Probing hemoglobin glyco-products by fluorescence spectroscopy

Maillard reaction products (MRPs) participate in reactions of carbohydrate intermediates with proteins, resulting in the formation of advanced glycation end-products (AGEs). Dietary Maillard reaction products are recognized as potential chemical modifiers of human proteins. We have investigated the reaction of isolated MRPs from an asparagine–glucose model system with hemoglobin (Hb) to elucidate the binding effect of the MRPs in hemoglobin by fluorescence spectrophotometry. The tryptophan-specific fluorescence obtained for glycated hemoglobin exhibited a Stokes effect since the wavelength of the emission peak was shifted to a higher wavelength than that of native Hb. The formation of new fluorescence emission features indicates the formation of modified hemoglobin species. Fluorescence spectroscopic studies provide evidence that the conformational changes in the β-Trp 37 moiety induce motion of the distal His 64 (E7) in the heme binding pocket. This results in the formation of inactive hemichrome forms of hemoglobin which are related to blood disorders.


Introduction
Maillard Reaction Products (MRPs) are attributes of thermally processed foods, contributing to their avor, aroma and texture. [1][2][3][4] The increased consumption of MRPs in recent years has pointed towards their implication in the development of processes such as diabetes and degenerative diseases. Advanced glycation end products (AGEs) lead to the formation of unstable, reactive intermediates that readily form intra-and intermolecular covalent crosslinks or glyco-oxidation products involved in the vascular and renal complications of diabetes mellitus. 5 People with diabetes and renal disease display elevated serum AGE levels and reduced urinary AGE excretion. Unreactive AGEs can readily form new crosslinks with low density lipoprotein (LDL) or collagen. It has been demonstrated that AGEs absorbed into the blood stream may represent a major source of chemically and biologically active toxins. These glycotoxins are only partially eliminated in the urine and may exert signicant reactivity in the body. Persons with impaired renal functions are unable to efficiency excrete glycotoxins in the urine, resulting in abnormally high AGE concentrations in the blood and tissues. Hemoglobin is the most abundant blood protein and consists of two a and b subunits which are non-covalently associated within erythrocytes as a 64.5 kDa tetramer. 5 The natural mechanisms of Hb uptake and decomposition, the latter of which leads to unloading of any bound compounds, can be employed to selectively monitor the Hb-bound complexes.
Recently, the isolation of reaction products of asparagine with reducing sugars at high pH and temperature was explored by high performance liquid chromatography (HPLC) coupling methods. [6][7][8] Spectroscopic analysis of selected Maillard reaction products demonstrated the appearance of asparaginesaccharide conjugates. 7 These were shown to induce the formation of a hemichrome species in hemoglobin, which obstructs the ligand binding site irreversibly. 8 The intrinsic uorescence of tryptophans (W) and tyrosines (Y) in Hb has been used as an important probe in understanding the dynamics and conformational changes induced by ligand binding and to elucidate the binding location and relevant parameters related to binding interactions. [9][10][11] Excitation with light at 280 nm reects contribution from both W and Y residues whereas excitation at 296 nm is used to distinguish the contribution of W residues alone without any uorescence energy transfer from the Y residues. The assignment of b37-Trp as the origin of the emitted uorescence in Hb when excited by light at 296 nm has been established providing solid evidence that this spectral feature is sensitive to conformational changes in Hb. [9][10][11] Anomalous emissions upon excitation at 280 nm have been attributed to a combination of tyrosine and tyrosinate emissions. Tryptophan uorescence measurements provide a reliable analytical tool to characterize the tryptophan environment which is related to the tertiary conformation. A rigid environment around the Trp and a change in polarity affects the emission of the Trp uorescence spectrum. A blue shi in the uorescence emission indicates the presence of a hydrophobic environment around the Trp and increased uorescence intensity indicates a more rigid environment. 9,10 The uorescence of advanced Maillard reaction products has also been investigated and the potential application in food sciences has been reported. [12][13][14][15] In this study, we decided to use fractions of an asparagine/ carbohydrate system to glycate hemoglobin in order to demonstrate that other glycated species may contribute towards the pool of glycated proteins in the blood. The reaction of the discrete MRPs originated from an asparagine-glucose model system with hemoglobin was investigated aer one day and one month incubation time by uorescence excitation/emission spectrophotometry. These specic time points were selected to demonstrate short and long-term exposure, respectively, of hemoglobin to these glycated species. The Trp-specic uorescence obtained for the glycated-Hb complexes exhibited Stokes effect which is attributed to the formation of Hb-MRPs complexes and to structural modication of Hb induced by the MRPs. We propose that the MRPs interact through hydrophobic and hydrogen bonding in the moiety of b-37 Trp, and the interaction perturbs the structural and functional properties of Hb. We suggest that the conformational changes at the N1H site have a direct effect in the O 2 binding site forming a hemichrome, and thus, b-37 Trp may be useful as an intrinsic probe to study the dynamic processes involved in the formation of hemichromes which are related to blood disorders. 5,8 Furthermore, comparison of the uorescence data in the presence and absence of the MRPs with UV resonance Raman data indicate that the microenvironment of b37Trp which includes aTyr140 which is located in the microenvironment of b37Trp, a43Tyr, and b145Tyr is involved in the R / T transition. 8

