A macromolecular transducer as illustrated by trout hemoglobin IV.

Abstract

Oxygen binding by trout Hb IV has been investigated as a function of pH up to 10 atmospheres (1 MPa) of pure 02. The results bring out an extreme proton-oxygen linkage, which gives rise to a Root effect. They are discussed in relation to the function of the hemoglobin as an oxygen pump. The system is of special interest as providing a prototype of a macromolecule acting as a transducer by coupling two allosterically linked reactions. The basic function of the hemoglobins is to ensure an adequate supply of oxygen to all parts of the organisms in which they occur. In order to accomplish this task they have developed, in the course of evolution, a common molecular mechanism based on the principle of ligand-linked conformational change in a multisubunit structure (1-3). Within the framework of this common mechanism, however, different hemoglobins, sometimes in the same organism, have acquired special features to meet special needs. This is illustrated in certain of the teleost fish, such as the rainbow trout (Salmo irideus), in which different hemoglobins, characterized by distinct properties, have been isolated and investigated (4). One of the components of trout blood, in fact the major one (trout Hb IV), has acquired very special properties that enable it to serve as a pump for injecting 02 into the swim bladder against a very high opposing pressure. This function, widespread among teleost fishes, was recognized long ago by Root (5) and was subsequently studied in the unfractioned hemolysate of benthic fishes and qualitatively analyzed by Scholander and van Dam (6).The object of this communication is the presentation of the oxygen equilibria of isolated trout Hb IV, which is responsible for this phenomenon. These equilibria are of interest not only because they illustrate the variations possible within the scope of an overall allosteric mechanism, but, even more, because this hemoglobin represents a type case of how a macromolecule, operating under steady-state conditions, can serve as a transducer.In view of the very low 02 affinity of trout Hb IV at acid pH, which is what makes the pump effective, the experiments called for the application of high partial pressures of 02; we developed, therefore, a special spectrophotometric cell capable of operating up to approximately 25 atmospheres (1 atm = 101 kPa), following the pioneering work of Scholander and van Dam (6). This makes it possible to undertake a complete thermodynamic characterization of 02 binding by this Hb, in the same way as was done in the case of CO (7). In that case, the thermodynamic analysis of the data obtained by calorimetric and equilibrium experiments provided clear evidence for the strong dependence of the allosteric equilibrium constant, Lo = (Ro)/(To), on the concentration of allosteric effectors, such as H+ and organic phosphates, according to prediction based The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C...

A polymerising Root‐effect fish hemoglobin with high subunit heterogeneity

“…5, the markedly low co-operativity of oxygen binding in the whole pH range and under all experimental conditions examined, with the Hill coefficient in the range 1.0-0.85 (being higher than 1 .O only at pH > 7.5), finds an explanation. The value of h,,, < 1 at pH < 7.0 can be accounted for by a pH-dependent stabilisation of the T state, accompanied by a marked subunit functional heterogeneity within the tetramer, a feature already found in other Root-effect fish hemoglobins (Noble et a]., 1970(Noble et a]., , 1986Brunori et al, 1978;Pennelly et al, 1978;Galdames-Portus et al, 1979;Morris and Gibson, 1982; Prisco et al, 1988). However, a rise in pH brings about the appearance in oxygenated hemoglobin of a predominance of the R state, as indicated by the autocatalytic shape of the oxygen-dissociation curve (see curve a in Fig.…”

“…In several fish hemoglobins, a large reduction in the oxygen affinity and co-operativity is observed at low pH values (and in the presence of allosteric effectors), so that these hemoglobins cannot be fully saturated even with pure oxygen at pressures of several Pascalles (Scholander and Van Dam, 1954;Brunori et al, 1978). According to the interpretation of this effect (called the Root effect) in the framework of the two-state model of Monod et al (1965), the T-R equilibrium is shifted by protons and/or other allosteric effectors towards the low-affinity conformation, thereby inhibiting the ligandlinked quaternary transitions and abolishing the co-operativity.…”

Multiple strategies for O2 transport: from simplicity to complexity

“…In addition, a pHdependent variation of the affinity for the T quaternary state can be observed, which is especially marked for O 2 binding, accompanied by a functional heterogeneity for the two types of subunits, which becomes clear-cut at moderately acid pH values (116). 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).…”

“…Thus, at moderately acid pH values (i.e., 56.5), trout HbIV is incompletely saturated even at atmospheric O 2 pressure, a phenomenon called 'Root effect' (93,108). Detailed investigation on the pH dependence for O 2 and CO binding (116,117) clearly envisaged a more complex behavior than for trout HbI. Thus, a marked pH dependence of the ligand binding isotherms is accompanied by a dramatic pH dependence of cooperativity, which underlies a linkage between H þ and the allosteric equilibrium (Fig.…”

The reaction of trout hemoglobins with isocyanides

“…that component characterized by a dramatic pH dependence of ligand binding, the Root effect, and involved in the secretion of 0 2 at the level of swimbladder) displays, at acid pH, a non-cooperative binding curve for both ligands and an extreme functional heterogeneity in the case of O2 [6]. In the case of trout HbIV the binding of both oxygen and carbon monoxide displays an exothermic character all along the equilibrium curve, thus resembling human HbA and differing from trout HbI.…”