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Methyglyoxal administration induces modification of hemoglobin in experimental rats: An in vivo study
Journal of Photochemistry & Photobiology, B: Biology 167 (2017) 82–88
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Methyglyoxal administration induces modification of hemoglobin in experimental rats: An in vivo study Sauradipta Banerjee National Brain Research Centre, NH-8, Manesar, Gurgaon, Haryana 122051, India
a r t i c l e
i n f o
Article history: Received 20 October 2016 Received in revised form 14 December 2016 Accepted 21 December 2016 Available online 26 December 2016 Keywords: Methylglyoxal Hemoglobin Diabetes mellitus Maillard-like reaction Advanced glycation end products Hydroimidazolone
a b s t r a c t Methylglyoxal, a highly reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. In the present study, the effect of methylglyoxal on experimental rat hemoglobin in vivo has been investigated with respect to structural alterations and amino acid modifications, after external administration of the α-dicarbonyl compound in animals. Different techniques, mostly biophysical, were used to characterize and compare methylglyoxal-treated rat hemoglobin with that of control, untreated rat hemoglobin. In comparison with methylglyoxal-untreated, control rat hemoglobin, hemoglobin of methylglyoxal-treated rats (32 mg/kg body wt. dose) exhibited slightly decreased absorbance around 280 nm, reduced intrinsic fluorescence and lower surface hydrophobicity. The secondary structures of hemoglobin of control and methylglyoxal-treated rats were more or less identical with the latter exhibiting slightly increased α-helicity compared to the former. Compared to control rat hemoglobin, methylglyoxal-treated rat hemoglobin showed higher stability. Peptide mass fingerprinting analysis revealed modifications of Arg-31α, Arg-92α and Arg-104β of methylglyoxal-treated rat hemoglobin to hydroimidazolone adducts. The modifications thus appear to be associated with the observed structural alterations of the heme protein. Considering the increased level of methylglyoxal in diabetes mellitus as well as its high reactivity, AGE-induced modifications may have physiological significance. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Reducing sugars react with proteins by Maillard reaction to form advanced glycation end products (AGEs) involving Schiff base and Amadori products [1]. The non-enzymatic 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]. Besides glucose, other glycating agents include fructose [3], glyoxal [4], methylglyoxal [5], 3deoxyglucosone [6] etc. Findings from our laboratory indicate that glycation of hemoglobin by glucose [7,8], fructose [9] and methylglyoxal [10] promote iron release and free radical-mediated oxidative reactions. Methylglyoxal (MG), a highly reactive α-oxoaldehyde, is mainly derived from triose phosphates D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate during glycolysis in eukaryotic cells, and its blood level increases in both type 1 and type 2 diabetes mellitus [11– 13]. The median concentration of MG is increased by 5–6-fold and 2– 3-fold in blood samples of diabetic patients with Type 1 and Type 2 diabetes mellitus, respectively [14], and the formation of MG-derived
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http://dx.doi.org/10.1016/j.jphotobiol.2016.12.028 1011-1344/© 2016 Elsevier B.V. All rights reserved.
AGEs is increased accordingly. MG-derived AGEs have been reported with different proteins namely insulin [5], human serum albumin [15], cytochrome c [16], α-synuclein [17], superoxide dismutase [18], etc. We have reported MG-induced structural alterations of myoglobin in recent studies [19,20], etc. In vitro reaction of hemoglobin with MG has been reported in some earlier studies. In a brief report, Chen et al. have shown that MG interacts with hemoglobin with modifications of arginine residues forming hydroimidazone (MG-H1) [21]. Gao and Wang have found that the sites and extents of MG modification of arginine residues are correlated with solvent accessibility of these residues [22]. We have recently reported interaction of MG with hemoglobin leading to modification of arginine residues and subsequent changes in structural and functional properties of the heme protein [10]. Besides several in vitro studies, a number of in vivo animal studies on MG have been published. The αoxoaldehyde has been reported to induce diabetes-like complications and retinal injury in experimental rats [23,24]. MG administration has been also shown to cause glucose intolerance, brain mitochondrial impairment and peritoneal fibrosis in rats [25–27]. Although in vitro interaction of MG with hemoglobin has been shown in previous studies, till date no studies describing the effect of MG on hemoglobin in vivo have been reported. Considering this, in the present study, we have administered MG in experimental rats and investigated its effect on rat
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2. Materials and methods
For ANS binding study, the samples (8 μM each) were incubated with 20 μM ANS for 10. minutes at room temperature and the fluorescence emission spectra (450–600 nm) were recorded with excitation at 370 nm.
2.1. Materials
2.7. CD study
MG, Sephadex G-100, acrylamide, Coomassie R250, sequencing grade trypsin, α-cyano-hydroxycinnamic acid matrix (CHCA), 1anilino-naphthalene-8-sulfonate (ANS), were purchased from Sigma Chemical Company, USA. All other reagents were AR grade and purchased locally.
CD spectra of the samples (3 μM each) were recorded in the far UV region (190–250 nm) in a spectropolarimeter (Jasco 600). The α-helical contents were calculated following the method of Chen et al. [30].
2.2. Methods
DSC study was carried out to measure thermal stability of samples. The melting profiles were recorded in a VP-DSC Microcalorimeter by heating the samples (1 °C/min) over a definite temperature range. Before introduction into the calorimetric cells, the protein samples were thoroughly degassed. Conformational stability of samples was measured by chemical denaturant-induced unfolding study. Fluorescence emission spectra (300–400 nm) of samples (3 μM each) were recorded with excitation at 280 nm after overnight incubation with 500 mM guanidine hydrochloride.
hemoglobin in vivo with respect to structure and stability changes, as well as site(s) and nature of amino acid modifications.
