Erythrocyte Organic Phosphates and Hemoglobin Function in Birds, Reptiles, and Fishes

AMER. ZOOL., 20:115-129 (1980) Erythrocyte Organic Phosphates and Hemoglobin Function in Birds, Reptiles, and Fishes1 RUSSELL E. ISAACKS AND DONALD R. HARKNESS Research Laboratories of the Veterans Administration Medical Center and the Department of Medicine, University of Miami School of Medicine, Miami, Florida, 33125 INTRODUCTION which bind to deoxyhemoglobins but not to oxyhemoglobins. In most mammalian red cells, the organic phosphate modulating hemoglobin oxygenation is 2,3 diphosphoglyceric acid (2,3-DPG), a glycolytic intermediate. The presence of unusually high concentrations of 2,3-DPG in porcine erythrocytes was first noted by Greenwald (1925) and subsequently recognized as a characteristic of most mammalian erythrocytes. Its function was unknown until 1967 when two laboratories independently reported that 2,3-DPG lowers the oxygen affinity of the hemoglobin tetramer by binding preferentially to deoxyhemoglobin (Benesch and Benesch, 1967; Chanutin and Curnish, 1967). Exceptions are the erythrocytes of sheep, goat, cow, and cat which have low concentrations of 2,3-DPG. However, the hemoglobins of each of the species have low oxygen affinities, apparently reducing the need for an organic phosphate modulator to facilitate oxygen release. In most 1 From the Symposium on Respiratory Pigments pre- mammals a relationship exists between an sented at the Annual Meeting of the American So- active glycolytic system and the metabolism ciety of Zoologists, 27-30 December 1978, at Rich- and function of the red cell, with the high Many interesting adaptations have occurred in nature which allow various species of animals to meet their metabolic requirements for oxygen. These needs for oxygen in relatively hypoxic environments can be met in several ways: the presence of particular blood hemoglobins with varying affinities for oxygen, the presence of gills and/or lungs, the distribution of capillaries, the rate of blood volume pumped by the heart, the mass of red blood cells per unit volume of blood (PCV), the concentration of hemoglobin within the cells, and the type and concentration of hemoglobin modulator(s). In addition, the unique allosteric properties of hemoglobin are such that H + , CO2, and increased temperature, features which pertain in the tissues, all promote the release of oxygen. The affinity of hemoglobins for oxygen is further regulated by the negatively charged organic phosphates present in the red cell mond, Virginia. 115 SYNOPSIS. Studies on the role of red blood cell organic phosphates in regulating oxygen transport and hemoglobin function in various species have emanated from several laboratories in the past few years. These data reveal that the organic phosphate composition of erythrocytes is certainly more diverse between classes of animals than previously recognized, that the kind and concentration of red cell organic phosphate modulator changes during development of the species, and that the role of organic phosphates in modulating hemoglobin function is variable. For example in birds we believe that the changes in amounts and distribution of the intraerythrocytic organic phosphates account for the sudden changes in whole blood oxygen affinity during development of the embryo and young bird as well as in the mature bird. In the loggerhead and green sea turtle, it appears that organic phosphate modulators regulate whole blood oxygen affinity during embryonic development but it is unlikely that whole blood oxygen affinity is controlled by organic phosphate modulation of hemoglobin function in the adult turtle. From the data now available on air-breathing fishes, it appears reasonable to consider that whole blood oxygen affinity may be regulated by a combination of organic phosphates; that is, the relative concentrations of the primary regulator (inositol-P2, 2,3-DPG, or inositol-P5) may be rather stable whereas the nucleotide triphosphate (ATP and GTP) concentrations may fluctuate depending upon physiological demands upon the animal for oxygen. 116 R. E. ISAACKS AND D. R. H A R K N E S S Shimizu, 1969); and 3) increases in organic phosphate concentrations without changes in the intrinsic oxygen affinities of hemoglobin during embryonic development (e.g., pigs) (Kim and Duhm, 1974). Two possible mechanisms had been previously considered to explain the decrease in whole blood oxygen affinity as the chick matures: 1) the replacement of molecular species of embryonic hemoglobin with adult-type hemoglobins (Hall, 1934) and 2) the continued decrease in the levels of red cell inorganic phosphate which might serve to regulate hemoglobin oxygen affinity (Huisman and Schillhorn Van Veen, 1964). When our work in this area began, one report (Oshima et al., 1964) had been published indicating that the level of inositol hexaphosphate (IHP) in the red cell increased during development of the young chick. The compound originally identified as IHP, or phytic acid, in the avian red cell (Rapoport, 1940) has since been identified as inositol 1,3,4,5,6 pentaphosphate (inositol-P5) (Johnson and Tate, 1969). Consequently, there is some ambiguity in the literature because most reports prior to the late sixties, and some even now, refer to the inositol polyphosphate in avian red cells as IHP or phytic acid (inositol-P6). In some of our initial work on the in vitro effects of organic phosphates on hemoglobin function, we like others utilized commercially available IHP, which does not occur to any extent in avian erythrocytes and in some cases contains impurities (other inositol polyphosphates). Therefore, we will use the term IHP or inositol-P6 in agreement with its referenced usage by other authors. In our own reports we have referred to the compound found in avian erythrocytes as inositol pentaphosphate (IPP) and in this paper I will use the designation inositol-P5. Organic phosphates of avian red cells and whole blood oxygen affinity From the outset, we recognized that a method was needed which would permit analysis of the major acid-soluble organic phosphates of the red cell with quantitative elution of either inositol-P5 or inositol-P0 and the simultaneous resolution of the concentrations of 2,3-DPG playing a major role in regulating hemoglobin function. Similarly, inositol pentaphosphate (inositol-P5) in erythrocytes of most mature avian species apparently regulates hemoglobin oxygenation in birds. Unusually high concentrations of inositol polyphosphates in avian erythrocytes were first reported by Rapoport (1940). Although little is known about the metabolism of inositolP5 in avian erythrocytes, it appears that a relationship between phosphate metabolism and oxygen transport exists. Whether the metabolism of inositol-P5 is related to glycolytic pathways in the red cell of birds has yet to be determined. Most of our work has involved correlations between erythrocyte organic phosphates and whole blood oxygen affinity during development of several speci (...truncated)