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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

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Fructose-induced modications of myoglobin: Change of structure from met (Fe3+ ) to oxy (Fe2+ ) form
Abhishek Bhattacherjee, Abhay Sankar Chakraborti
Department of Biophysics, Molecular Biology and Bioinformatics, University College of Science, 92, Acharya Prafulla Chandra Road, Kolkata 700009, India

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We studied structural modications of metmyoglobin (Mb) after short-term (6 days) and long-term (30 days) glycation by fructose (fructation). Fructation caused gradual changes in the structure of the protein with respect to increased absorbance at 280 nm, enhanced uorescence emission (with excitation at 285 nm), increased surface accessible tryptophan residues and reduced -helix content and change in tertiary structure. However, long-term fructation changed Mb to oxymyoglobin (MbO2 ), as demonstrated by different spectroscopic (absorption, uorescence, circular dichroic and electron paramagnetic resonance) studies and triuoperazine-induced oxygen release experiment. Fructation appeared to modify Arg139 to arg-pyrimidine, which exhibited antioxidative activity and might be involved in the conversion of met (Fe3+ ) to oxy (Fe2+ ) form of myoglobin. 2010 Published by Elsevier B.V.

Article history: Received 30 September 2010 Received in revised form 4 November 2010 Accepted 9 November 2010 Available online xxx Keywords: Myoglobin Fructation Diabetes mellitus Advanced glycation end products Heme iron Arg-pyrimidine

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1. Introduction Protein modication by non-enzymatic glycation is a complex series of reactions, collectively called Maillard reaction, which may be signicant with increased level of blood sugar over prolonged periods of time in diabetes. The key step in the modication of proteins by glucose is Schiff base formation, followed by Amadori rearrangement [1]. The Amadori products then undergo oxidative cleavage resulting in the formation of advanced glycation end products (AGEs) [2]. The rst indication that a simple chemical reaction between glucose and free amino groups on protein can lead to irreversible modication came with the characterization of hemoglobin A1c (HbA1c ), which has the N-terminus (valine) of the -chains linked to glucose [3]. Findings from our laboratory indicate that HbA1c exhibits structural and functional changes, and it may be a source of iron-mediated free radicals and oxidative stress in diabetic condition [47]. Glycation also induces modications of another heme containing protein myoglobin [8,9], and is also asso-

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Abbreviations: Mb, metmyoglobin; MbO2 , oxymyoglobin; HbA1c , hemoglobin A1c ; AGE, advanced glycation end product; Fr-Mb6, 6 days fructated myoglobin; Fr-Mb30, 30 days fructated myoglobin; CD, circular dichroism; EPR, electron paramagnetic resonance; TFZ, triuoperazine. Corresponding author. Tel.: +91 33 2350 8386x327; fax: +91 33 2351 9755. E-mail addresses: ascbmbg@caluniv.ac.in, abhay chakraborti@yahoo.co.in (A.S. Chakraborti). 0141-8130/$ see front matter 2010 Published by Elsevier B.V. doi:10.1016/j.ijbiomac.2010.11.003

ciated with oxidative reactions. Glycated heme proteins may thus be associated with eliciting oxidative stress in diabetes mellitus, which is considered as a free radical mediated disease [10]. Although glucose-induced protein glycation has been emphasized in most studies involving Maillard reaction, fructose-induced glycation, known as fructosylation or fructation, is also possible and is considered a highly probable event in diabetic condition [11]. Fructose is more potent in glycation and AGE formation than glucose [12]. Moreover, glucose to fructose shunt via polyol pathway becomes more active in diabetic condition resulting in increased concentration of fructose [13]. Fructose-induced Maillard reactions may thus play a signicant role in the pathogenesis of diabetic complications. Recent studies including one from our laboratory indicate that fructose interacts with hemoglobin causing structural and functional alterations of the protein [1416]. The ndings are signicant, but at preliminary stage. We have, therefore, studied time-dependent structural changes of a heme protein induced by fructose to understand the short-term and long-term effects of non-enzymatic fructation. In the present study, we have used myoglobin, a simple monomeric heme protein, which is very similar with or subunit of tetrameric hemoglobin ( 2 2 ) in several respects. After in vitro fructation of metmyoglobin for different periods of time, the fructated proteins have been separated and compared with the structural properties of the normal protein. The study demonstrates that fructation induces progressive changes in the structure of myoglobin, leading to its conversion from metmyoglobin to oxymyoglobin. Arg-pyrimidine, the AGE formed by

