The mini-hemoglobins in neural and body wall tissue of the nemertean worm, Cerebratulus lacteus.

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.

Assembly of the gigantic hemoglobin of the earthworm Lumbricus terrestris. Roles of subunit equilibria, non-globin linker chains, and valence of the heme iron.

The extracellular hemoglobin of the earthworm Lumbricus terrestris has four major kinds of O2-binding chains: a, b, and c (forming a disulfide-linked trimer), and chain d. Non-heme, non-globin structural chains, "linkers," are also present. Light-scattering techniques have been used to show that the ferrous CO-saturated abc trimer and chain d form an (abcd)4 complex of 285 kDa at neutral pH. Formation of the full-sized 4-MDa molecule requires the addition of linker chains in the proportion of two linkers per (abcd)4 and occurs much more rapidly in the presence of 10 mM calcium. This stoichiometry is supported not only by direct quantitative analysis of the intact hemoglobin but also by the fact that the addition of 50% of the proposed stoichiometric quantity of linkers results in the conversion of 50% of the (abcd)4 to full-sized molecules. Isolated CO-saturated abc trimers self-associate to (abc)2 and higher aggregates up to an apparent limit of (abc)10 approximately 550 kDa. The CO-saturated chain d forms dimers, (d)2, and tetramers, (d)4. Oxidation of the (abcd)4 complex with ferricyanide causes complete dissociation of chain d from the abc trimer, but addition of CN- maintains the (abcd)4 complex. Valence hybrids have also been studied. The ferrous CO-saturated abc trimer and met (ferric) chain d also associate to form (abcd)4, but the met abc trimer and ferrous CO-saturated chain d do not. Oxidation of the abc trimer and chain d to the ferric form causes the formation of a characteristic hemichrome spectrum with a maximum at 565 nm and a shoulder near 530 nm. These results show that interactions between the abc trimer and chain d are strongly dependent on the ligand and valence state of the heme iron. Light-scattering measurements reveal that oxidation of the intact Hb produces a significant drop in molecular mass from 4.1 to 3.6 MDa. Inclusion of CN- prevents this drop. These experiments indicate that oxidation causes the Hb to shed subunits. The observations provide an explanation for the wide variations in the molecular mass of L. terrestris Hb that have been observed previously.

Origin of a "bridge" intron in the gene for a two-domain globin.

Red cells of the clam Barbatia reeveana express two hemoglobins, one composed of 16- to 17-kDa chains and the other of 35-kDa chains. The nucleotide sequence of the cDNA encoding the 35-kDa chain shows that the polypeptide has two very similar heme-binding domains, which are joined without use of an additional bridging sequence. Two novel introns occur in the gene for the two-domain globin: one, the "precoding" intron, is located two bases 5' from the start codon, and the other, a "bridge" intron, separates the DNA sequences encoding the two domains. Close correspondence exists between the 3' end of the precoding intron and the 3' end of the bridge intron and between parts of the 3' noncoding region of the cDNA for the two-domain globin and the 5' end of the bridge intron. These observations indicate that the bridge intron arose by unequal crossing-over between two identical or very similar genes for a single-domain globin. This conclusion, together with the proposal that exons were initially independent "minigenes" [Gilbert, W. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 901-905], suggests that many introns may have evolved from the 5' noncoding region of one gene and/or the 3' noncoding region of a second gene. This hypothesis implies that splice junctions would be associated with the original NH2 and COOH termini of proteins and provides an explanation for the observation that splice junctions usually map to protein surfaces. They do so because most NH2- and COOH-terminal residues are usually located on or near the surfaces of proteins.