Isohemoglobin differentiation in the bimodal-breathing amazon catfish Hoplosternum littorale

The bimodal gill(water)/gut(air)-breathing Amazonian catfish Hoplosternum l ittorale that frequents hypoxic habitats uses "mammalian" 2,3-diphosphoglyc erate (DPG) in addition to "piscine" ATP and GTP as erythrocytic O-2 affini ty modulators. Its electrophoretically distinct anodic and cathodic hemoglo bins (Hb(An) and Hb(Ca)) were isolated for functional and molecular charact erization. In contrast to HbAn, phosphate-free Hb(Ca) exhibits a pronounced reverse Bohr effect (increased O-2 affinity with decreasing pH) that is ob literated by ATP, and opposite pH dependences of K-T (O-2 association const ant of low affinity, tense state) and the overall heat of oxygenation. Dose -response curves indicate small chloride effects and pronounced and differe ntiated phosphate effects, DPG < ATP < GTP < IHP, Hb(Ca)-O-2 equilibria ana lyzed in terms of the Monod-Wyman-Changeux model show that small T state bo nd energy differences underlie the differentiated phosphate effects. Synthe tic peptides, corresponding to N-terminal fragment of the cytoplasmic domai n of trout band 3 protein, undergo oxygenation-linked binding to Hb(Ca), su ggesting a metabolic regulatory role for this hemoglobin. The amino acid se quences for the alpha and beta chains of Hb(Ca) obtained by Edman degradati on and cDNA sequencing show unusual substitutions at the phosphate-binding site that are discussed in terms of its reverse Bohr effect and anion sensi tivities.