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fructation of arginine residues, may be involved in the conversion of met (Fe3+ ) to oxy (Fe2+ ) form of myoglobin. 2. Materials and methods 2.1. Materials Horse heart myoglobin (Mb), d() fructose and triuoperazine (TFZ) were purchased from Sigma Chemical Co., USA. BioRex 70 resin (200400 mesh) and 1,2-cyclohexanedione were obtained from BioRad, India, and Alfa Acer, USA, respectively. Other chemicals were of analytical grade and purchased locally. The standard arg-pyridine was obtained as a gift from Dr. R. Nagaraj, Case School of Medicine, Ohio, USA. 2.2. In vitro fructation of Mb Purchased Mb, which is mostly in met form, was dissolved in 50 mM potassium phosphate buffer, pH 6.0 and its concentration was determined using 408 nm = 116 mM1 cm1 [17]. For in vitro fructation, 100 M Mb solution was mixed with 100 M fructose solution (nal volume 1.0 ml). The solutions were sterile-ltered to maintain aseptic condition. The mixture was divided into several parts and incubated at 25 C for different days (3, 6, 9, 12, 18, 24 and 30 days). For control sets, only Mb or fructose solutions were incubated for the respective time periods. After incubation, amount of free fructose left in the samples was estimated following the method of Roe [18]. 100 l sample was mixed with 12.5 l each of 10% ZnSO4 and 0.5 M NaOH. After centrifugation, the supernatant was collected. 30% HCl and 0.1% alcoholic resorcinol (25 l each) were added to the supernatant and was incubated at 80 C for 15 min. The absorbance was taken at 505 nm and the amount of fructose consumed by the protein was determined. 2.3. Separation of fructated Mb (Fr-Mb) and unchanged Mb After in vitro fructation, Fr-Mb and Mb present in the reaction mixtures were separated following the method previously described for separation of Mb glycation mixture [8]. Briey, two fractions (fractions I and II) were separated from incubation samples by elution buffers 50 mM potassium phosphate of pH 6.6 and pH 7.0 in a BioRex 70 ion exchange column (7.0 cm 1.0 cm), pre-equilibrated with 50 mM potassium phosphate buffer, pH 6.0. Fraction I, isolated as fructose-modied Mb from different incubation samples, was denoted as Fr-Mb3, Fr-Mb6, Fr-Mb12, etc. Fraction II appeared to be unchanged Mb. The characteristics of fraction II and freshly prepared Mb solutions were almost similar. Comparisons were, therefore, shown here between Fr-Mb fractions and freshly prepared Mb solution. In most of the experiments, Fr-Mb6 and Fr-Mb30 were used as the short-term (6 days) and long-term (30 days) fructated proteins, respectively, for comparing structural properties with the normal protein, Mb. The concentrations of Fr-Mb6 and Fr-Mb30 were determined using 408 nm = 116 mM1 cm1 and 418 nm = 128 mM1 cm1 , respectively, and are used for nding concentrations of Mb and MbO2 , respectively [17]. 2.4. Spectroscopic studies Absorption spectral (250600 nm) analysis of 3 M each of Mb, Fr-Mb6, Fr-Mb30 and MbO2 was done using UV/VIS Spectrophotometer (Hitachi U2000). MbO2 was prepared from Mb according to the method of Dixon and McIntosh [19]. Fluorescence emission spectra of the protein fractions (3 M) were recorded with excitation at 285 nm in a spectrouorimeter (Hitachi F-3010). Surface accessibility of tryptophan residues in Mb,