Animal experiments were performed in accordance with regulations specified and monitored by the Institutional Ethics Committee. Male Wister rats (weighing 70–80 g) were divided in four groups - I, II, III and IV, consisting of four rats in each group. Group I rats were not treated with MG and denoted as control group. Groups II, III and IV rats were respectively injected with 8, 16 and 32 mg/kg body weight MG intravenously and were denoted as MG-treated groups. All animals were fed with standard diet and water ad libitum. Whole blood was collected from each group after one week by heart puncture and hemolysates prepared. 2.3. Separation of hemoglobin by size-exclusion column chromatography Hemolysates prepared from different groups of rats were subjected to native PAGE. Total hemoglobin was purified from hemolysates using Sephadex G-100 column chromatography [28] and subjected to native gel electrophoresis. The concentration of hemoglobin was determined from Soret absorbance using an extinction coefficient (ε415nm) of 125 mMˉ1 cmˉ1 (heme basis) [29]. 2.4. MALDI-TOF mass spectrometric study Hemoglobin obtained from control group I and MG-treated group IV rats were subjected to mass spectral analysis using the linear positive ion mode of MALDI-TOF MS after digestion with sequencing-grade trypsin in solution at 37 °C for 16 h using enzyme: protein ratio 1:100 (w/w). The digested samples (0.5 μl each) were loaded directly to the MALDI plate, mixed with 0.5 μl of saturated CHCA solution (prepared in 50% acetonitrile and 0.1% trifluoroacetic acid) and allowed to dry and crystallize. Mass spectra were recorded in a 4800 Proteomics Analyzer (MALDI-TOF/TOF mass spectrometer, Applied Biosystems) using the linear positive ion mode of MALDI-TOF MS at 20 kV acceleration voltage. Identification of MG-modified peptides and specific MG-derived AGE adducts was performed as described earlier [19,20]. Purified hemoglobin of group I and group IV rats were used for further experiments. 2.5. Absorbance spectroscopy Absorbance spectra of control and MG-treated rat hemoglobin (3 μM each) were recorded in a UV/VIS Spectrophotometer (Hitachi U 2000) using 1 ml quartz cuvette of path length 1 cm in the region 250– 600 nm, taking 3 μM protein in each case. 2.6. Spectrofluorimetric study Fluorescence emission spectra of purified hemoglobin of control and MG-treated rats were recorded in the region 320–400 nm with excitation at 280 nm in a spectrofluorimeter (Hitachi F-3010). Protein concentration was adjusted to 3 μM for recording each spectrum.
2.8. Stability studies
3. Results and Discussion 3.1. Separation of hemoglobin The native gel profile of hemolysates obtained from different groups of rats (I–IV) was found to be more or less identical, as shown in Fig. 1A (lanes 1–4). Hemoglobin was purified from the hemolysates of different groups by size-exclusion chromatography. Fig. 1B shows gel electrophoretic profile of hemoglobin purified from different groups. Purified hemoglobin of group I and group IV rats were subjected to further experiments. 3.2. Mass spectrometric study The tryptic mass fingerprint spectrum of control (group I) rat hemoglobin is shown in Fig. 2A. In the spectrum of MG-treated (group IV) rat hemoglobin (Fig. 2B), the peptides with m/z values 1141.67 Da, 1180.64 Da and 1583.79 Da indicated modifications of Arg-92α, Arg104β and Arg-31α to hydroimidazolone (MG-H1) adducts, respectively. The modified peptides were absent in control rat hemoglobin. All modifications were confirmed by MSMS fragmentation of the modified peptides (data not shown) using Collision Induced Dissociation [5,20]. Results are summarized in Table 1. The observation was found to be similar to previously published reports on MG-hemoglobin interaction studies, where the reactive α-oxoaldehyde was found to modify arginine residues of the heme protein to hydroimidazolone adducts in vitro [10,21,22]. Among the modified arginines in hemoglobin, Arg-104 in the β chain is most accessible to solvent (103 Å2 in surface exposable area), followed by Arg-92 in α chain (40 Å2 in surface exposable area), as estimated from the web-based program GETAREA [22]. Thus the solvent accessibility and hence MG modification of these residues may be correlated with their exposed surface area as reported earlier. Arg-104 β is located in an alpha-helical region whereas Arg-92 α is located in a nonhelical region of the heme protein with no distinct secondary structure (based on the structure of oxyhemoglobin- PDB ID: 1GZX). On the other hand, Arg-31 in α chain (located in alpha-helix) is buried inside the protein and its surface exposable area is 3.8 Å2. The observation that MG was found to modify Arg-31 in α chain is consistent with previous studies where the reactive α-oxoaldehyde modified arginine residues of the yeast protein enolase located in deep crevice at the dimer interface of the protein structure with reduced surface exposure
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Fig. 1. Native PAGE experiments. (A) Gel electrophoretic profile of hemolysates obtained from different groups of rat. Lane 1: control group I; lanes 2–4: MG-treated groups II–IV. (B) Gel electrophoretic profile of hemoglobin purified from different groups of rats. Lane 1: control group I; lanes 2–4: MG-treated groups II–IV. 15 μl protein at a concentration of 15 μM was loaded in each well.
Fig. 2. Mass spectrometric studies. Tryptic mass fingerprint spectra of control (group I) rat hemoglobin (A) and MG-treated (group IV) rat hemoglobin (B). In the spectrum of MG-treated rat hemoglobin, AGE-modified peptides are marked (☆).
