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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)

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 (z 7 4 , in a region where the Root effect is not operative, comparison of the kinetics of the various components (Hb trout I, 11 and IV) shows considerable similarities between them. For Hb trout I (and 11) the time course of the reaction with both 0, 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 0, 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 a t least for 0, the effect of pH on the ''off' kinetic constants is much more marked.

Four protein protease inhibitors (I, II, III, IV) having low molecular weights (10600–6500) and basic isoelectric points were isolated by affinity chromatography from bovine spleen. Inhibitor IV was identified as the basic pancreatic trypsin inhibitor (Kunitz inhibitor); the presence and distribution of components I, II and III vary in the different bovine organs. Spleen inhibitors I, II, III and IV were purified by ion‐exchange chromatography: they form 1:1 complexes with trypsin and inhibit enzymatic activity of trypsin, chymotrypsin and kallikrein. Inhibitors I, II and III contain carbohydrate moieties (7–4%) covalently bound to the polypeptide chain. Specific basic pancreatic trypsin inhibitor antiserum has shown the complete identity between inhibitor IV and the basic pancreatic trypsin inhibitor, while partial cross‐reactivity between the basic pancreatic trypsin inhibitor and inhibitors I, II and III can be seen from a double immunodiffusion test.

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