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Fr-Mb6 and Fr-Mb30 was estimated from the dynamic quenching of the protein uorescence by using acrylamide as a neutral quencher. The tryptophan uorescence of the protein (3 M) was quenched by adding increasing concentration of the quencher, from which Lehrer plots [20] and SternVolmer plots [21] were constructed to measure the surface accessibility of tryptophan residues of the proteins. For AGE estimation, the uorescence emission spectra (420520 nm) of the fractions (3 M) were recorded with excitation at 370 nm [22]. Circular dichroic (CD) spectra of the fractions (6 M) were recorded in the regions 200250 nm, 250350 nm and 400600 nm in Jasco 600 spectropolarimeter, and -helical contents of the proteins were determined according to the method of Geraci and Parkhurst [23]. The percentages of conformation and random coil were calculated using the program K2D [24]. Electron paramagnetic resonance (EPR) spectra of Mb, FrMb fractions and MbO2 (10 M) were recorded at 100 C in a Jeol EPR spectrometer (JES-FA100) with instrument settings: frequency = 9.23 GHz, center magnetic eld = 290 mT, Mod = 100 kHz, time constant = 0.3 s, power = 0.99 mW, sweep time = 2.0 min and amplitude = 1600. Both frequency and magnetic eld of EPR signals were used to calculate g values [25].

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2.5. Oxygraph experiments Oxygen release from Mb, Fr-Mb6, Fr-Mb30 and MbO2 by TFZ was measured in an oxygraph machine (Hansatech instruments, England), following previously published method [6]. The change in partial pressure due to released oxygen in the protein solution (2 ml, 10 M) in a stoppered cell was detected by the membrane covered oxygen electrode tted with the cell. The output signal was recorded at 25 C in the oxygraph chart as a function of time. The phosphate buffer alone or the protein sample in the absence of TFZ showed no change in the output signal. Considering 250 nmol dissolved oxygen present in 1 ml buffer [26], the oxygraph chart was calibrated in terms of nmol oxygen release from the change in output signal due to the total depletion of free oxygen from 2 ml buffer when 0.1 gm sodium dithionite was added.

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2.6. Molecular modeling studies The downloaded protein data bank le Mb (PDB ID-1DWR) and the ligand le of fructose (PDB ID-2051) were read in AutoDockTools (ADT) version 4 [27]. A three-dimensional box (grid) was created, in which the protein molecule and ligand were enclosed. The AutoGrid 4 job was run to create one map for every atom type in the ligand. AutoDock and Pymol were used to calculate the binding energies and for viewing the molecular graphics, respectively, in the docked ligandprotein complexes. Ramachandran plot was prepared to test the validity of the model used for the study [28].

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2.7. Fructation of Mb after modication by 1,2-cyclohexanedione Arginine residues of Mb were modied by reaction with 1,2cyclohexanedione [29]. The reaction mixture (1 ml) containing 7 mg Mb and 1 mg 1,2-cyclohexanedione in 0.2 N NaOH was incubated at 25 C for 18 h and was dialyzed against water. The chemically modied Mb thus prepared was used for in vitro fructation reaction and free fructose remained in the reaction mixtures was determined. For control experiment, Mb was treated similarly in the absence of 1,2-cyclohexanedione and was used for fructation reaction.