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Table 1 Assignment of modified amino acid residues in MG-treated (group IV) rat hemoglobin. Observed mass (Da)
Theoretical mass (Da)
Peptide sequence
Mass increase(Da)
AGE identified
Modified residue
1141.67
1087.71
54
MG-H1
Arg-92α
1180.64
1126.56
54
MG-H1
Arg-104β
1583.79
1529.73
LR*VDPVNFK (91-99) LHVDPVNFR* (96-104) VGAHAGEYGA EALER* (17-31)
54
MG-H1
Arg-31α
The specific AGEs are indicated in the table and the modified amino acid residues are marked (*).
[31,32]. Similarly, MG has found to modify arginine residues of human serum albumin with low surface exposure area in a previous study [15]. This indicated that arginine residues located interior of the protein with low surface exposure can have solvent accessibility and susceptibility to modification. The reactive α-oxoaldehyde, glyoxal has also been found to modify Arg-31α of hemoglobin both under in vitro and in vivo conditions as reported in earlier studies from our lab [33,34]. Very recently, we have found MG to modify arginine residues of hemoglobin, namely, Arg-31α, Arg-92α, Arg-40β and Arg-104β to hydroimidazolone (MG-H1) adducts in vitro, resulting in subsequent changes of the heme protein [35]. However, in contrast to in vitro reports, in the present study MG was not found to modify either Arg-40 of β chain (located in 3/10 helix) or Arg-141 of α chain (non-helical region) of hemoglobin. This may be explained by the fact that glycation in vivo is site-specific whereby only a few amino acid residues are consistently modified in contrast to in vitro glycation which is a more complex and heterogeneous process.
Additionally, the extent and nature of modification is also regulated by concentrations of the reacting substances (MG, hemoglobin), reaction conditions, etc. Arg-30 of β chain (located in alpha-helix) of hemoglobin (3.6 Å2 in surface exposable area) was also not found to be modified in our present study, an observation consistent with previous findings. 3.3. Spectroscopic studies Fig. 3A shows the absorption spectra of hemoglobin of control and MG-treated rats. As shown in the figure, the absorbance spectrum of control rat hemoglobin exhibited Soret peak around 415 nm along with the presence of Q bands (absorbance peaks at 540 and 580 nm) characteristic of oxygenated Fe2 +-hemoglobin (oxyhemoglobin) (trace a) [36,37]. In oxyhemoglobin, O2 is bound to the Fe2+ on the opposite side of the porphyrin ring from the His ligand, so that Fe2+ is octahedrally coordinated. The absorbance spectrum of MG-treated rat hemoglobin were found to be more or less identical except around the
Fig. 3. Spectroscopic studies. (A) Absorbance spectra of control rat hemoglobin (trace a) and MG-treated rat hemoglobin (trace b) recorded in the 250–600 nm region. Protein concentration was kept at 3 μM for recording each spectrum. (B) Fluorescence emission of 3 μM each of control rat hemoglobin (trace a) and MG-treated rat hemoglobin (trace b) recorded in the region 320–400 nm with excitation at 280 nm. (C) Fluorescence emission of ANS in presence of control rat hemoglobin (trace a) and MG-treated rat hemoglobin (trace b). The protein samples (8 μM each) were incubated with 20 μM ANS for 10 min at room temperature before recording the fluorescence emission. The spectrum of each sample was recorded in the region 450–600 nm with excitation at 370 nm. (D) CD spectra of 3 μM each of control rat hemoglobin (trace a) and MG-treated rat hemoglobin (trace b) recorded in the region 190–250 nm.
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aromatic region where the latter exhibited slightly decreased absorbance (trace b) in comparison with the former (trace a), possibly indicating MG-induced subtle changes in tertiary structure of the heme protein. Fluorescence emission of MG-treated rat hemoglobin (trace b) was found to be lower than that of control rat hemoglobin (trace a) (Fig. 3B). The fluorescence λmax was found to be around 330 nm in both cases. A decrease in fluorescence emission without shift in λmax may occur due to increased exposure of aromatic residues to solvent molecules that collide with the fluorophores and quench the fluorescence energy [38,39]. However, in absence of shift in emission maxima, it is difficult to suggest the change in solvent accessibility of aromatic residues of MG-treated rat hemoglobin. Decrease in fluorescence emission without shift in λmax may also occur due to increased energy transfer (quenching) to heme moiety or neighbouring amide groups due to decrease in distance between heme moiety and aromatic residues on protein modification [38,40]. The extrinsic fluorescent dye ANS is charged, hydrophobic and used for studying surface hydrophobicity of proteins [41]. It is sulfonated naphthalene with aniline group, where the naphthalene backbone and aniline ring are hydrophobic, but sulfonate backbone has negative charge. The sulfonate group of ANS interacts with positively charged amino acids and the aromatic ring with that of the apolar groups, and the complementary interaction of both groups is the key mechanism of binding to proteins. ANS is strongly fluorescent when bound to proteins and non-fluorescent when surrounded by water. ANS binding was studied to determine the effect of MG on the surface hydrophobicity of the heme protein. Fig. 3C shows the fluorescence emission of ANS in presence of control and MG-treated rat hemoglobin. As shown in the figure, fluorescence intensity of ANS was found to be lower when bound to MG-treated rat hemoglobin (trace b) than when bound to control rat hemoglobin (trace a), suggesting a reduction of surface hydrophobicity of the heme protein on MG modification. Changes in tertiary structure of proteins may alter exposure of hydrophobic amino acid residues and ANS binding [41,42]. The CD spectra of control and MG-treated rat hemoglobin are shown in Fig. 3D. As shown in the figure, MG modification in vivo was not found to affect the native, secondary structure of hemoglobin significantly. MG-treated rat hemoglobin exhibited marginal increase in α helicity (76%) (trace b) compared to control rat hemoglobin (74%) (trace a). As reported in some previous in vitro studies, MG-induced modification has not been found to appreciably alter the secondary structures of several proteins, namely, αA-crystallin, insulin, α-lactalbumin, etc. [43, 44], an observation similar to our present in vivo finding. The related αoxoaldehyde, glyoxal, was also not found to affect the secondary structure of hemoglobin in vitro, as reported by us earlier [33]. In a recent study from our lab, MG modification was found not to significantly perturb the secondary structure of the heme protein in vitro [35]. 