A comparative approach to protein-and ligand-dependence of the Root effect for fish haemoglobins

Abstract

Ligand-binding equilibria, kinetics and (13)C n.m.r. spectra of bound (13)CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the (13)CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O(2) strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the alpha and beta chains in the tetramer were obtained for both haemoglobins from (13)CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.

Allosteric Effect of Water in Fish and Human Hemoglobins

“…These Hbs bind a mutually similar but smaller number of water molecules than the anodic Hbs during the deoxy to oxy transition (ϳ17-31, when betaine is used as solute; Table I) despite marked differences in their sensitivity to organic phosphates, which is lacking in trout HbI and pronounced in eel HbC (9,25).…”

The reaction of trout hemoglobins with isocyanides

“…The Bohr effect in fish hemoglobins may be assigned to two different contributions, one of quaternary origin and linked to the pH dependence of the allosteric constant (Lo), the other of tertiary origin and hence local to the subunits constituting the tetrameric molecule. In the case of 0 2 , tertiary heterotropic effects and low-affinityhigh affinity pH-dependent quaternary transition have been clearly demonstrated [2,6,17], along with a marked functional heterogeneity, as is possibly the case with iPrNC and tBuNC. Thus, the overall functional behaviour is a complex average of both contributions as is also suggested by the different pH dependence of affinity displayed by the three ligands (Fig.…”

Multiple strategies for O2 transport: from simplicity to complexity

“…This behavior cannot be accounted for the simple MWC model and it requires the introduction of additional elements, such as subunit functional heterogeneity and the existence of a nesting of tertiary structures within a given quaternary state (117). A marked subunit functional heterogeneity is not infrequent in fish Hbs for both O 2 and CO binding, allowing us to distinguish clearly the behavior of the two chains (119); in some cases, it has been possible to fully attribute this subunit functional heterogeneity to variations of the stereochemistry of the proximal bond (120).…”