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2.8. High performance liquid chromatographic (HPLC) separation of AGEs formed in long-term fructated Mb Fructation-induced AGEs were separated essentially following the method of Wilker et al. [30] with few modications. Briey, after 30 days incubation of Mb (100 M) with fructose (100 M), the reaction mixture (500 l) was treated with equal volume of 10% trichloroacetic acid (TCA). The precipitated protein was collected, lyophilized and hydrolyzed with 6N HCl (200 l) at 110 C for 16 h. The sample was then lyophilized and dissolved in water (200 l) followed by ltration through a 0.45 m nylon syringe lter. The ltrate (100 l) was injected into C18 reverse phase analytical column (250 mm 4.60 mm, Phenomenex, USA). The solvents used were: solvent A water:orthophosphoric acid (99:1) and solvent B acetonitrile:water:orthophosphoric acid (60:39:1). A linear solvent gradient (03 min 0% B, 325 min 30% B, 2535 min 35% B, 3545 min 60% B, 4550 min 100% B, 5055 min 50% B, 5565 min 0% B) was used with a ow rate 1.0 ml/min. The column efuent was monitored at 330 nm. Two fractions (I and II) were separated from Fr-Mb30 hydrolysate. The standard arg-pyrimidine was also subjected to HPLC and the eluant appeared to be the same with the fraction II. 2.9. Spectrophotometric and Fourier transformed infra red (FT-IR) study of fraction II Fraction II was used for spectral and FT-IR analysis by using UV/VIS Spectrophotometer and PerkinElmer Spectrum 100, respectively. Standard arg-pyrimidine was also similarly characterized. 2.10. Assay of antioxidant property and reducing power of fraction II The antioxidant property of fraction II was assayed according to the method of Prieto et al. [31]. The sample (10 M, 300 l) was incubated with 3 ml of a reagent mixture containing 0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate at 95 C for 90 min. After cooling, the absorbance of the solutions was recorded at 695 nm. The antioxidant activity was expressed as the number of equivalents of gallic acid, used as the standard. The reducing power of fraction II was quantied by the method of Yen and Chen [32]. The reaction mixture (1 ml) containing the sample (10 M) and potassium ferricyanide (1%) was incubated at 50 C for 20 min. After addition of TCA solution (nal concentration 10%), the mixture was centrifuged. The supernatant (125 l) was mixed with 125 l each of distilled water and ferric chloride (0.1%) solution. The mixture was incubated at 37 C for 10 min and absorbance was measured at 700 nm. The absorbance indicative of reducing power was compared with that of gallic acid standard. The antioxidant property and reducing power were also tested with arg-pyrimidine. 3. Results and discussion Several proteins namely hemoglobin [1416], crystallins [33], glyceraldehydes 3-phosphate dehydrogenase, catalase, superoxide dismutase [34], albumin [35] and aspartate amino transferase [36] have been reported to be the targets of fructose modication. Although glycation of Mb has been reported from our laboratory [8,9], there has been no study on its fructation. Myoglobinuric renal failure in diabetic condition is known [37,38], and it may result from free radical damage involving free iron release [39]. Glycation of Mb induces iron-mediated free radical damage of cell constituents [8], indicating its harmful association with pathological complications. Compared to glucose, its ketose isomer fructose is more

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reactive in Maillard reaction [12], because fructose has higher concentration of acyclic forms in aqueous solutions [40]. Moreover, glucose to fructose shunt via polyol pathway becomes more active in diabetic condition [13], causing increased fructose concentration. Considering these facts, it seems worthwhile to study the fructation of Mb. Here we have reported how fructation changes the structure of Mb in a time-dependent manner. 3.1. In vitro fructation of Mb and separation of the fructated proteins After reaction of Mb with fructose for different number of days, the amounts of fructose consumed were calculated from free sugar levels in the samples. The fructose consumption increased proportionately up to 18 days, after which the consumption did not change (Fig. 1a). After in vitro fructation, fructated myoglobin fraction and remaining unchanged protein Mb were separated by ion exchange chromatography. Fractions I and II were eluted with phosphate buffer pH 6.6 and pH 7.0, respectively, as shown in the elution proles of 6 days and 30 days incubation mixtures (Fig. 1b). The elution proles of intermediate reaction mixtures also gave two fractions (not shown). Fresh Mb solution was eluted with phosphate buffer pH 7.0 at the same position of fraction II, indicating the fraction being unchanged Mb. Fraction I was, therefore, considered to be the fructated protein. The fructation reaction done at neutral pH also exhibited almost similar kinetic prole (not shown) with that obtained at pH 6.0 (Fig. 1a). However, since the buffers of pH 6.6 and 7.0 were used for separation of the fractions (I and II) by ion exchange chromatography, the fructation reaction was done at a lower pH 6.0. AGE formation in Mb due to fructation was studied by exciting the separated fructated proteins at 370 nm [21]. As shown in Fig. 1c, the emission maxima at 443 nm of the fructated proteins increased progressively, indicating increased AGE formation with the extent of fructation. Although the extent of fructation did not increase after about 18 days of incubation under our experimental condition (Fig. 1a), AGE formation, which requires conversion of Amadori products through a number of steps [41], continued for a much longer period. 3.2. Spectroscopic studies with normal and fructated Mb fractions 3.2.1. Absorption spectroscopy Absorption spectra (250600 nm) of Mb and Fr-Mb6 fractions were almost similar with exception of slight increase in absorbance around 280 nm region for the fructated protein (Fig. 2a). The Soret peaks obtained with both fractions were around 410 nm. Fr-Mb30, on the other hand, exhibited several differences in the absorption spectrum. Absorbance at 280 nm region further increased, Soret peak shifted to around 420 nm and Q bands appeared. Increase in absorbance of the fructated proteins near 280 nm may represent the changes in the environment of tyrosine and tryptophan residues by exposing their aromatic side chains. However, increased absorbance of the modied proteins in UV regions may also be due to the production of UV-absorbing substances like ketoamines, dicarbonyls and further developed AGEs [42]. The shifting of Soret peak and appearance of Q bands indicate oxy-form of myoglobin, which was also evident from the spectrum of MbO2 . The oxygen content of Fr-Mb30 was calculated from the Huang and Redeld equation [43]: oxygen content (%) = 12.73 9.92(A560 /A540 ) 100/3.77 + 3.88(A560 /A540 ), where A560 and A540 indicate absorbances at 560 nm and 540 nm, respectively, and the oxygen content was found to be approximately 95%.