3.4. Stability studies DSC thermograms of the samples are shown in Fig. 4(A, B). Comparison of the melting profiles of control and MG-treated rat hemoglobin revealed slightly higher thermal stability of the latter (Tm value of 75.50 °C) (Fig. 4B) with respect to the former (Tm value of 73.67 °C) (Fig. 4A). MG modification thus appeared to enhance the thermal stability of experimental rat hemoglobin in vivo. Guanidine hydrochloride is a powerful denaturing agent, which denatures proteins by preferentially interacting with the backbone CONH groups and other polar groups in the side chains by forming multiple Hbonds [45,46], leading to the salting in of such groups in the aqueous solution. Intrinsic fluorescence measurement of the samples following overnight incubation with the denaturant showed λmax value of around 341 nm for control rat hemoglobin (trace a) and around 338 nm for MGtreated rat hemoglobin (trace b) (Fig. 4C). A lower red shift of λmax
Fig. 4. Stability studies. Thermal stability was detected by DSC study. DSC thermograms of control rat hemoglobin (A) and MG-treated rat hemoglobin (B) recorded over a definite temperature range. Conformational stability was detected by denaturant-induced unfolding study. (C) Fluorescence emission of control rat hemoglobin (a) and MGtreated rat hemoglobin (b) (3 μM each) recorded in the region 300–400 nm with excitation at 280 nm after overnight incubation with guanidine hydrochloride (500 mM).
indicated that the extent of unfolding of MG-treated rat hemoglobin was lower compared to control rat hemoglobin. Thus MG-treated rat hemoglobin exhibited higher resistance to chemical-induced denaturation compared to control rat hemoglobin. MG modification was thus found to enhance the stability of experimental rat hemoglobin in vivo without significant perturbation of its structure. The observation is supported by previous in vitro studies where MG has been found to enhance the stability of several proteins without affecting their structure. Thermal unfolding of protein generally initiates with local changes in tertiary structure associated with very little or no changes in overall
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secondary structural conformation, followed by significant perturbation of secondary structure with increasing temperature resulting in complete unfolding of the protein. Generally for heme proteins, thermal unfolding begins with changes in tertiary structure near the heme region affecting the interaction of heme with the neighbouring aromatic residues lining the heme pocket followed by global changes in secondary structure associated with complete removal of heme from the heme active site [40,47]. MG-induced changes in tertiary structure of the heme protein (as seen from results of intrinsic fluorescence) may be associated with changes in distance between the heme moiety and neighbouring aromatic residues, as described earlier. MG-induced changes may thus alter the interaction of heme with the surrounding residues lining the heme pocket which may affect the initiation of protein unfolding (generally associated with local changes in tertiary structure around the heme moiety including its interaction with neighbouring residues) and overall stability of the heme protein. In other words, increased stability of the heme-globin linkage and altered steric pattern due to MG-induced changes in tertiary structure of the heme protein may explain its increased thermal stability. Lower thermal stability of the heme proteins, hemoglobin and myoglobin has been found to be associated with lowering of stability of heme-globin linkage on glycation [7,48]. In recent studies from our lab, both glyoxal and MG were found to increase the stability (thermal or conformational) of hemoglobin in vitro by mild changes in tertiary structure of the heme protein [33,35]. The reactive oxoaldehyde has also been reported to enhance the thermodynamic stability of αA-crystallin by subtle change of tertiary structure without alteration of its secondary structure [49]. MG-mediated slight decrease in surface hydrophobicities of insulin, α-lactalbumin and γ-crystallin resulted in increased resistance to thermal and chemical stresses without significant structural alterations [44]. Electrostatic interactions and optimisation of charge-charge interactions have been reported to play a major role in enhancing the thermal stability of globular proteins [50–52]. Glycation-induced neutralization of positive charges was found to be associated with increase in secondary structural content and stability of α-crystallin [53]. On the other hand, glycation of human serum albumin and the proteolytic enzymes, trypsin or chymotrypsin has been found to increase their stability without significant change of secondary structure [54,55]. MG-induced modification of arginine residues of αA-crystallin to neutral hydroimidazolone adducts has been reported to enhance the stability of the protein without major structural perturbations [43,49]. In our present study, MG-induced modification of arginine residues to neutral AGE adducts also enhanced the stability of the heme protein, an observation similar to previous studies. Glyoxal or MG-derived hydroimidazolones have also been found to enhance the stability of hemoglobin in recent in vitro studies. Previous studies reported MG to interact with hemoglobin resulting in modification of arginine residues and formation of MG-H1 adducts in vitro [21,22], leading to changes in structural and functional properties of the heme protein, including enhanced free iron-mediated oxidative reactions [10]. In the present study, we have found MG to modify arginine residues of experimental rat hemoglobin to MG-H1 in vivo, resulting in changes of structure and stability of the heme protein. The in vivo experimental observations add to the previous in vitro studies for a detailed understanding of the interaction of MG with hemoglobin. Considering the increased level of MG in diabetes mellitus as well as its high reactivity, MG-induced structural modifications of the heme protein by MG-H1 may have physiological significance. However, further studies are necessary to understand how MG-derived AGE-mediated structural modifications of the heme protein are associated with change of its functional properties, particularly under in vivo physiological conditions. Acknowledgments S.B. received a research fellowship [Grant No. 09/028(0802)/2010EMR-1] from the Council of Scientific and Industrial Research, New