Sample preparation of MRPs
Solutions of glucose and asparagine (0.2 M, pH 8.0) were prepared in phosphate buffer (50 mM, pH 8.0). Samples were heated in closed screw-capped tubes at 180 C in a heating oven (Memmert, Germany) for 2 hours.

HPLC-fraction collector analysis of MRPs
The HPLC instrumental setup and the separation of individual MRP fractions were described in detail earlier. 7,8 The HPLC experimental setup consisted of a Varian 218 Prepstar Solvent Delivery Module, an Agilent Manual FL-Injection Valve, an Agilent 1260 Innity Variable Wavelength Detector (VWD) and an Agilent 440 LC Fraction Collector. HPLC analysis was performed on a 4.6 Â 250 mm, 5 mm particle size, Zorbax SB-Aq analytical column (Agilent Technologies). Maillard reaction products (MPRs) were analyzed under aqueous conditions using a previously reported method. 7 Water at a ow rate of 0.5 ml min À1 was used isocratically as the mobile phase at room temperature. Maillard reaction products (MPRs) were detected at 200 nm.

HPLC-fraction collector analysis of MRPs
Hemoglobin from bovine blood (Sigma) at approximately 1 mM concentration in 50 mM phosphate buffer (pH 8.0) was diluted hundred-fold to avoid inner lter effects during uorescence measurement. The diluted hemoglobin was immediately mixed with each individual LC fraction (2 : 1 v/v) from the reaction between asparagine and glucose at 180 C. The solutions were mixed and kept at room temperature. The uorescence spectra of the mixtures were collected up to 30 days incubation.

Fluorescence analysis
The Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, USA) controlled from a PC using the Agilent Technologies WinFLR soware (version 1.2) was used for obtaining the uorescent signal. Fluorescence values of the native and glycated hemoglobin samples were measured using a clear four-sided quartz cuvette in the spectrophotometer sample compartment. The excitation and emission slit widths for both the excitation and emission monochromators were set with the value of 5.0 nm. The Excitation-Emission Matrix (EEM) measurement and processing were generated by scanning emission spectra from 320 nm to 500 nm at 1 nm intervals, with 5 nm increments of the excitation wavelengths from 260 nm to 310 nm. The Milli-Q water blank was subtracted from the sample EEM spectra.