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Fig. 1. In vitro fructation of Mb. (a) Fructation of Mb as a function of time. After different days of incubation of mixtures containing 100 M each of Mb and fructose at 25 C, amount of free fructose left in the samples was estimated to nd the extent of fructation. The results are mean SD of four sets of experiments. (b) The elution prole of the fructated and unchanged Mb fractions. The fractions were separated from 6 days and 30 days incubation mixtures by ion exchange chromatography (BioRex 70) using different pH (6.6 and 7.0) of 50 mM phosphate buffer. Freshly prepared Mb solution was also eluted under identical condition. (c) Fluorescence emission spectra (excitation 370 nm) representing AGE formation in Mb and separated Fr-Mb fractions.

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3.2.2. Fluorescence spectroscopy When excited at 285 nm, Mb and fructated fractions exhibited emission maxima at 340 nm. However, the intrinsic uorescence of the fructated proteins was signicantly higher compared to that of Mb (Fig. 2b). The extent of emission increased with extent of fructation as shown by higher emission of Fr-Mb30 than that of Fr-Mb6. Enhanced emission of the fructated proteins with excitation at 285 nm may be correlated with increased absorbance of the modied proteins around 280 nm.

To measure the surface accesibilility of tryptophan residues in Mb and fructated proteins, tryptophan uorescence quenching was studied with addition of increasing concentration of acrylamide. Lehrer plots were constructed using the relation [20]: F0 / F = 1/f + 1/fK1/[Q], where f is the fraction of tryptophan quenched, F = F0 F, where F0 and F are the uorescence intensities without and with the quencher (Q), respectively, and K is the dynamic quenching constant. From Lehrer plots (Fig. 2c), the surface accessible tryptophan residues, as measured from f (estimated

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Fig. 2. Spectrophotometric and spectrouorimetric studies of different protein fractions. (a) Absorption spectra of Mb, Fr-Mb6, Fr-Mb30 and MbO2 . 3 M protein fractions were taken in 1 ml cuvette with pathlength 1 cm. (b) Fluorescence emission spectra (excitation at 285 nm) of the proteins (3 M) in 1 ml cuvette with pathlength 1 cm. (c) Lehrer plots of Mb, Fr-Mb6 and Fr-Mb30 for the determination of surface accessibility of tryptophan residues to acrylamide for quenching interactions. The quenched uorescence of the protein at 334 nm (excitation 285 nm) with successive additions of acrylamide was measured for the plots. The results are mean SD of four different experiments.