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Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Methyglyoxal administration induces modification of hemoglobin in experimental rats: An in vivo study Sauradipta Banerjee National Brain Research Centre, NH-8, Manesar, Gurgaon, Haryana 122051, India
a r t i c l e
i n f o
Article history: Received 20 October 2016 Received in revised form 14 December 2016 Accepted 21 December 2016 Available online 26 December 2016 Keywords: Methylglyoxal Hemoglobin Diabetes mellitus Maillard-like reaction Advanced glycation end products Hydroimidazolone
a b s t r a c t Methylglyoxal, a highly reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. In the present study, the effect of methylglyoxal on experimental rat hemoglobin in vivo has been investigated with respect to structural alterations and amino acid modifications, after external administration of the α-dicarbonyl compound in animals. Different techniques, mostly biophysical, were used to characterize and compare methylglyoxal-treated rat hemoglobin with that of control, untreated rat hemoglobin. In comparison with methylglyoxal-untreated, control rat hemoglobin, hemoglobin of methylglyoxal-treated rats (32 mg/kg body wt. dose) exhibited slightly decreased absorbance around 280 nm, reduced intrinsic fluorescence and lower surface hydrophobicity. The secondary structures of hemoglobin of control and methylglyoxal-treated rats were more or less identical with the latter exhibiting slightly increased α-helicity compared to the former. Compared to control rat hemoglobin, methylglyoxal-treated rat hemoglobin showed higher stability. Peptide mass fingerprinting analysis revealed modifications of Arg-31α, Arg-92α and Arg-104β of methylglyoxal-treated rat hemoglobin to hydroimidazolone adducts. The modifications thus appear to be associated with the observed structural alterations of the heme protein. Considering the increased level of methylglyoxal in diabetes mellitus as well as its high reactivity, AGE-induced modifications may have physiological significance. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Reducing sugars react with proteins by Maillard reaction to form advanced glycation end products (AGEs) involving Schiff base and Amadori products [1]. The non-enzymatic 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]. Besides glucose, other glycating agents include fructose [3], glyoxal [4], methylglyoxal [5], 3deoxyglucosone [6] etc. Findings from our laboratory indicate that glycation of hemoglobin by glucose [7,8], fructose [9] and methylglyoxal [10] promote iron release and free radical-mediated oxidative reactions. Methylglyoxal (MG), a highly reactive α-oxoaldehyde, is mainly derived from triose phosphates D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate during glycolysis in eukaryotic cells, and its blood level increases in both type 1 and type 2 diabetes mellitus [11– 13]. The median concentration of MG is increased by 5–6-fold and 2– 3-fold in blood samples of diabetic patients with Type 1 and Type 2 diabetes mellitus, respectively [14], and the formation of MG-derived
E-mail addresses: [email protected], [email protected].
http://dx.doi.org/10.1016/j.jphotobiol.2016.12.028 1011-1344/© 2016 Elsevier B.V. All rights reserved.
AGEs is increased accordingly. MG-derived AGEs have been reported with different proteins namely insulin [5], human serum albumin [15], cytochrome c [16], α-synuclein [17], superoxide dismutase [18], etc. We have reported MG-induced structural alterations of myoglobin in recent studies [19,20], etc. In vitro reaction of hemoglobin with MG has been reported in some earlier studies. In a brief report, Chen et al. have shown that MG interacts with hemoglobin with modifications of arginine residues forming hydroimidazone (MG-H1) [21]. Gao and Wang have found that the sites and extents of MG modification of arginine residues are correlated with solvent accessibility of these residues [22]. We have recently reported interaction of MG with hemoglobin leading to modification of arginine residues and subsequent changes in structural and functional properties of the heme protein [10]. Besides several in vitro studies, a number of in vivo animal studies on MG have been published. The αoxoaldehyde has been reported to induce diabetes-like complications and retinal injury in experimental rats [23,24]. MG administration has been also shown to cause glucose intolerance, brain mitochondrial impairment and peritoneal fibrosis in rats [25–27]. Although in vitro interaction of MG with hemoglobin has been shown in previous studies, till date no studies describing the effect of MG on hemoglobin in vivo have been reported. Considering this, in the present study, we have administered MG in experimental rats and investigated its effect on rat
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2. Materials and methods
For ANS binding study, the samples (8 μM each) were incubated with 20 μM ANS for 10. minutes at room temperature and the fluorescence emission spectra (450–600 nm) were recorded with excitation at 370 nm.
2.1. Materials
2.7. CD study
MG, Sephadex G-100, acrylamide, Coomassie R250, sequencing grade trypsin, α-cyano-hydroxycinnamic acid matrix (CHCA), 1anilino-naphthalene-8-sulfonate (ANS), were purchased from Sigma Chemical Company, USA. All other reagents were AR grade and purchased locally.
CD spectra of the samples (3 μM each) were recorded in the far UV region (190–250 nm) in a spectropolarimeter (Jasco 600). The α-helical contents were calculated following the method of Chen et al. [30].
2.2. Methods
DSC study was carried out to measure thermal stability of samples. The melting profiles were recorded in a VP-DSC Microcalorimeter by heating the samples (1 °C/min) over a definite temperature range. Before introduction into the calorimetric cells, the protein samples were thoroughly degassed. Conformational stability of samples was measured by chemical denaturant-induced unfolding study. Fluorescence emission spectra (300–400 nm) of samples (3 μM each) were recorded with excitation at 280 nm after overnight incubation with 500 mM guanidine hydrochloride.
hemoglobin in vivo with respect to structure and stability changes, as well as site(s) and nature of amino acid modifications.
