Search

The burrowing brittle star Hemipholis elongata (Say) possesses hemoglobin-containing coelomocytes (RBCs) in its water vascular system. The RBCs, which circulate between the arms and body, are thought to play a role in oxygen transport. The hemoglobin of adult animals has a moderate affinity for oxygen (P(50) = 11.4 mm Hg at pH 8, 20 degrees C, measured in cellulo) and exhibits cooperativity (Hill coefficient > 1.7). The hemoglobin of juveniles has a higher affinity (P(50) = 2.3 mmHg at pH 8.0, 20 degrees C) and also exhibits cooperativity. The oxygen-binding properties of the hemoglobin are relatively insensitive to pH, temperature, and hydrogen sulfide. Adult hemoglobin is a heterogeneous mixture composed of three major fractions. The combined results of electrospray mass spectrometry and oxygen-binding experiments performed on purified fractions indicate that the native hemoglobin is in the form of homopolymers. A partial amino acid sequence (about 40 amino acids) of adult hemoglobin reveals little homology with holothurian hemoglobins.

Hemoglobin (Hb) occurs in circulating red blood cells, neural tissue, and body wall muscle tissue of the nemertean worm, Cerebratulus lacteus. The neural and body wall tissue each express single major Hb components for which the amino acid sequences have been deduced from cDNA and genomic DNA. These 109-residue globins form the smallest stable Hbs known. The globin genes have three exons and two introns with splice sites in the highly conserved positions of most globin genes. Alignment of the sequences with those of other globins indicates that the A, B, and H helices are about one-half the typical length. Phylogenetic analysis indicates that shortening results in a small tendency of globins to group together regardless of their actual relationships. The neural and body wall Hbs in situ are half-saturated with O2 at 2.9 and 4.1 torr, respectively. The Hill coefficient for the neural Hb in situ, approximately 2.9, suggests that the neural Hb self-associates in the deoxy state at least to tetramers at the 2-3 mM (heme) concentration estimated in the cells. The Hb must dissociate upon oxygenation and dilution because the weight-average molecular mass of the HbO2 in vitro is only about 18 kDa at 2-3 microM heme concentration. Calculations suggest that the Hb can function as an O2 store capable of extending neuronal activity in an anoxic environment for 5-30 min.

Heat-shocked organisms are known to produce not only "heat shock proteins" but also diadenosine tetraphosphate (Ap4A) and related compounds that may act as "alarmones" that alert the cell to the onset of metabolic stress. We found that Ap4A is synthesized in chicken erythrocytes and that the Ap4A level in the whole blood of heat-stressed birds increases about 10-fold. In searching for alarmone receptors, we found that the diadenosine polyphosphates bind preferentially with high affinity to the deoxy conformation of hemoglobin in a ratio of one/tetramer. The binding affinity of this new class of effectors of hemoglobin function is directly related to the number of phosphates which bridge the nucleotide moieties, with the most dramatic in vitro effect on oxygen affinity being shown by Ap6A. Decreasing effects are brought about by diadenosine penta-, tetra-, tri-, di-, and monophosphates. The association constant for Ap4A binding to deoxygenated human hemoglobin at pH 7.25 is 26 microM-1, close to that for 2,3-diphosphoglycerate. At 100-fold excess over heme, Ap4A increases the P50 of stripped Hb A in 0.05 M HEPES buffer at pH 7.25, 20 degrees C, from 0.85 to 6.03 mm Hg. The binding, which markedly enhances the Bohr effect, involves the beta chain anion-binding site. The kinetics of both ligand binding and dissociation are affected, with a greater quantitative effect on the oxygen dissociation process. Although the low concentration of the diadenosine polyphosphates in red cells precludes a physiologically significant modulation of oxygen delivery, competition with the ATP- and NAD(P)H-binding sites on hemoglobin or regulatory enzymes may prove to be of adaptive significance.

An oxygen store based on the reversible combination of oxygen with hemoglobin or related hemoproteins could be of considerable value to organisms or tissues faced with intermittent hypoxia or anoxia. It would be of interest, therefore, to know the physical and chemical requirements and limitations of useful storage. A model is presented which predicts storage time from physical, chemical, and geometric properties of the store. Storage time is arbitrarily defined as the time for 50% of the stored O2 to diffuse from a fully-loaded store when the store is exposed to a zero O2 environment. Results are presented in the form of simple approximate equations and in graphical form and indicate that: (1) a wide range of storage times can be obtained with reasonable biological parameters; (2) storage times approaching 1,000 s can be achieved even with stores of microscopic dimension; (3) biological O2 storage is predominantly diffusion-limited, although reaction-limited behavior is not impossible; and (4) the major effect of co-operativity appears to be on the form of O2 release: hemoglobins without co-operativity release O2 at a constantly declining rate, whereas those with high co-operativity release O2 at a constant rate. The model is applicable not only to hemoglobin-based O2 storage, but also to any reaction-diffusion system in which one substance can be "stored" by reversible combination with some other substance which acts as the store.