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Fig. 3. CD spectroscopic studies of different protein fractions. (a) CD spectra of Mb, Fr-Mb6 and Fr-Mb30 in far UV region. 6 M protein fractions were taken in 1 ml cuvette with pathlength 1 mm. (b) Percentage of -helix, conformation and random coil in different protein fractions. Distribution of conformation and random coil were calculated from the CD spectra in far UV region by using K2D software. (c) CD spectra of Mb, Fr-Mb6 and Fr-Mb30 in near UV region. 6 M protein fractions were used in 1 ml cuvette with pathlength 1 mm. (d) CD spectra of 6 M each of Mb, Fr-Mb6, Fr-Mb30 and MbO2 in visible region using 1 ml cuvette with pathlength 1 mm.

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from the intersection of the linear plots with F0 / F axis) were found to be approximately 46%, 60% and 71%, for Mb, Fr-Mb6 and FrMb30, respectively. To verify this further, SternVolmer plots were constructed for quenching of the proteins by acrylamide using the relation [21]: F0 /F = 1 + Ksv [Q], where Ksv is the quenching constant, a measure of surface accessibility of tryptophan residues. From SternVolmer plots (plots not shown) the surface accessible tryptophan residues were found to be approximately 30%, 41% and 58% for Mb, Fr-Mb6 and Fr-Mb30, respectively. Mb contains two tryptophans (Trp7 and Trp14), which are partially exposed. Fructation enhances their exposure, justifying increased absorbance around 280 nm as well as enhanced protein uorescence with excitation at 285 nm. Similar structural modication was obtained with glycation of Mb [9]. 3.2.3. CD spectroscopy Fructation-induced structural changes of Mb were studied by CD spectral analysis. Fig. 3a exhibits spectra at far UV region (200250 nm). Changes in the ellipticity at 222 nm in the CD spectra are useful probes for visualizing -helix contents. Compared to Mb, the fructated proteins Fr-Mb6 and Fr-Mb30 showed gradual decrease in negative ellipticity in the region 210225 nm. Molar ellipticity [] value obtained using the relation [23]: [] = [Mr W]/10lc, where c (g/ml) is the concentration of protein, (mdeg), obtained directly from dichrograph chart, is the observed rotation, l (cm) is the pathlength and Mr W is the mean residual molecular weight 110 of the amino acid. The -helical contents of protein samples were estimated according to the relation: Fraction of -helix = ([]222 + 2340)/30,300, where []222 is the ellipticity at 222 nm. The -helical contents of Mb, Fr-Mb6 and Fr-Mb30 appeared to be approximately 83%, 75% and 57%, respectively. The reduced -helix contents in fructated proteins over Mb is probably due to alteration in the spatial arrangement of intra-molecular amino acid residues causing an increased volume of the protein. The percentages of conformation and random coils were found to be

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approximately 0 and 17 in Mb, 0 and 25 in Fr-Mb6 and 16 and 27 in Fr-Mb30, respectively, as shown in Fig. 3b. The reduced -helix contents in fructated proteins are thus compensated by increase in random coiling suggesting an increased volume of the protein due to fructation. Long-term fructation product Fr-Mb30 exhibits to transfer, which has also been reported for reaction of hemoglobin with fructose [15]. The induced beta sheet in Fr-Mb30 may tend to aggregate the modied protein. However, the amount of the fraction used in the experiments was quite low and probably below the critical concentration for aggregation to be observed. Besides secondary structure, fructation also induces change in the tertiary structure of myoglobin, as demonstrated by the difference in CD spectra of Mb and fructated proteins in near UV region (250320 nm) shown in Fig. 3c. The ellipticity of Mb around 270 nm decreased after 6 days fructation, and shifted to 282 nm after 30 days fructation. The peak at 294 nm decreased gradually with the extent of fructation. These indicate a change in the tertiary structure of the protein. CD spectra in visible region (400450 nm) shown in Fig. 3d indicated Soret peak position of Fr-Mb30 at 420 nm, characteristic of the oxymyoglobin, while those of Fr-Mb6 and Mb were at 410 nm, as expected for the met protein. CD spectrum of MbO2 in this region also exhibited peak position at 420 nm, indicating similarity with Fr-Mb30. 3.2.4. EPR spectroscopy The EPR spectra of the protein fractions are shown in Fig. 4. Both Mb and Fr-Mb6 fractions exhibited similar spectra, showing two peaks corresponding to g values 5.62 (high-spin signal) and 2.097 (low spin and radical signal). However, Fr-Mb30 and MbO2 fractions did not show high spin iron signal. There was only one peak in their spectra corresponding to g values 2.178 and 2.032 for FrMb30 and MbO2 , respectively, indicating low spin signal for both the proteins. Mb exhibits large EPR signal due to binding of water molecule to the sixth coordination position of its ferric iron atom

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Fig. 4. EPR spectra of Mb, Fr-Mb6, Fr-Mb30 and MbO2 . The sample (10 M) volume used was 20 l. JEOL FA-SERIES software was used to calculate g.

