Characterization of the hemoglobins of the Australian lungfish Neoceratodus forsteri (Krefft)

Abstract

We examined for the first time the hemoglobin components of the blood of the Australian lungfish, Neoceratodus forsteri and their functional responses to pH and the allosteric modulators adenosine triphosphate (ATP), guanosine triphosphate (GTP), 2,3-bisphosphoglyceric acid (BPG) and inositol hexaphosphate (IHP) at 25 °C. Lysates prepared from stripped, unfractionated hemolysate produced sigmoidal oxygen equilibrium curves with high oxygen affinity (oxygen partial pressure required for 50% hemoglobin saturation, p₅₀ = 5.3 mmHg) and a Hill coefficient of 1.9 at pH 7.5. p₅₀ was 8.3 and 4.5 mmHg at pH 6 and 8, respectively, which corresponded to a modest Bohr coefficient (Δlog p₅₀/ΔpH) of − 0.13. GTP increased the pH sensitivity of oxygen binding more than ATP, such that the Bohr coefficient was − 0.77 in the presence of 2 mmol L^− 1 GTP. GTP was the most potent regulator of hemoglobin affinity, with concentrations of 5 mmol L^− 1 causing an increase in p₅₀ from 5 to 19 mm Hg at pH 7.5, while the order of potency of the other phosphates was IHP > ATP > BPG. Three hemoglobin isoforms were present and each contained both α and β chains with distinct molecular weights. Oxygen affinity and pH-dependence of isoforms I and II were essentially identical, while isoform III had a lower affinity and increased pH-dependence. The functional properties of the hemoglobin system of Neoceratodus appeared consistent with an active aquatic breather adapted for periodic hypoxic episodes.

Introduction

Lungfishes derive from an ancient lineage of air-breathing dipnoan fishes first appearing in the fossil record 380 million years ago (Campbell and Barwick, 1990). There are six extant species of lungfishes: four African species in the genus Protopterus (Family Protopteridae), one South American species, Lepidosiren paradoxa (Family Lepidosirenidae), and one Australian species, Neoceratodus forsteri (Family Ceratodontidae). They are all bimodal breathers and possess both gills and lungs, the lungs being homologous with the pulmonary gas exchange organs of higher vertebrates (Lomholt et al., 1975, Brainerd, 1994, Graham, 2006). During the alternation of water breathing and air breathing, the pattern of circulation oscillates between that of a fish and a tetrapod (Fishman et al., 1985). In addition, molecular studies point to a link between lungfishes and early tetrapod evolution and thus the critical transition from water- to air-breathing that led to a vertebrate conquest of terrestrial habitats (Meyer and Wilson, 1990, Brinkmann et al., 2004).

The impressive abilities of Protopterus and Lepidosiren to survive for long periods in hypoxic or anoxic water, or in mud-lined cocoons when pools dry out, are supported by diversion of the branchial circulation to the pulmonary gas exchange surfaces to enable air breathing (Johansen et al., 1976, Abe and Steffensen, 1996, Jucá-Chagas, 2004). In contrast to these obligate air-breathers with paired lungs, Neoceratodus is predominantly a water-breather, has a single lung, and is less well able to survive extended periods out of water (Grigg, 1965, Lenfant et al., 1966, Fritsche et al., 1993). Neoceratodus may therefore be considered the most primitive of the extant Dipnoi and represents an important stage in the transition from water to air-breathing. It occurs naturally in slow flowing waters of the Burnett and Mary River systems in South-Eastern Queensland. During dry periods when aquatic oxygen levels decline sharply, Neoceratodus is reported to supplement its oxygen uptake by breathing air (Gannon et al., 1983). Field observations by Grigg (1965) however, linked air-breathing in Neoceratodus to periods of physical activity, an idea only recently resurrected as an adaptation for air-breathing (Farmer and Jackson, 1998, Clark et al., 2007, Wells et al., 2007). The Queensland lungfish does not build a mud-lined cocoon like the African species, and cannot survive for more than a few days out of water (Kind, 2003).

Several studies have examined aspects of the blood oxygen transport system that support air-breathing in lungfishes. During estivation the African lungfish Protopterus amphibious increased hematocrit and hemoglobin concentration by 50%, reflecting a raised oxygen carrying capacity, while the concentration of erythrocyte organic phosphates decreased thus leading to a sharp increase in whole blood-oxygen affinity where p₅₀ (oxygen partial pressure required for 50% hemoglobin saturation) changed from 33 to 9 mmHg (Johansen et al., 1976). The increase in oxygen affinity has been interpreted as providing improved oxygenation during estivation in a low oxygen environment. The red blood cells of the African lungfish Protopterus aethiopicus, and the South American lungfish Lepidosiren paradoxa contain high concentrations of adenosine and guanosine triphosphates (ATP and GTP) together with an inositol polyphosphate (IP2), a potent category of allosteric regulators of hemoglobin oxygen affinity occurring in birds (Bartlett, 1978, Isaacks et al., 1978). Two major hemoglobin components, each with similar sensitivity to GTP, are present in Protopterus amphibious, and a single GTP-sensitive hemoglobin occurs in Protopterus annectens (Weber et al., 1977).

Although the erythrocytes of Neoceratodus forsteri contain both ATP and GTP, IP2 was not detected (Isaacks and Kim, 1984), which could reflect an earlier evolutionary stage of facultative air-breathing. To date, no detailed studies have appeared on the hemoglobin components of the blood of Neoceratodus or their functional responses to pH and organic phosphate modulators. In view of both the interesting phylogeny of this lungfish species, and its contrasting behaviour of facultative air-breathing, we have examined aspects of the hemoglobin system in Neoceratodus, and in particular, structure-function relationships of the highly adaptable hemoglobin protein.