified peptide with m/z value 2880.87 Da was also fragmented by CID to obtain sequence information and locate arginine modification. The peptide sequence 96–120 of b chain appeared to be LHVDPENFR⁄EVLIQLFTGHPETLEK, with Arg-104 modified to MG-H1 (CID mass spectrum not shown). The results are summarized in Table 1. Spectroscopic studies Absorption spectra of HbA0 and MG-Hb were recorded in the region 250–650 nm (Fig. 2a). The spectrum of MG-Hb was found to be almost similar with that of HbA0, with a slight decrease in the absorbance of MG-Hb spectrum around 280 nm (Fig. 2a inset). When excited at 285 nm, both HbA0 and MG-Hb showed similar profile in the emission spectra (300–400 nm) (Fig. 2b). However, the arbitrary fluorescence emission of MG-Hb was much less than that of HbA0. Comparison with other protein concentrations even having absorbance as low as 0.05 at 285 nm showed decreased protein fluorescence of MG-Hb than HbA0, suggesting a correlation of MG-induced protein modification with observed tryptophan fluorescence. MG-induced conformational change of hemoglobin was studied by CD spectral analysis. Fig. 2c exhibits spectra of HbA0 and MG-Hb at far UV region (200–250 nm). Compared to HbA0, MG-Hb showed an increase in negative ellipticity in the region 210–225 nm. Molar ellipticity [h] values were obtained using the relation [29]: [h] = [MrW]/10.l.c, where c (g/ml) is the concentration of the protein, h (mdeg), obtained directly from the dichrograph chart, is the observed rotation, l (cm) is the path length and MrW is the mean residual molecular weight 110 of the amino acid. The a -helical contents of HbA0 and MG-Hb were estimated according to the relation [30]: Fraction of a -helix = ([h]222 + 2340)/ 30300, where [h]222 is the ellipticity at 222 nm. The a -helical contents of HbA0 and MG-Hb were found to be approximately 75% and 82%, respectively. Both HbA0 and MG-Hb exhibited more or less identical spectra in near UV region (250–350 nm) and visible region (400– 600 nm) (not shown). Thermal denaturation study For thermal denaturation study, HbA0 and MG-Hb samples were subjected to DSC analysis. A representative experiment is shown in Fig. 3. The thermograms (heat flow vs. temperature) exhibited 73 °C as the melting temperature (Tm) for HbA0 and 62 °C for MG-Hb, depicting higher thermolabile nature of the modified protein.
Fig. 1. The elution profiles of MG-modified HbA0 and unchanged HbA0 fractions separated by ion-exchange chromatography (–). After in vitro reaction of HbA0 (100 lM) with MG (100 lM) at 25 °C for 3 days, unchanged and modified fractions were separated in Bio-Rex-70 column by stepwise increase of NaCl concentrations in PB. NaCl concentrations 0, 0.05, 0.15 and 0.3 M were used to separate fractions I, II, III and IV, respectively. Absorbances of eluted fractions at 415 nm were plotted against fraction numbers. The elution profile of control HbA0 subjected to similar conditions of ion-exchange chromatography (d–d). Inset: Nondenaturing PAGE (10%) of freshly prepared HbA0 (lane 1) and fractions I (lane 2) and IV (lane 3) isolated after in vitro reaction of HbA0 with MG.
Iron release experiment and iron-mediated oxidative reactions Iron release from HbA0 and MG-Hb was studied by incubating the protein fractions with different concentrations of H2O2 at 37 °C for 1 h and the amount of free iron was estimated by ferrozine reaction, as shown in Fig. 4a. H2O2 induced iron release from both HbA0 and MG-Hb fractions. However, more iron was released from MG-Hb than from HbA0.