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[44]. On the other hand, deoxymyoglobin having no unpaired electrons, and MbO2 having even number of unpaired electrons, do not show high spin signals in EPR spectra. 3.3. TFZ-induced oxygen release experiments TFZ interacts with MbO2 and HbO2 , and releases oxygen from the heme proteins [6,45]. TFZ-induced oxygen release from Mb, Fr-Mb6 and Fr-Mb30 was measured in an oxygraph machine. The output signal was recorded in the oxygraph chart as a function of time. In the absence of TFZ, no oxygen was released from the protein fractions. Fig. 5a is the representative oxygraph chart for release of oxygen from Fr-Mb30 due to gradual addition of TFZ (25, 37, 49, 61, 73 and 85 M) at 5 min interval. The percentage of oxygen release from Fr-Mb30 increased gradually up to the addition of 61 M TFZ, after which it was leveled off (Fig. 5b). Fr-Mb30 thus behaved as oxy form, from which oxygen was released by interaction with the drug. On the other hand, under identical experimental condition, no oxygen was released from either Fr-Mb6 or Mb (not shown). Long-term fructation thus induces a change in myoglobin from met form to oxy form, which is evident from the characteristics of Fr-Mb30: (a) the position of Soret peak around 420 nm as well as appearance of the Q band in the absorption spectrum, (b) peak posiFig. 5. TFZ-induced oxygen release from Fr-Mb30. (a) Representative oxygraph chart of the release of oxygen from 2 ml Fr-Mb30 (10 M), using TFZ of different nal concentrations at 5 min interval. (b) Percentage of oxygen release from FrMb30 by different concentrations of TFZ in the oxygraph experiments. Results are mean SD of four different experiments.

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tion around 420 nm in the CD spectrum in visible region, (c) only low spin iron signal in the EPR spectrum and (d) TFZ-induced oxygen release. Moreover, these characteristics are in well agreement with those of MbO2. 3.4. Molecular modeling studies Using the software AutoDock 4 [27], docking was performed. Fructose docking in a specic pocket of Mb is shown in Fig. 6a. Fig. 6b shows possible hydrogen bondings (numbered 14) between fructose and nearby amino acid residue Arg139 of the helix H of Mb. Gibbs free energy of binding was minimized to 8.89 kcal/mole. The model used for docking was tested by Ramachandran plot (Fig. 6c), in which almost all points were found

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Fig. 6. Molecular modeling study. (a) Space lling model showing Mb (PDB ID-1DWR) with docked fructose (PDB ID-2051). (b) Ribbon model of a part of H helix of Mb showing Arg139 (ball and stick model) as the probable site of modication by fructose (space lling model). Numbers 14 indicate possible hydrogen bonds between fructose and different atoms of Arg139. (c) Ramachandran plot showing validity of the model constructed from PDB ID-1DWR.

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A. Bhattacherjee, A.S. Chakraborti / International Journal of Biological Macromolecules xxx (2010) xxxxxx Table 1 Antioxidant and reducing properties of fraction II and arg-pyrimidine. Sample Arg-pyrimidine (1 M) Fraction II (1 M) Antioxidant property (gallic acid equivalent) 551 mM 534 mM Reducing power (gallic acid equivalent) 688 mM 643 mM 7

The results are mean of three experiments (SD < 10%).

Fig. 7. Fructation of Mb and Arg-modied Mb. Arg residues in Mb are modied by 1,2-cyclohexanedione, as shown in the box. Mb and modied Mb were subjected to fructation reaction. Extents of fructose consumption are mean SD of three different experiments.

