Avian hemoglobins have attracted much attention in view of the unique oxygen transport characteristics. The present study describes the primary structure of minor hemoglobin component HbD from Tufted duck (Aythya fuligula), a migratory bird seen in Pakistan during the winter season. Separation of the polypeptide subunits was achieved by ion exchange chromatography in the presence of 8 M urea. Molecular masses of the intact protein as well as peptides obtained from chemical and enzymatic cleavages were determined by electrospray ionization mass spectrometry. The sequence was studied by automatic Edman degradation of the native chains and their tryptic/hydrolytic fragments in a gas-phase sequencer. Comparison of the hemoglobin sequence with the corresponding sequences of Anseriform representatives and other avian species shows residues like alpha(D)23 Asp, alpha(D)120 Asp as being specific to Tufted duck. The three-dimensional structure analyzed with the protein structure modeling package, WHAT IF, using the crystal structure coordinates of chicken hemoglobin (PDB code = 1hbr) shows alpha(D)34 Val, alpha(D)38 Gln, and alpha(D)94 Asp as possible mediators offering alternate pathway for oxygen uptake and release thereby leading to distinct hypoxia tolerance in the Tufted ducks. Results are discussed with reference to function and evolution in the Anseriform representatives. (C) 2004 Elsevier Inc. All rights reserved.