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Table 1 Assignment of modified amino acid residues. Observed mass (Da) 1141.67 2880.87
Theoretical mass (Da) 1087.71 2826.51
Peptide sequence ⁄
LR VDPVNFK (91–99) LHVDPENFR⁄EVLIQLFTGHPETLEK (96–120)
Mass increase (Da)
AGE formed
Modified residue (R⁄)
54 54
MG-H1 MG-H1
Arg-92a Arg-104b
Fig. 3. Differential scanning calorimeter thermograms (heat flow vs. temperature) of HbA0 and MG-Hb. 40 lM protein fractions (750 ll) were used for the DSC scans.
both H2O2 and protein samples (lanes 3 and 6), as evident from the prominent form II DNA in the lanes. MG-Hb and H2O2 together (lane 3) exerted more effect than HbA0 and H2O2 combination (lane 6). Besides form II, form III DNA, which arises due to further breakdown of DNA, appeared in lane 3. DFO prevented both H2O2/MGHb and H2O2/HbA0–mediated DNA breakdown considerably, as shown in lanes 4 and 7, respectively.
Enzyme activities Hemoglobin possesses enzyme-like activities. It interacts with H2O2 to yield a potent oxidant ferrylhemoglobin, which is capable
Fig. 2. (a) The representative absorption spectra of 12 lM HbA0 ( ) and MG-Hb () in the region 250–650 nm. Inset: The same spectra of HbA0 ( ) and MG-Hb () in 250–300 nm region with enlarged scale. (b) The representative fluorescence emission spectra with excitation at 285 nm of 6 lM HbA0 ( ) and MG-Hb ( ) in the wavelength region 300–400 nm. (c) The representative CD spectra of 3 lM HbA0 ( ) and MG-Hb ( ) in far UV region (200–250 nm).
DNA (plasmid) degradation by HbA0 and MG-Hb was studied in the absence or presence of H2O2. After incubation, the samples were subjected to gel electrophoresis (Fig. 4b). When plasmid DNA was incubated with MG-Hb (lane 2) or HbA0 (lane 5), DNA degradation occurred to some extent in comparison with that of only DNA (lane 1). However, degradation was more pronounced in the presence of
Fig. 4. (a) H2O2-induced iron release from HbA0 (j) and MG-Hb (d). Hemoglobin samples (40 lM and 250 ll) were incubated at 37 °C for 1 h with varying concentrations of H2O2. After protein precipitation with TCA, the supernatant was used to estimate free iron by ferrozine reaction. The results (expressed as lg iron released) are mean ± SEM of three individual sets of experiments. (b) H2O2mediated plasmid DNA degradation by HbA0 and MG-Hb. Reaction systems containing DNA and different components, as indicated, were incubated at 37 °C for 1 h, after which 10% glycerol was added and the samples were subjected to agarose (1%) gel electrophoresis and ethidium bromide staining. This is a representative experiment from three different experiments. Lanes: 1. DNA, 2. DNA + MG-Hb, 3. DNA + MG-Hb + H2O2, 4. DNA + MG-Hb + H2O2 + DFO, 5. DNA + HbA0, 6. DNA + HbA0 + H2O2, 7. DNA + HbA0 + H2O2 + DFO.
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of oxidizing a wide variety of electron donors resembling peroxidase-like activities [27]. Peroxidase activities of the hemoglobin fractions were assayed using o-dianisidine. In comparison with HbA0, MG-Hb exhibited less peroxidase activity as shown in Fig. 5a. Hemoglobin also exhibits esterase-like activity [28]. The rate of p-NPA hydrolysis by hemoglobin samples was followed to estimate their esterase activities. As shown in Fig. 5b, MG-Hb exhibited more p-NPA hydrolysis over time than HbA0, suggesting the potentiation effect of MG-induced modification on the esterase activity of the heme protein. The rate of p-NPA hydrolysis by HbA0 and MG-Hb were found to be pH dependent (Fig. 5c). Compared to HbA0, MG-Hb-mediated esterase activity became more pronounced with gradual increase in pH.