Results and discussion
Maillard reactions are highly inuenced by pH and temperature. 4 Model systems similar to reactions naturally occurring in processed foods that have undergone high temperature treatments are useful in resembling more complex chemical systems occurring in foods. Maillard reaction rate is highly increased by the combination of factors such as slightly alkaline pH and high temperatures. 7,8 In this context, we have chosen pH 8 and a temperature of 180 C in order to produce these Maillard reaction intermediates in sufficient concentrations for subsequent isolation. Our choice of pH aimed to keep a balance between our model Maillard system and the physiological pH of hemoglobin in human blood which is slightly alkaline.
The HPLC chromatograms of the asparagine-glucose reactions are shown in Fig. 1. A major signal at Rt ¼ 5.7 min is present which corresponds to the excess unreacted asparagine and other smaller chromatographic peaks arising from individual MRPs. Fraction 1 corresponds to the Schiff base and Fractions 2, 3 and 4 have been characterized to originate from the Amadori product, decarboxylated Amadori product and acrylamide, respectively. 7 These fractions were incubated with Hb in a one day and one month period and their effect on hemoglobin was probed by uorescence spectroscopy.
Proteins react with glucose to form a stable Amadori product through Schiff base adducts that are transformed to advanced glycation end products (AGEs) some of which exhibit uorescence. 14-16 Therefore, their uorescence intensities indicate of the extent of AGE-modied proteins. These excitation-emission maxima are distinct from tryptophan uorescence in proteins. A worthy feature of intrinsic protein uorescence in Hb is the high sensitivity of the local environment of b-37 Trp. [9][10][11] Alterations in the emission spectra of tryptophan may occur in response to conformational transitions, subunit association, substrate binding, and/or denaturation. Fig. 2 shows the uorescence excitation-emission matrix (EEM) of native hemoglobin (pH 8.0). Hb displays weak uorescence under these conditions when maximally excited at 280 nm. The contour map enables the identication of peaks within a multi-component sample matrix. The different colors are associated with intensity with the deep red being the most intense. It is evident that for native hemoglobin the maximum excitation/emission is 280 nm/338 nm (Fig. 2). Excitation at 280 nm will excite both tyrosine and tryptophan residues. The signals from the 280 nm excitation are three times more intense and thus small changes can be determined more reliably. The uorescence spectra shown in Fig. 2 are in good agreement with those previously reported. 9,10 Upon excitation at 296 nm, the Trp residues are selectively excited and the majority of the uorescence observed in Hb has been previously assigned to the b-37 Trp residue. The observed difference in uorescence intensity between the T and R states in Hb has been attributed to the changes occurring at b-37 Trp. Therefore, the Trp-37 in the bchains of Hb is the uorophoric moiety of Hb and can serve as the marker residue for monitoring the dynamics of Hbcomplexes. It is known that a-140Tyr which is located in the microenvironment of b-37 Trp, b-42 Tyr, and b-145 Tyr undergo conformational changes during the R / T transition. A dramatic change in uorescence intensity could result from altered Tyr / Trp and Trp / heme energy-transfer pathways. 9,10 Fig. 3 shows the uorescence emission spectra of the reactions of Hb with the Schiff base (Fraction 1), Amadori product (Fraction 2), decarboxylated Amadori product (Fraction 3) and acrylamide (Fraction 4) aer 1 day of incubation. The Raman peak of H 2 O which is present in all the uorescence spectra is always located of 3400-3600 cm À1 lower in energy than the excitation wavelength. In the 260-295 nm excitation range, the emission maxima were located at 348 nm (Fig. 3A) for the Schiff base (Fraction 1) and approximately at 342 nm ( Fig. 3B-D) for the Amadori product (Fraction 2), decarboxylated Amadori product (Fraction 3) and acrylamide (Fraction 4). The uorescence obtained for these glycated Hb complexes exhibited a Stokes effect since the wavelength of the emission peak is shied 10 nm in the Hb-Schiff base complex and 4 nm in the Hb-Amadori complex, Hb-decarboxylated Amadori product and Hb-acrylamide complexes. The lowering of this energy level can be associated to conformational changes of Hb. From our experimental results aer 1 day incubation it was evident that all 4 fractions achieve the initial binding to hemoglobin as there is a conformational change in the hemoglobin molecule as depicted by the uorescence spectral shis. The origin of the uorescence when excited by light at 280 nm is attributed to tyrosine and tryptophan emissions. The decreased intensity that accompanied the emission shi serves as evidence that this conformational change arises from b-37 Trp at the a 1 b 2 interface when the Hb-complexes are excited by light at 296 nm. 10 No emissions are observed in the 300-310 nm excitation range. It is generally accepted that when the tryptophan residue becomes hydrogen bonded or increasingly exposed to water, its uorescent emission shis to longer wavelengths. Therefore, the increased hydrophilicity can arise as a result of a conformational change coupled to increased exposure of b-37 Trp to the solvent. The observed behavior of the Hb complexes with respect to the contribution of the b-37 Trp environment at the  a 1 b 2 interface is similar to that observed during the R / T transition. 9,10 Fig. 4 illustrates the uorescent spectra of the hemoglobin-MRP mixtures aer 1 month incubation. There is a noticeable shi to longer wavelengths in the emission spectra of the Hb-Amadori complex, Hb-decarboxylated Amadori product and Hb-acrylamide complexes as compared to those spectra observed aer one day of incubation, shown in Fig. 3B-D, whereas that of the Hb-Schiff base complex does not show any further change in uorescent characteristics as that observed in the spectra in Fig. 3A aer one day of incubation. This may denote that there was no signicant Hb-AGE formation in this case. On the contrary, for the other 3 fractions there are further uorescence shis aer this time frame, demonstrating further structural change in hemoglobin and Hb-AGE formation. The 6 nm shi of the Hb-Amadori complex and Hbdecarboxylated Amadori product indicates that prolonged incubation times induce a further conformational change at the b-37 Trp microenvironment and the 11 nm shi of the Hbacrylamide complex indicates strong H-bonded interactions and conformational changes in the moiety of the b-37 Trp residue. The appearance of a new strong emission feature around 395 nm at the excitation range of 295-310 nm is attributed to uorescence quenching effect on tryptophan uorescence. This effect was oen utilized to demonstrate ligand-protein interactions. 16 A similar 390 nm emission band was recently identied from uorescent intensity maps of degenerated cartilage from osteoarthritis patients. The authors discussed that this was a result of non-enzymatic glycation reaction which is followed by the formation of several types of uorescent crosslinks (AGEs). 17 Hb-AGE uorescent crosslinks are likely to result in loss of protein functionality as it is evident from the Hb-acrylamide uorescent prole.
Glycation process results in two different products: early and advanced glycation endproducts (AGEs). Hb-AGEs formation is a slower process and occurs aer prolonged exposure to glycated species. These events are more predominant aer 1 month incubation based on their relative intensities as illustrated by uorescence mapping of incubated hemoglobin in Fig. 5. In Fig. 5 the uorescence mapping aer one month of  incubation of hemoglobin with the LC fractions 1-4 are presented. The uorescent chemical mapping analysis when compared with the corresponding analysis of Hb shown in Fig. 2 demonstrates the effect of MRPs in the moiety of b-37 Trp residue and shows that all the Hb-MRPs complexes are highly heterogeneous systems.
Shis in uorescence emission and uorescence quenching effects are indicative of protein modication in this relatively simple model system. Hb contains three tryptophan residues, but only b-37 Trp is located at the interface between the two dimers. The b-37 Trp residues are located in helices C of the b-subunits and are involved in contacts with the segments FG of the a-subunits at the interdimeric a 1 b 2 and a 2 b 1 interfaces of the hemoglobin tetramer. 18 The intrinsic uorescence signal of Hb arises from the indole group of tryptophan b-37 and has been used to determine the changes in the quaternary structure of Hb upon ligand binding. 19 In addition, the energetic changes that occur on ligand binding in human hemoglobin are determined by measurements of the exchange rates of the indole proton of b-37Trp (C3) and the increased exchange rates of the indole proton of b-37Trp determine ligand binding. The interface regions experience a change in structural free energy upon ligand binding and these hinge regions are involved in the transmission of free energy during its allosteric transition. 19 Results that involve Trp-37 were also obtained to establish the effectiveness of natural drug delivery mechanisms and investigate the interaction between the drug and it natural carrier Hb. It is thus well established that b-37 Trp serves as good optical probe for monitoring ligand binding far away ($15 A) from the oxygen binding site. 11 Hemichromes are formed when hemoglobin undergoes conformational changes resulting in the formation of a sixcoordinated Fe 3+ low-spin His-Fe-His species. [20][21][22] Hemoglobin A (HbA) in humans can form hemichromes even under physiological conditions as a result of pH and temperature alterations, and in the autoxidation of oxyHb. 20 We have recently demonstrated that tetrameric Hbs form a partial hemichrome state upon addition of MRPs compounds. The results presented here in conjunction with our previous observations strongly indicate that Trp-37 plays a critical role in the formation of the hemichrome. 8,[20][21][22] Obviously, the Maillard reaction species act as external ligands to a protein site near the b-37 Trp moiety, triggering the formation of six-coordinated bishistidyl hemichrome species. 23,24 The coordination of the distal His 64 (E7) to the iron produces a scissoring motion in helices E and F which results in modications in the tertiary structure of the tetramer, mainly in the ab interface which is critical in the transition from the R to the T state. 23 Modication of the EF fragment exposes the heme to a more solvent-exposed position narrowing the heme pocket as the E-and F-helices move toward each other. 8,23,24

