Effect of nonenzymatic glycation on functional and structural properties of hemoglobin
Introduction
The central identifying feature of diabetes mellitus is chronic and substantial elevation of the circulating glucose concentration. The increased blood glucose stimulates nonenzymatic glycation of proteins namely, serum albumin [1], α-crystallin [2], collagen [3], low-density lipoprotein [4], hemoglobin [5] etc. The key step in the modification of proteins by glucose is Schiff base formation, followed by Amadori rearrangement [6]. The Amadori product can then undergo oxidative cleavage, resulting in the formation of advanced glycation end products (AGEs) [7]. The first indication that a very simple chemical reaction between glucose and free amino groups on protein can lead to irreversible modification came with the characterization of hemoglobin A1c (HbA1c), which has the N-terminus of the β chain (valine) linked to glucose [6]. HbA1c concentration is proportionately increased in diabetic patients with ambient hyperglycemia and reflects the extent as well as management of diabetic condition [8]. Several reports have been made on glycation-induced structural and functional modification of hemoglobin [5], [9], [10], [11], [12], [13], [14], [15], [16]. From computer modeling and electron paramagnetic resonance spectral studies, allosteric structure of glycated hemoglobin was speculated to be fixed in ‘T’ or Tough state [9], [10]. Using ESR spectroscopic study, Watala et al. [10] reported the decreased mobility of the lysine residue in glycated hemoglobin and suggested a change in the conformation of the molecule. Compared to HbA0, a reduced peroxidase activity of HbA1c was reported by both Khoo et al. [12] and Kar and Chakraborti [13]. A modulation mechanism linked to structural change of the protein was suggested. Compared to HbA0, HbA1c was reported to exhibit moderately high oxygen affinity [14]. According to Inouye et al. [15], glycation of hemoglobin via chronic hyperglycemia was significantly correlated with cholesterol peroxidation in erythrocytes. Hemoglobin glycation was suggested to induce oxygen-derived free radicals causing oxidative damage to endogenous molecules, including cholesterol.
Although free radicals and associated oxidative stress have been implicated in eliciting diabetes mellitus [17], [18], the mechanism of increased formation of free radicals in diabetes is not yet clear. Findings from our laboratory indicate that HbA1c may be a source of free radicals and oxidative stress [13], [16]. Ferrous iron with six coordination states is bound in heme pocket of hemoglobin. Under specific circumstances, iron can be liberated from the heme and ligated to another moiety, probably distal histidine (E7) near heme pocket. This iron has been termed ‘mobile reactive iron’ [19], which can catalyze Haber–Weiss reaction producing free radicals, particularly hydoxyl (OH) radicals, and in turn, may damage different cell constituents [20]. We have shown that free reactive iron level in purified hemoglobin, isolated from blood of diabetic patients is proportionately increased with increased level of blood glucose [16]. This iron may cause increased level of free radicals, which have been suggested to be involved in pathological complications of diabetes mellitus [13], [16]. In a recent study, Cussimanio et al. [21] have demonstrated that hemoglobin and myoglobin are extremely susceptible to damage by glucose in vitro through a process that leads to complete destruction of heme group. The iron released from heme destruction enhances AGE formation.
H2O2 is known to induce iron release from hemoglobin [22]. We have shown that H2O2 promotes more iron release from HbA1c than that from HbA0 and iron-mediated free radical reactions namely, lipid peroxidation and deoxyribose degradation are more pronounced with HbA1c in comparison with HbA0 (13). These results are also in agreement with our recent findings, which correlate glycation-induced modification of myoglobin and a mechanism of iron-mediated increased formation of free radicals [23]. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress in diabetic patients. The modified functional properties of glycated heme proteins—hemoglobin and myoglobin may thus be correlated with increased free radical reactions.
To verify the correlation further, we have undertaken the present study to understand hemoglobin-catalyzed iron-mediated free radical reactions with respect to DNA breakdown, protein degradation and carbonyl formation. The findings confirm the role of hemoglobin as a source of catalytic iron and its modification by glycation enhances further this functional property of the protein. Glycation also stimulates oxidation of hemoglobin, which may further complicate oxidative reactions in diabetic condition. Besides functional modification, we have also studied glycation-induced structural modification of hemoglobin with respect to conformations, surface accessible tryptophan residues, thermal stability and stability of heme-globin linkage. The findings in this communication suggest that glycation causes both structural and functional modifications, which may be associated with pathophysiological complications of diabetes mellitus.
Section snippets
Materials
Sephadex G-25, sephadex G-100, human serum albumin (HSA), bovine serum albumin (BSA), agarose, ethidium bromide, alanine dehydrogenase, nicotinamide adenine dinucleotide (NAD+), hydrazine hydrate, dinitrophenylhydrazine (DNPH), acrylamide, desferrioxamine (DFO), mannitol, thiobarbituric acid (TBA) and hemin chloride were purchased from Sigma Chemical, USA. BioRex-70 resin (200–400 mesh) was purchased from BioRad, India. Other chemicals were of analytical grade and purchased locally.
Preparation of hemoglobin, separation of HbA1c and HbA0 from hemoglobin and their characterization
Human blood
H2O2-induced HbA0 and HbA1c-catalyzed DNA breakdown
H2O2-induced hemoglobin-catalyzed DNA breakdown was studied (Fig. 2). DNA was incubated with HbA0 or HbA1c in the presence or absence of H2O2 and subjected to gel electrophoresis. In the presence of H2O2 only, DNA was not degraded (Lane 1: only DNA, Lane 2: DNA+H2O2). HbA0 (Lane 3) or HbA1c (Lane 5) alone could not degrade DNA. However, DNA degradation was clearly evident with conversion of form I to form II in the presence of both H2O2 and protein (Lanes 4 and 6). HbA1c and H2O2 together (Lane
Discussion
In hemoglobin, ferrous iron is completely domesticated and tamed within protoporphyrin cage. Under certain conditions, this iron is liberated and may be detected by reaction with ferrozine [19]. We have shown earlier that free iron level in purified hemoglobin isolated from blood of diabetic patients is proportionately increased with increased level of blood glucose or extent of the disease condition [16]. We have also found that ferrozine-detected iron level is increased proportionately in in
Acknowledgements
S.S. is a Senior Research Fellow of the Indian Council of Medical Research, New Delhi. A part of the study was supported by a grant from the University Grants Commission, New Delhi. Thanks are due to Prof. C.K. DasGupta for permission to use the Hitachi F-3010 Spectrofluorimeter and Prof. U. Chaudhuri for helpful discussions.
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