Discussion Our finding together with the existing reports [18,19] suggests that MG reacts with hemoglobin. Mass spectrometric studies reveal that Arg-92 in a -chains and Arg-104 in b -chains of HbA0 are modified by MG to MG-H1 adducts (Supplemental Fig. S1 and Table 1). Chen et al. [18] have shown that incubation of 100 lM hemoglobin with 500 lM MG at 37°C for 24 h results in modifications of Arg-31 in the a -chains and Arg-30, 40 and 104 residues in the b -chains of the protein, with the latter two being the most reactive sites. Gao and Wang [19] have reported that Arg-92 and 141 residues in the a -chains and Arg-40 and 104 residues in the b -chains are hotspot sites of modification by MG in the reaction mixtures containing hemoglobin and different concentrations of MG (10 lM–200 mM) at 37 °C for 24 h. Our study differs from the existing reports in several respects. Instead of total hemoglobin, we have used HbA0 (100 lM) for reaction with MG (100 lM) under different condition (25 °C for 3 days). Moreover, the modified protein (MG-Hb) has been separated and used for its characterization. However, Arg-104 b appears to be the common site of modification by MG in all these studies. Compared to other arginines, Arg-104 b is the most surface accessible residue of hemoglobin [19], and is the most preferable target for modification by MG. Compared to HbA0, MG-Hb exhibits increased electrophoretic mobility possibly due to the loss of positive charge (Fig. 1 inset), decreased absorbance around 280 nm (Fig. 2a), reduced fluorescence emission with excitation at 285 nm (Fig. 2b) and increased a -helix content (Fig. 2c), indicating more compact structure of the modified protein. The thermal stability of HbA0 decreases due to interaction with MG (Fig. 3). MG-modification of a -crystallin has also been reported to suggest structural changes by partial unfolding, decreased tryptophan fluorescence intensity, reduced thermal stability [13] and increased ellipticity [31]. On the other hand, studies from our laboratory indicate that compared to normal hemoglobin and myoglobin, glucose or fructose-modified heme proteins exhibit reduced thermal stability, but increased fluorescence emission and reduced level of a -helix contents representing increased volume of the proteins [4,5,32]. Thus although hexoses and MG induce modifications through Maillard reactions, the observed effects on the molecules are different. Iron in hemoglobin is completely domesticated and tamed within protoporphyrin cage and further surrounded by globin boundary. Under certain conditions, iron is liberated from heme moiety and loosely bound to another moiety [24]. Iron release exhibits a positive correlation with the extent of in vitro reaction of MG with HbA0 (data not shown). H2O2 is known to induce iron release from hemoglobin [33,34]. However, it causes more iron release from MG-Hb than from HbA0 (Fig. 4a). Release of trace metals such as iron and copper in biological systems may be significant, because they may be a source of reactive oxygen
Fig. 5. (a) Peroxidase-like activities of HbA0 (j) and MG-Hb (d). The reaction mixture (1 ml) contained 50 mM citrate buffer, pH 5.4, 0.5 lM HbA0 or MG-Hb and 0.002% o-dianisidine. The reaction was initiated by addition of 17.6 mM H2O2. The absorbance at 450 nm was monitored. The results are mean ± SEM of four individual experiments. (b) Esterase activities of HbA0 (j) and MG-Hb (d). Hydrolysis of p-NPA (1.5 mM) was monitored at 400 nm in a reaction mixture containing 3.5 lM protein sample in 50 mM phosphate buffer, pH 7.4. Hydrolysis of p-NPA by buffer alone was also monitored (N). The results are mean ± SEM of four individual experiments. (c). Effect of pH on the rate of p-NPA hydrolysis by HbA0 (j) and MG-Hb (d). Hemoglobin sample used was 3.5 lM. The results are mean ± SEM of three sets of experiments after correction for buffer hydrolysis.