Animal experiments were performed in accordance with regulations specified and monitored by the Institutional Ethics Committee. Male Wister rats (weighing 70–80 g) were divided in four groups - I, II, III and IV, consisting of four rats in each group. Group I rats were not treated with MG and denoted as control group. Groups II, III and IV rats were respectively injected with 8, 16 and 32 mg/kg body weight MG intravenously and were denoted as MG-treated groups. All animals were fed with standard diet and water ad libitum. Whole blood was collected from each group after one week by heart puncture and hemolysates prepared. 2.3. Separation of hemoglobin by size-exclusion column chromatography Hemolysates prepared from different groups of rats were subjected to native PAGE. Total hemoglobin was purified from hemolysates using Sephadex G-100 column chromatography [28] and subjected to native gel electrophoresis. The concentration of hemoglobin was determined from Soret absorbance using an extinction coefficient (ε415nm) of 125 mMˉ1 cmˉ1 (heme basis) [29]. 2.4. MALDI-TOF mass spectrometric study Hemoglobin obtained from control group I and MG-treated group IV rats were subjected to mass spectral analysis using the linear positive ion mode of MALDI-TOF MS after digestion with sequencing-grade trypsin in solution at 37 °C for 16 h using enzyme: protein ratio 1:100 (w/w). The digested samples (0.5 μl each) were loaded directly to the MALDI plate, mixed with 0.5 μl of saturated CHCA solution (prepared in 50% acetonitrile and 0.1% trifluoroacetic acid) and allowed to dry and crystallize. Mass spectra were recorded in a 4800 Proteomics Analyzer (MALDI-TOF/TOF mass spectrometer, Applied Biosystems) using the linear positive ion mode of MALDI-TOF MS at 20 kV acceleration voltage. Identification of MG-modified peptides and specific MG-derived AGE adducts was performed as described earlier [19,20]. Purified hemoglobin of group I and group IV rats were used for further experiments. 2.5. Absorbance spectroscopy Absorbance spectra of control and MG-treated rat hemoglobin (3 μM each) were recorded in a UV/VIS Spectrophotometer (Hitachi U 2000) using 1 ml quartz cuvette of path length 1 cm in the region 250– 600 nm, taking 3 μM protein in each case. 2.6. Spectrofluorimetric study Fluorescence emission spectra of purified hemoglobin of control and MG-treated rats were recorded in the region 320–400 nm with excitation at 280 nm in a spectrofluorimeter (Hitachi F-3010). Protein concentration was adjusted to 3 μM for recording each spectrum.
2.8. Stability studies
3. Results and Discussion 3.1. Separation of hemoglobin The native gel profile of hemolysates obtained from different groups of rats (I–IV) was found to be more or less identical, as shown in Fig. 1A (lanes 1–4). Hemoglobin was purified from the hemolysates of different groups by size-exclusion chromatography. Fig. 1B shows gel electrophoretic profile of hemoglobin purified from different groups. Purified hemoglobin of group I and group IV rats were subjected to further experiments. 3.2. Mass spectrometric study The tryptic mass fingerprint spectrum of control (group I) rat hemoglobin is shown in Fig. 2A. In the spectrum of MG-treated (group IV) rat hemoglobin (Fig. 2B), the peptides with m/z values 1141.67 Da, 1180.64 Da and 1583.79 Da indicated modifications of Arg-92α, Arg104β and Arg-31α to hydroimidazolone (MG-H1) adducts, respectively. The modified peptides were absent in control rat hemoglobin. All modifications were confirmed by MSMS fragmentation of the modified peptides (data not shown) using Collision Induced Dissociation [5,20]. Results are summarized in Table 1. The observation was found to be similar to previously published reports on MG-hemoglobin interaction studies, where the reactive α-oxoaldehyde was found to modify arginine residues of the heme protein to hydroimidazolone adducts in vitro [10,21,22]. Among the modified arginines in hemoglobin, Arg-104 in the β chain is most accessible to solvent (103 Å2 in surface exposable area), followed by Arg-92 in α chain (40 Å2 in surface exposable area), as estimated from the web-based program GETAREA [22]. Thus the solvent accessibility and hence MG modification of these residues may be correlated with their exposed surface area as reported earlier. Arg-104 β is located in an alpha-helical region whereas Arg-92 α is located in a nonhelical region of the heme protein with no distinct secondary structure (based on the structure of oxyhemoglobin- PDB ID: 1GZX). On the other hand, Arg-31 in α chain (located in alpha-helix) is buried inside the protein and its surface exposable area is 3.8 Å2. The observation that MG was found to modify Arg-31 in α chain is consistent with previous studies where the reactive α-oxoaldehyde modified arginine residues of the yeast protein enolase located in deep crevice at the dimer interface of the protein structure with reduced surface exposure
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Fig. 1. Native PAGE experiments. (A) Gel electrophoretic profile of hemolysates obtained from different groups of rat. Lane 1: control group I; lanes 2–4: MG-treated groups II–IV. (B) Gel electrophoretic profile of hemoglobin purified from different groups of rats. Lane 1: control group I; lanes 2–4: MG-treated groups II–IV. 15 μl protein at a concentration of 15 μM was loaded in each well.
Fig. 2. Mass spectrometric studies. Tryptic mass fingerprint spectra of control (group I) rat hemoglobin (A) and MG-treated (group IV) rat hemoglobin (B). In the spectrum of MG-treated rat hemoglobin, AGE-modified peptides are marked (☆).
