Lamprey Hemoglobin

Abstract

Lampreys and hagfish, the most primitive living vertebrates, have hemoglobins (Hbs) 1 distinct from those of all others in the class. Indeed, the low isoelectric points of the Hbs caused Svedberg and Eriksson (2) to identify them with invertebrate Hbs for which they had revived the term erythrocruorin.2 They found the Hbs to be monomeric, like myoglobins, rather than the tetramers characteristic of other vertebrates. However, Wald and Riggs (6) found that oxygen equilibria of lamprey (Petromyzon marinus) Hb were strongly pH-dependent (Bohr effect), a surprising result at the time because oxygen equilibria of the monomeric myoglobins were pH-independent. Furthermore, they found that the oxygen equilibrium at pH 6.8 was slightly cooperative, with a Hill coefficient of 1.2, but dismissed this result as an artifact because cooperativity is impossible with monomeric Hbs. Briehl (7) resolved this paradox by showing that deoxygenated lamprey Hb self-associates. Protonation accompanying the association accounts for the Bohr effect of O 2 binding. Behlke and Scheler (8) showed with sedimentation velocity measurements that ligated Hb from a similar species of lamprey (Lampetra fluviatilis) also self-associates, but only at low pH. Although the metHb was found to be monomeric, addition of metHb ligands such as azide or cyanide causes dimer formation at low pH (9). This property, oxidationinduced dissociation and ligand-dependent reassociation, absent in other vertebrate Hbs, but found in the Hbs of organisms of five invertebrate phyla, is another indication of the functional relationship of lamprey and invertebrate Hbs (10). Andersen and Gibson (11,12) showed by kinetic analysis of ligand binding by the Hb of P. marinus that the results could be accurately described by a model in which protonation of a single site per monomer with pK ϭ 6.0 accompanied the formation of dimers of low O 2 affinity. There was no need to include self-association beyond the dimer, so they concluded that higher aggregates may not be physiologically important. This conclusion was reinforced by sedimentation equilibria of the deoxy-Hb which indicated only very weak dimer-tetramer association. However, the conclusion that tetramers are physiologically insignificant was based on extrapolation of kinetic and sedimentation measurements at 10 -60 M concentration to millimolar concentrations. Dohi et al. (13) extended the sedimentation measurements to concentrations of 3 mM with Hb from another species of lamprey, Entosphenus japonicus. Their results suggested that as much as 85% of the deoxy-Hb within red cells of this species would be tetrameric. The amino acid composition (13) of E. japonicus globin is virtually identi-

Crystalline Ligand Transitions in Lamprey Hemoglobin

“…A Comparison of the Mutational and Crystallographic Analyses-An important test of the proposed structural mechanism for regulation of lamprey hemoglobin has been mutagenesis carried out by Qui et al (21). Nine mutants were expressed and subjected to sedimentation equilibrium and oxygen binding experiments to probe the role of individual residues in the assembly and regulation of oxygen affinity.…”

Hagfish Hemoglobins

“…2). The substitution Tyr-30 3 Phe in itself is sufficient to inhibit dimerization in recombinant lamprey hemoglobin, because it disrupts the interface hydrogen bonding network (41). The presence of Lys-71 in place of arginine in both HbI and HbIII and of Ser-75 in place of glutamic acid in HbI can likewise have a destabilizing effect on the dimeric assemblage.…”

“…Only HbII is able to self-associate and to combine with both HbI and HbIII, as shown by ultracentrifugation (Table II) The requirement of HbII in the formation of M. glutinosa oligomers is intriguing. A possible explanation can be proposed on the basis of the crystal structure of deoxygenated, dimeric P. marinus HbV (12) and of the self-association properties of the mutant proteins constructed by Qiu et al (41). In the deoxygenated lamprey hemoglobin dimer the contact area at the interface is rather restricted, 478 Å 2 (Fig.…”

“…The decreased tendency to dimerize of HbII relative to lamprey hemoglobin (K 1, 2 1.7 ϫ 10 2 versus 6.3 ϫ 10 5 M Ϫ1 (41)) can be accounted for by the Asn-79 3 Gln substitution coupled to the replacement of Tyr-27 by a methionine residue. It is relevant in this connection that the N79D and N79H mutants of P. marinus HbV are unable to polymerize (41).…”

“…In lamprey HbV the Bohr effect is due to an unusual cluster of four glutamyl residues formed at the dimer interface by the side chains of Glu-31 and Glu-75 of both subunits (12). The vicinity of these residues in the deoxy state raises their pK a values from 4.5 to 6.0 and enables them to share a Bohr proton (41). In lamprey hemoglobin, bicarbonate does not behave as an allosteric effector (22).…”