Conclusions
Maillard reaction products (MRPs) which are absorbed in the blood stream represent a major source of chemically and biologically active toxins. These dietary glycotoxins in high local concentration and time bind irreversibly to Hb. Intrinsic uorescence activity accompanied by uorescent mapping was employed in this study to provide experimental evidence regarding the relationship of intrinsic Hb uorescence and the chemical events that follow interactions with glycotoxins originated form the Maillard reaction products. The data presented here demonstrate that the primary interactions of the MRPs with b-37 Trp forming the initial Hb-MRPs complexes are followed by major modications under prolonged period of time forming the nal Hb-MRPs. The present data and those previously reported suggest that b-37 Trp plays a major role in forming the inactive Hb hemichrome complexes, as the result of the presence of the Maillard reaction products near the b-37 Trp moiety of Hb. The implication of the non-enzymatic glycation reaction is the formation of uorescent cross linked species (AGEs) as a result of the inactive hemichrome form of hemoglobin. 25 The small number of Maillard reactions being addressed in model reactions in this investigation may point towards the need of a larger in vivo study that will extend the applicability of this uorescence technique in combination with structure sensitive techniques such as FTIR and Raman spectroscopies to a more heterogeneous in vivo system. [26][27][28][29][30]

Conflicts of interest
There are no conicts to declare.