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Table 1 Assignment of modified amino acid residues in MG-treated (group IV) rat hemoglobin. Observed mass (Da)
Theoretical mass (Da)
Peptide sequence
Mass increase(Da)
AGE identified
Modified residue
1141.67
1087.71
54
MG-H1
Arg-92α
1180.64
1126.56
54
MG-H1
Arg-104β
1583.79
1529.73
LR*VDPVNFK (91-99) LHVDPVNFR* (96-104) VGAHAGEYGA EALER* (17-31)
54
MG-H1
Arg-31α
The specific AGEs are indicated in the table and the modified amino acid residues are marked (*).
[31,32]. Similarly, MG has found to modify arginine residues of human serum albumin with low surface exposure area in a previous study [15]. This indicated that arginine residues located interior of the protein with low surface exposure can have solvent accessibility and susceptibility to modification. The reactive α-oxoaldehyde, glyoxal has also been found to modify Arg-31α of hemoglobin both under in vitro and in vivo conditions as reported in earlier studies from our lab [33,34]. Very recently, we have found MG to modify arginine residues of hemoglobin, namely, Arg-31α, Arg-92α, Arg-40β and Arg-104β to hydroimidazolone (MG-H1) adducts in vitro, resulting in subsequent changes of the heme protein [35]. However, in contrast to in vitro reports, in the present study MG was not found to modify either Arg-40 of β chain (located in 3/10 helix) or Arg-141 of α chain (non-helical region) of hemoglobin. This may be explained by the fact that glycation in vivo is site-specific whereby only a few amino acid residues are consistently modified in contrast to in vitro glycation which is a more complex and heterogeneous process.
Additionally, the extent and nature of modification is also regulated by concentrations of the reacting substances (MG, hemoglobin), reaction conditions, etc. Arg-30 of β chain (located in alpha-helix) of hemoglobin (3.6 Å2 in surface exposable area) was also not found to be modified in our present study, an observation consistent with previous findings. 3.3. Spectroscopic studies Fig. 3A shows the absorption spectra of hemoglobin of control and MG-treated rats. As shown in the figure, the absorbance spectrum of control rat hemoglobin exhibited Soret peak around 415 nm along with the presence of Q bands (absorbance peaks at 540 and 580 nm) characteristic of oxygenated Fe2 +-hemoglobin (oxyhemoglobin) (trace a) [36,37]. In oxyhemoglobin, O2 is bound to the Fe2+ on the opposite side of the porphyrin ring from the His ligand, so that Fe2+ is octahedrally coordinated. The absorbance spectrum of MG-treated rat hemoglobin were found to be more or less identical except around the
Fig. 3. Spectroscopic studies. (A) Absorbance spectra of control rat hemoglobin (trace a) and MG-treated rat hemoglobin (trace b) recorded in the 250–600 nm region. Protein concentration was kept at 3 μM for recording each spectrum. (B) Fluorescence emission of 3 μM each of control rat hemoglobin (trace a) and MG-treated rat hemoglobin (trace b) recorded in the region 320–400 nm with excitation at 280 nm. (C) Fluorescence emission of ANS in presence of control rat hemoglobin (trace a) and MG-treated rat hemoglobin (trace b). The protein samples (8 μM each) were incubated with 20 μM ANS for 10 min at room temperature before recording the fluorescence emission. The spectrum of each sample was recorded in the region 450–600 nm with excitation at 370 nm. (D) CD spectra of 3 μM each of control rat hemoglobin (trace a) and MG-treated rat hemoglobin (trace b) recorded in the region 190–250 nm.
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aromatic region where the latter exhibited slightly decreased absorbance (trace b) in comparison with the former (trace a), possibly indicating MG-induced subtle changes in tertiary structure of the heme protein. Fluorescence emission of MG-treated rat hemoglobin (trace b) was found to be lower than that of control rat hemoglobin (trace a) (Fig. 3B). The fluorescence λmax was found to be around 330 nm in both cases. A decrease in fluorescence emission without shift in λmax may occur due to increased exposure of aromatic residues to solvent molecules that collide with the fluorophores and quench the fluorescence energy [38,39]. However, in absence of shift in emission maxima, it is difficult to suggest the change in solvent accessibility of aromatic residues of MG-treated rat hemoglobin. Decrease in fluorescence emission without shift in λmax may also occur due to increased energy transfer (quenching) to heme moiety or neighbouring amide groups due to decrease in distance between heme moiety and aromatic residues on protein modification [38,40]. The extrinsic fluorescent dye ANS is charged, hydrophobic and used for studying surface hydrophobicity of proteins [41]. It is sulfonated naphthalene with aniline group, where the naphthalene backbone and aniline ring are hydrophobic, but sulfonate backbone has negative charge. The sulfonate group of ANS interacts with positively charged amino acids and the aromatic ring with that of the apolar groups, and the complementary interaction of both groups is the key mechanism of binding to proteins. ANS is strongly fluorescent when bound to proteins and non-fluorescent when surrounded by water. ANS binding was studied to determine the effect of MG on the surface hydrophobicity of the heme protein. Fig. 3C shows the fluorescence emission of ANS in presence of control and MG-treated rat hemoglobin. As shown in the figure, fluorescence intensity of ANS was found to be lower when bound to MG-treated rat hemoglobin (trace b) than when bound to control rat hemoglobin (trace a), suggesting a reduction of surface hydrophobicity of the heme protein on MG modification. Changes in tertiary structure of proteins may alter exposure of hydrophobic amino acid residues and ANS binding [41,42]. The CD spectra of control and MG-treated rat hemoglobin are shown in Fig. 3D. As shown in the figure, MG modification in vivo was not found to affect the native, secondary structure of hemoglobin significantly. MG-treated rat hemoglobin exhibited marginal increase in α helicity (76%) (trace b) compared to control rat hemoglobin (74%) (trace a). As reported in some previous in vitro studies, MG-induced modification has not been found to appreciably alter the secondary structures of several proteins, namely, αA-crystallin, insulin, α-lactalbumin, etc. [43, 44], an observation similar to our present in vivo finding. The related αoxoaldehyde, glyoxal, was also not found to affect the secondary structure of hemoglobin in vitro, as reported by us earlier [33]. In a recent study from our lab, MG modification was found not to significantly perturb the secondary structure of the heme protein in vitro [35]. 