species (ROS). The accumulation of ROS in cells leads to various forms of oxidative modifications of proteins (carbonylation or nitro-modifications), lipids (hydroperoxide lipid derivatives) and DNA (adducts and breaks) leading to loss of their molecular functions [35]. OH radicals are produced by Fenton reaction: Fe2+ + H2O2 = Fe3+ + OH + OH [33]. More iron released from MG-Hb than HbA0 leading to increased formation of OH radicals by Fenton reaction may explain the increased breakdown of DNA by the modified protein (Fig. 4b). Inhibition of the reaction by DFO indicates that this is an iron-dependent reaction. In the presence of H2O2, both HbA0 and MG-Hb induce lipid peroxidation and deoxyribose degradation, and MG-Hb appears to be more effective than HbA0 in these reactions (data not shown). MG-H1 modifications in several proteins have been associated with physiological
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abnormalities like oxidative stress, dyslipidemia, mitochondrial dysfunction, cell detachment, and apoptosis [16,17]. However, there has been no study on the effect of MG-H1 modification of hemoglobin. Our finding suggests that MG-induced iron release from hemoglobin is another source of ROS generation in the development of MG toxicity, and is in agreement with our earlier reports on glucose and fructose toxicities of heme proteins [3–5,36]. Hemoglobin samples isolated from streptozotocin-induced diabetic rats also exhibit enhanced iron-dependent oxidative reactions of the cell constituents [6,7]. MG, being a much stronger reactive agent than its parent hexoses, may have significant contribution to the observed pathological effect in diabetic condition. However, in contradiction with existing reports on harmful effects of AGEs, we have shown in a recent study that fructose-induced arginine modification (arg-pyrimidine) in myoglobin exhibits antioxidative activity and is involved in the conversion of met (Fe3+) to oxy (Fe2+) form of the protein [37]. AGE-mediated effects should, therefore, be further explored. The decreased peroxidase activity exhibited by MG-Hb (Fig. 5a) may be associated with a modulation mechanism linked to structural change of the modified protein. The change may cause reduced ferryl formation or may interfere with entry of the secondary substrate molecule o-dianisidine to the heme pocket leading to decreased peroxidase activity. Compared to HbA0, a reduced peroxidase activity of HbA1c has been reported by using o-dianisidine [3] and 5-aminosalicylic acid [38]. On the other hand, glycated myoglobin exhibits more peroxidase activity than normal myoglobin, due to enhanced ferryl formation in the modified protein [36]. Increased esterase activity of MG-Hb (Fig. 5b) is in agreement with that of glycated or fructated heme proteins [39,5]. Human hemoglobin has 38 histidyl residues of which His2b is very crucial for the catalytic activity [40]. His2b is in close proximity to the bound MG as found in molecular modeling study (not shown), and may be influenced by the modified arginine residue MG-H1, leading to a potentiating effect on the catalytic activity of hemoglobin. The difference between esterase activities of HbA0 and MG-Hb at physiological pH appears to be small. However, considering high concentration of hemoglobin within erythrocytes, total esterase activity may be considerably high, particularly in uncontrolled diabetes with increased level of MG. The increased esterolytic activity is associated with vascular alterations, including impaired structure and function of erythrocytes in diabetes [41] . The pH dependence of the esterase activity (Fig. 5c) indicates that the catalysis is related to deprotonation of the appropriate side chains with increase in pH, and is also consistent with the participation of histidine residues in this process. In a related study, Ahmed et al. [9] have shown that at high pH (pH 9.4) MG-treated human serum albumin exhibits higher esterase activity than the native protein. Conclusion MG induces modification of arginine residues forming MG-H1 with consequent change in the structural and functional properties of hemoglobin, including enhanced free iron-mediated oxidative reactions. Considering the increased level of MG in diabetes mellitus as well as its high reactivity, MG-induced modifications of hemoglobin may be associated with the oxidative stress and pathological complications of the metabolic disorder. Acknowledgments T.B and A.B received research fellowships [Grant Nos. F.3-20/ 2002 (SR-II) and F.4-1/2006 (BSR)/5-16/2007(BSR), respectively]
from the University Grants Commission, New Delhi. S.B received a research fellowship [No. 09/028(0802/2010-EMR-1] from the Council of Scientific and Industrial Research, New Delhi. The study was supported by financial assistances from the Department of Science and Technology, New Delhi [Grant No. DST/SR/FST/LSI-286/ 2006(c)] and the University Grants Commission, New Delhi [Grant No. UGC (DSA) F.4-1/2009 (SAP-II)]. We are thankful to Prof. U. Chaudhuri, Ex-Professor, Department of Biophysics, Molecular Biology and Bioinformatics, Calcutta University and Dr. D. Mukherjee, Saha Institute of Nuclear Physics, Kolkata for helpful discussions.
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