Isohemoglobin differentiation in the bimodal-breathing amazon catfish **Hoplosternum littorale**

Abstract The bimodal gill(water)/gut(air)-breathing Amazonian catfish Hoplosternum littorale that frequents hypoxic habitats uses “mammalian” 2,3-diphosphoglycerate (DPG) in addition to “piscine” ATP and GTP as erythrocytic O2affinity modulators. Its electrophoretically distinct anodic and cathodic hemoglobins (HbAn and HbCa) were isolated for functional and molecular characterization. In contrast to HbAn, phosphate-free HbCa exhibits a pronounced reverse Bohr effect (increased O2 affinity with decreasing pH) that is obliterated by ATP, and opposite pH dependences ofK T (O2 association constant of low affinity, tense state) and the overall heat of oxygenation. Dose-response curves indicate small chloride effects and pronounced and differentiated phosphate effects, DPG < ATP < GTP < IHP. HbCa-O2 equilibria analyzed in terms of the Monod-Wyman-Changeux model show that small T state bond energy differences underlie the differentiated phosphate effects. Synthetic peptides, corresponding to N-terminal fragment of the cytoplasmic domain of trout band 3 protein, undergo oxygenation-linked binding to HbCa, suggesting a metabolic regulatory role for this hemoglobin. The amino acid sequences for the α and β chains of HbCa obtained by Edman degradation and cDNA sequencing show unusual substitutions at the phosphate-binding site that are discussed in terms of its reverse Bohr effect and anion sensitivities.