3.4. Stability studies DSC thermograms of the samples are shown in Fig. 4(A, B). Comparison of the melting profiles of control and MG-treated rat hemoglobin revealed slightly higher thermal stability of the latter (Tm value of 75.50 °C) (Fig. 4B) with respect to the former (Tm value of 73.67 °C) (Fig. 4A). MG modification thus appeared to enhance the thermal stability of experimental rat hemoglobin in vivo. Guanidine hydrochloride is a powerful denaturing agent, which denatures proteins by preferentially interacting with the backbone CONH groups and other polar groups in the side chains by forming multiple Hbonds [45,46], leading to the salting in of such groups in the aqueous solution. Intrinsic fluorescence measurement of the samples following overnight incubation with the denaturant showed λmax value of around 341 nm for control rat hemoglobin (trace a) and around 338 nm for MGtreated rat hemoglobin (trace b) (Fig. 4C). A lower red shift of λmax
Fig. 4. Stability studies. Thermal stability was detected by DSC study. DSC thermograms of control rat hemoglobin (A) and MG-treated rat hemoglobin (B) recorded over a definite temperature range. Conformational stability was detected by denaturant-induced unfolding study. (C) Fluorescence emission of control rat hemoglobin (a) and MGtreated rat hemoglobin (b) (3 μM each) recorded in the region 300–400 nm with excitation at 280 nm after overnight incubation with guanidine hydrochloride (500 mM).
indicated that the extent of unfolding of MG-treated rat hemoglobin was lower compared to control rat hemoglobin. Thus MG-treated rat hemoglobin exhibited higher resistance to chemical-induced denaturation compared to control rat hemoglobin. MG modification was thus found to enhance the stability of experimental rat hemoglobin in vivo without significant perturbation of its structure. The observation is supported by previous in vitro studies where MG has been found to enhance the stability of several proteins without affecting their structure. Thermal unfolding of protein generally initiates with local changes in tertiary structure associated with very little or no changes in overall
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secondary structural conformation, followed by significant perturbation of secondary structure with increasing temperature resulting in complete unfolding of the protein. Generally for heme proteins, thermal unfolding begins with changes in tertiary structure near the heme region affecting the interaction of heme with the neighbouring aromatic residues lining the heme pocket followed by global changes in secondary structure associated with complete removal of heme from the heme active site [40,47]. MG-induced changes in tertiary structure of the heme protein (as seen from results of intrinsic fluorescence) may be associated with changes in distance between the heme moiety and neighbouring aromatic residues, as described earlier. MG-induced changes may thus alter the interaction of heme with the surrounding residues lining the heme pocket which may affect the initiation of protein unfolding (generally associated with local changes in tertiary structure around the heme moiety including its interaction with neighbouring residues) and overall stability of the heme protein. In other words, increased stability of the heme-globin linkage and altered steric pattern due to MG-induced changes in tertiary structure of the heme protein may explain its increased thermal stability. Lower thermal stability of the heme proteins, hemoglobin and myoglobin has been found to be associated with lowering of stability of heme-globin linkage on glycation [7,48]. In recent studies from our lab, both glyoxal and MG were found to increase the stability (thermal or conformational) of hemoglobin in vitro by mild changes in tertiary structure of the heme protein [33,35]. The reactive oxoaldehyde has also been reported to enhance the thermodynamic stability of αA-crystallin by subtle change of tertiary structure without alteration of its secondary structure [49]. MG-mediated slight decrease in surface hydrophobicities of insulin, α-lactalbumin and γ-crystallin resulted in increased resistance to thermal and chemical stresses without significant structural alterations [44]. Electrostatic interactions and optimisation of charge-charge interactions have been reported to play a major role in enhancing the thermal stability of globular proteins [50–52]. Glycation-induced neutralization of positive charges was found to be associated with increase in secondary structural content and stability of α-crystallin [53]. On the other hand, glycation of human serum albumin and the proteolytic enzymes, trypsin or chymotrypsin has been found to increase their stability without significant change of secondary structure [54,55]. MG-induced modification of arginine residues of αA-crystallin to neutral hydroimidazolone adducts has been reported to enhance the stability of the protein without major structural perturbations [43,49]. In our present study, MG-induced modification of arginine residues to neutral AGE adducts also enhanced the stability of the heme protein, an observation similar to previous studies. Glyoxal or MG-derived hydroimidazolones have also been found to enhance the stability of hemoglobin in recent in vitro studies. Previous studies reported MG to interact with hemoglobin resulting in modification of arginine residues and formation of MG-H1 adducts in vitro [21,22], leading to changes in structural and functional properties of the heme protein, including enhanced free iron-mediated oxidative reactions [10]. In the present study, we have found MG to modify arginine residues of experimental rat hemoglobin to MG-H1 in vivo, resulting in changes of structure and stability of the heme protein. The in vivo experimental observations add to the previous in vitro studies for a detailed understanding of the interaction of MG with hemoglobin. Considering the increased level of MG in diabetes mellitus as well as its high reactivity, MG-induced structural modifications of the heme protein by MG-H1 may have physiological significance. However, further studies are necessary to understand how MG-derived AGE-mediated structural modifications of the heme protein are associated with change of its functional properties, particularly under in vivo physiological conditions. Acknowledgments S.B. received a research fellowship [Grant No. 09/028(0802)/2010EMR-1] from the Council of Scientific and Industrial Research, New
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