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The functional properties of human hemoglobin and of isolated α- and β-chains with free SH groups blocked by 2-chloromercuri-4-nitrophenol, 4-chloromercuri-2-nitrophenol and p-chloromercuribenzoate have been determined. The oxygen affinity of all the hemoglobin derivatives is slightly higher than that of the untreated protein; the shape of the oxygen equilibrium curve is pH dependent (except for p-chloromercuribenzoate reacted hemoglobin). The alkaline Bohr effect is similar to that of normal hemoglobin, but in the acid range it appears to be modified to a different extent in the various cases. Thus the properties of the modified hemoglobins appear to depend on the details of structure of the organic mercurial reacted with the β93-SH group. The results with isolated β-chains show that the presence of the organic mercurials on F9(93), but not on G14(112) is associated with a decrease in oxygen affinity and a large increase in the oxygen dissociation velocity constant. [ABSTRACT FROM AUTHOR]

The ligand-binding kinetics of the isolated component from trout (Salmo irideus) blood has been extensively investigated ; it appears to be fully consistent with their equilibrium behaviour, described previously. At alkaline pH (&assymp; 7.5), in a region where the Root effect is not operative, comparison of the kinetics of the various components (Hb trout I, II and IV) shows considerable similarities between them. For Hb trout I (and II) the time course of the reaction with both O2 and CO is practically pH independent. On the other hand Hb trout IV, characterized by a Root effect, displays a strong dependence on pH of the kinetics of ligand binding. Thus in the pH region where the Root effect becomes operative there is a large change of the character, as well as of the speed, of the kinetics with both O2 and CO. The Root effect characteristic of Hb trout IV appears to originate, from a kinetic point of view, in a pH-dependent change of both the “on” and “off” constants, although at least for O2 the effect of pH on the “off” kinetic constants is much more marked. [ABSTRACT FROM AUTHOR]

This paper reports some physico-chemical and functional properties of two of the isolated hemoglobin components from trout (Salmo irideus) blood (Hb trout I and Hb trout IV). Both hemoglobins in the oxygenated state are tetrameric at pH between 7 and 7.8 and protein concentration above &assymp; 0.1 mg/ml. The tetramer is extremely stable towards dissociation into subunits and the stability constants, estimated by differential gel filtration, are between 10 and 50 times larger than that of human hemoglobin in phosphate buffer. A very small degree of dissociation is also suggested by the very small extent of hybrid formation even in concentrated KI or triethanolamine. The dichroic properties of both Hb trout I and Hb trout IV show distinct features in the wavelength region between 300 and 200 nm. Hb trout IV resembles human hemoglobin in the shape of the CO spectrum and in the values of the reduced ellipticity (θ); furthermore upon deoxygenation the value of θ at 222 nm becomes more negative by about 50%. On the ether hand Hb trout I shows a different circular dichroism spectrum and in particular the ellipticity at 222 nm is lower than that of Hb trout IV. Moreover the spectrum is insensitive to the presence of heme ligands. Hb trout IV binds human haptoglobin (Hp) as shown by quenching of the fluorescence of the tryptophanyl residues of Hp upon addition of hemoglobin. The apparent stoichiometry of complex formation corresponds to two Hb tetramers per Hp molecule (while for human hemoglobin the stoichiometry corresponds to &assymp; one tetramer per Hp). A clear cut effect of Hp binding is observed on the kinetics of O2 and especially CO combination as studied by flash photolysis. The effect of several third components on the O2 equilibrium of both Hb trout I and IV has been investigated. In general the effect of adding ions or other molecules to a solution of lib trout I is very minor. On the other hand Hb trout IV is more susceptible to changes in solvent composition. Of particular importance is the action of ATP, the physiological organic phosphate, which is without effect on Hb trout I, while it has a clear effect on Hb trout IV. The addition of ATP shifts the Root effect, the characteristic property of Hb trout IV, to higher pH values, thereby acting as one of the allosteric effectors. [ABSTRACT FROM AUTHOR]