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inside the plot, indicating the validity of the model used for the study [28]. 3.5. Fructation experiment of chemically modied Mb To verify the possibility for Arg residues being the potential target of fructation, 1,2-cyclohexanedione-modied Mb was used for fructation reaction. 1,2-cyclohexanedione reacts with the guanidium group of Arg residues [29], as shown in the box of Fig. 7. The extents of fructation in chemically modied Mb appeared to be very low after both short-term (6 days) and long-term (30 days) incubation, compared to that in normal Mb (Fig. 7). Arg modication by 1,2-cyclohexanedione thus prevented fructation, suggesting the residue as the potential target of fructation. However, there are reports suggesting lysine residues of bovine serum albumin [32] and hemoglobin [16] as the targets of fructose. 3.6. Separation and characterization of AGEs formed in Fr-Mb30 AGEs were separated from acid hydrolyzate of long-term fructated Mb (Fr-Mb30) by using HPLC, and monitored by absorbance

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at 330 nm (Fig. 8a). Under our experimental condition, two fractions were eluted at 8.349 min (fraction I) and 9.902 min (fraction II). For control experiment, Mb was incubated for 30 days and treated similarly for the preparation of acid hydrolyzate. However, it did not exhibit any characteristic absorbance in the region 325335 nm indicating the absence of the AGEs in the unmodied protein. Arg-pyrimidine and pentosidine are important AGEs formed by Arg modication and have characteristic absorbances in the region 320330 nm [30]. Since both arg-pyrimidine and pentosidine have absorbance within a narrow region of wavelength, two of the fractions eluted in the chromatogram of the hydrolysate of Fr-Mb30 might be for these two AGEs. The standard arg-pyrimidine when subjected to HPLC under the same experimental condition, eluted at 9.90 min (inset of Fig. 8a), indicating the same position as that of fraction II. The spectral and FT-IR analysis, as shown in Fig. 8b and c, suggest the similarity of the standard arg-pyrimidine and fraction II. All these experiments indicate that fraction II obtained from Fr-Mb30 is arg-pyrimidine. Fraction II exhibited antioxidant property, as evident by converting Mo (VI) to Mo (V). It also showed reducing activity, as demonstrated by Fe3+ to Fe2+ conversion (Table 1). The activities, expressed in terms of gallic acid, were found to be quite comparable with those of standard arg-pyrimidine. The ndings further indicate that fraction II is arg-pyrimidine. Sreejayan et al. [46] have also shown that arg-pyrimidine reduces ferric ions to ferrous ions and by acting as an antioxidant scavenges free radicals. On the other hand, Kim et al. [47] have shown that methylglyoxal induces cellular damage and oxidative DNA damage by increasing argpyrimidine accumulation in human lens epithelial cells. A critical

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Fig. 8. Separation and characterization of AGE formed in Fr-Mb30. (a) Separation of fractions from acid hydrolysate of long-term fructated Mb (Fr-Mb30) by HPLC. Inset: Chromatogram of standard arg-pyrimidine. (b) Absorption spectra (300360 nm) of fraction II and standard arg-pyrimidine. (c) FT-IR spectra (4000400 cm1 ) of fraction II and standard arg-pyrimidine.

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evaluation of the effects of arg-pyrimidine is, therefore, necessary to understand the role of the AGE under physiological conditions. Our studies reveal that long-term fructation induces formation of arg-pyrimidine, which, in turn, reduces Mb(Fe3+ ) to Mb(Fe2+ ). The latter species (deoxymyoglobin) binds with oxygen to form MbO2 . 4. Conclusion Long-term fructation of Mb causes AGE formation together with conversion from met to oxy form. These two processes may be related as the AGE (arg-pyrimidine) formation may change the redox state of the heme iron forming MbO2 . Acknowledgements A.B. gets a fellowship from the RFSMS scheme of the University Grants Commission, New Delhi. A part of the study was supported by a grant from the Council of Scientic and Industrial Research, New Delhi [grant no. 38(1129)/03/EMR-II]. Thanks are due to the DBT Center of Bioinformatics, Presidency College, Kolkata for assistance in the molecular modeling studies. References
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