Studies on the Properties of Fish Hemoglobins Molecular Properties and Interaction with Third Components of the Isolated Hemoglobins from Trout (Salmo irideus)

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 ∼ 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 5%. On the other 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 ∼ one tetramer per Hp). A clear cut effect of Hp binding is observed on the kinetics of O 2 and especially CO combination as studied by flash photolysis. The effect of several third components on the O 2 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 Hb 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.