The allosteric properties of hemoglobins, especially their responses to ligands other than oxygen, vary widely in different classes of vertebrates. Knowing the stereochemistry of the cooperative effects in human hemoglobin, one can infer the stereochemical basis of these variations from the changes in amino acid sequence. The results indicate that the tertiary and quaternary structures of deoxy- and oxyhemoglobin have remained almost invariant during vertebrate evolution and that most of the amino acid replacements between species are functionally neutral. Adaptations leading to responses to new chemical stimuli have evolved by only a few (one to five) amino acid substitutions in key positions. Once such a response has become superfluous, it may be inactivated, not necessarily by a reversal of one of the original substitutions but by any other that happens to inhibit it.

Species adaptation in a protein molecule.

This chapter describes the hemoglobin (Hb) structure and function in fishes. Hb of most vertebrates is a tetrameric globular protein consisting of two α and two β polypeptide chains, each having an oxygen-binding heme. The oxygen affinity of Hb varies with globin structure, giving rise to species differences that relate to differences in primary structure. The hydrogen ion equilibria of Hb are fundamental to both the structure and the physiological function of the protein. The exchange of H + between protein and solvent is important for blood CO 2 transport and it makes Hb an effective nonbicarbonate buffer that limits fluctuations in blood pH upon acid or base additions. The acidic carboxyl groups of glutamic acid and aspartic acid residues are negatively charged at physiological pH and are titrated only when pH falls below 6. Basic groups, such as the guanidyl group of arginine and the amino group of lysine side chains, are positively charged at physiological pH and release H + only when the pH exceeds above 9. It is found that the fixed acid Haldane effect is generally large in teleosts, but differs among species.

1 Hemoglobin Structure and Function

In 1931 Root reported that the presence of CO2 markedly reduced the oxygen content of the blood of toadfish, sea robin, and mackerel, and caused the oxygen dissociation curve of the hemoglobin to become “nearly asymptotic with respect to the abscissa before saturation is complete”, which is still a valid description of the Root effect (cf. Brittain 1987). The same effect is obtained by acid addition to the blood (Root and Irving 1943).

The Physiology of the Root Effect

Considering the presently available data it is clear that the Root effect represents an exaggerated alkaline Bohr effect which occurs in the absence of a normal acid Bohr effect and is associated with a loss of oxygen binding capacity at low pH. Undoubtedly at the molecular level the presence of a Ser residue at position F9(94) beta in these haemoglobin is of primary importance. No Root effect haemoglobin has yet been identified which lacks this substitution. On the other hand however many haemoglobins are known which possess this Ser residue and at the same time lack a Root effect. Other factors arising from interactions at other sites in the haemoglobin molecule are obviously sufficient to negate the otherwise stabilizing effect of this critical Ser residue. The loss of cooperativity of Root effect systems as the pH is lowered is readily explained as due to stabilization of the low affinity T state to such a degree that the switch to the high affinity R state is suppressed even in the fully liganded molecule. The observation of Hill coefficients of less than unity requires that within the T state chain heterogeneity exists such that the alpha and beta chain haems demonstrate significantly different affinities for ligand. The physiological role of Root effect haemoglobins is demonstrably not inevitably linked to the swim bladder but more probably arose from the need to oxygenate the poorly vascularized retina of many fishes.(ABSTRACT TRUNCATED AT 250 WORDS)

Max F. Perutz

Papers

Kinetic and Equilibrium Studies on the Role of the β-147 Histidine in the Root Effect and Cooperativity in Carp Hemoglobin