Increased fructose concentration in diabetes mellitus causes fructation of several proteins. Here we have studied fructose-induced modifications of hemoglobin. We have demonstrated structural changes in fructose-modified hemoglobin (Fr-Hb) by enhanced fluorescence emission with excitation at 285 nm, more surface accessible tryptophan residues by using acrylamide, changes in secondary and tertiary structures by CD spectroscopy, and increased thermolability by using differential scanning calorimetry in comparison with those of normal hemoglobin, HbA0. Release of iron from hemoglobin is directly related with the extent of fructation. H2O2-induced iron release from Fr-Hb is significantly higher than that from HbA0. In the presence of H2O2, Fr-Hb degrades arachidonic acid, deoxyribose and plasmid DNA more efficiently than HbA0, and these processes are significantly inhibited by desferrioxamine or mannitol. Thus increased iron release from Fr-Hb may cause enhanced formation of free radicals and oxidative stress in diabetes. Compared to HbA0, Fr-Hb exhibits increased carbonyl formation, an index of oxidative modification. Functional modification in Fr-Hb has also been demonstrated by its decreased peroxidase activity and increased esterase activity in comparison with respective HbA0 activities. Molecular modeling study reveals Lys 7(α), Lys 127(α) and Lys 66(β) to be the probable potential targets for fructation in HbA0. © 2008 Elsevier B.V. All rights reserved.