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

Abstract

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 a t pH between 7 and 7.8 and protein concentration above x 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 K I or triethanobmine.The dichroic properties of both Hb trout I and Hb trout IV show distinct features in the wavelength region between 300 and 200 nm. H b trout IV resembles human hemoglobin in the shape of the CO spectrum and in the values of the reduced ellipticity ( 6 ) ; furthermore upon deoxygenation the value of 8 a t 222 nm becomes more negative by about 5O/,. On the other hand H b trout I shows a different circular dichroism spectrum and in particular the ellipticity a t 222 nm is lower than that of H b 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 H b tetramers per H p molecule (while for human hemoglobin the stoichiometry corresponds to one tetramer per Hp). A clear cut effect of H p binding is observed on the kinetics of 0, and especially CO combination as studied by flash photolysis.The effect of several third components on the 0, equilibrium of both Hb trout I and IV has been investigated. I n 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.It was shown in the previous papers of this series property of Hb trout IV, i.e. of that component which is present in the blood in larger amounts (%65O/, of the total). On the other hand the 0, binding properties of both Hb trout I and Hb trout I1 are invariant with pH, although the equilibrium curve is markedly cooperative (n = 2.5).The functional differences between the various components have been used to interprete the physiological role played by each of them in the economy of the organism [4]. It is clear that knowledge Eur. J. Biochem. 39 (1973)

Hemoglobin components from trout (Salmo irideus): Determination of the carboxyl and amino terminal sequences and their functional implications

“…His 2 and His 143 @) appear to be missing in Hb Trout IV. Addition of ATP, the physiologically important phosphate in fish, to Hb Trout IV decreases the oxygen affinity only about half as much as in human hemoglobin [20] and, in general, the effect of organic phosphates on the 0, binding of Hb Trout IV is considerably smaller than that observed with human hemoglobin [5,21]. Similar correlations were pointed out by Powers and Edmundson [ 171 in the case of Cs.…”

Global Allostery Model of Hemoglobin

“…The highly sensitive pH dependence of the O 2 binding curves exhibited by Hb with BZF ϩ IHP Ϯ Cl Ϫ (Figs. 1F, 2F, and 4) reminds us of the Root effect of some fish Hbs in terms of the exaggerated pH sensitivity of P 50 accompanied by decreasing cooperativity at acidic pH values (49). This behavior of Hb must be created primarily by the Mode R mechanism, by which the O 2 affinity of oxy-Hb (K R ) is reduced as much as 3 orders of magnitude upon acidification.…”

Multiple strategies for O2 transport: from simplicity to complexity

“…This effect turned out to be so (109). The lack of heterotropic interactions has been related to the substitution of some key residues involved in the interaction of organic phosphates in human Hb (110). marked that at pH 5 6.5 no quaternary transition occurs and it was possible for the first time to obtain a T-liganded Hb (at least for the CO-bound form), which was characterized by a variation of the spectral properties (118).…”

“…The structural basis for the lack of heterotropic interactions has been proposed to rely on the substitution of some key residues involved in the interaction of organic phosphates (110) (Fig. 3).…”