M Brunori

STUDIES ON FUNCTIONAL PROPERTIES OF FISH HEMOGLOBINS .2. OXYGEN EQUILIBRIUM OF ISOLATED HEMOGLOBIN COMPONENTS FROM TROUT BLOOD

Homogeneous components of trout hemoglobin (Salmo irideus) have been isolated by column chromatography. n nThe oxygen equilibrium of the two main components has been investigated. The oxygen affinity and the shape of the ligand equilibrium curve is independent of pH for component I. On the other hand, component IV is characterized by a very large Bohr effect, to which a considerable change in the shape of the oxygen equilibrium curve with pH is associated. n nThe different oxygen-binding behavior of the isolated components can explain data obtained with the whole blood and in particular the contribution of the various proteins to the Root effect. n nThe dependence on pH of the apparent ΔH for oxygenation has been measured for both components. Component I is characterized by a fairly low (ΔH ~ −3 kcal/mole) and pH independent value of the enthalpy change, while for component IV the apparent ΔH decreases from ~ −14 kcal/mole at pH near 9 to ~ −7 kcal/mole at pH 7.

Distribution of the haemoglobin components of trout blood among the erythrocytes: Observations by single-cell spectroscopy

The distribution of haemoglobin components in erythrocytes has been determined by single-cell spectrophotometry. In the case of trout blood, which contains three major haemoglobin components, it has been shown that all erythrocytes contain the component characterized by the Root effect (Hb trout IV) in a proportion very similar to that found in the haemolysate. The method described here also appears to have great possibilities in the study of the distribution, among and within the erythrocytes, of abnormal human haemoglobin.

NMR studies of 13CO-hemoglobin. α and β chain identification

In a preceding paper ~1] it was shown that the resonance signal o f a3CO bound to the homes o f human hemoglobin wa~ split wk~e only a single resonance lhae was present in ~ e case o f aBCO my@oh re . It was sngges~.ed tlam ~lae two resonance lines of hemoglobin n~fight e or~esgond to 1 3 C O bound Io dae ~ and t~ dae ~ chains of ~ e molecule. These ~ u l ~ s have been confirmed and more accurme values o f chem~caa sibAft have been assigned [2] to ~he ~wo resonance s igna l s . T h e rneasnrer .aen.~ of 13C 0 r e s o n a n c e m i g h ~ • hhen .offer ~ e possibility to m.ea~re directly the d i e ~iibution o f the CO b,oand ,to hemo~ob in beW, e~n the ~ and the ~ chains provided ,that ~t ~s known which ~esonance signal c oliesp.onds to which chahn. A ,en*ative assigrm~ent hs~ been a~ade on fl~e b~is of kin,e~c da~a I2]. With this aim we havle d v t e ~ i n e d the N~ spectnma o f , h e ]3:CO derivatives of .the i sohIed hem, Ggl,obin a and ~ oh:fins. The data obtain,ed are ;eporte,d in the pr,esent note.

Nuclear magnetic resonance quadrupole relaxation studies of chloride binding to the isolated hemoglobins from trout (Salmo irideus)

NMR studies of chloride binding to the main components of trout blood, Hb Trout I and Hb Trout IV, indicate that although the affinity of chloride is high for both hemoglobins, the characteristics of the binding process are markedly differnet. In Hb Trout IV chemical exchange at the chloride binding site(s) is fast and quadrupole effects determine the linewidth; chloride binding has a definite pH dependence, but there is no significant oxygen linkage. In contrast Hb Trout I represents a unique case of slow chemical exchange, which may depend on unusual stereoche mical characteristics of the chloride binding site; chloride binding is pH independent, but shows a significant oxygen linkage, which may be attributed to changes of the lifetime of chloride at the binding site. The chloride binding properties displayed by Hb Trout I and IV have been compared with those of normal and modified human hemoglobins and discussed in terms of the structural differences in the C- and N-terminal regions of the alpha- and beta-chains.

Spectral properties and reactivity towards azide of Dicrocoelium dendriticum met-hemoglobin

The spectroscopic properties of Dicrocoelium dendriticum met-hemoglobin, investigated between pH 3.8 and 10.5, display two proton-induced transitions with apparent pK values of 8.1 and 4.7. The spectral changes, over the pH region 6.5 to 10.5, correspond to the high to low spin transition usually observed in ferric hemoproteins. The relaxation time (tau = 3.2 ms at the pK) associated with this transition is closely similar to that observed in Aplysia limacina met-myoglobin, but approximately 1000-fold slower than that of sperm whale met-myoglobin. The spectral changes associated with the more acid transition are in the opposite direction and have not been resolved by the temperature jump method. The rate constants for the reaction of azide with Dicrocoelium dendriticum met-hemoglobin were measured between pH 3.8 and 6.7 by the temperature jump method. Two kinetic schemes, both consistent with the pH dependence of the apparent rate constant for binding of azide, were identified. No objective way of discriminating between the two models is available. However, one of them is amenable to a physical interpretation based on a comparison with the structural, kinetic and spectral properties of other monomeric hemoproteins.

Functional properties of partially oxidized trout hemoglobins

This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The O2 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However, the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the pH dependence of the apparent on constant in the oxidation mixtures is similar to that characteristic of the native molecule. The results of the O2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is an increase in the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.

Kinetic properties of intermediates in hemoglobin from trout Salmo irideus

Understanding of the reaction mechanism in the binding of ligand to ferrous hemoglobin has greatly profited from successful attempts to prepare and characterize partially ligated intermediates. The availability of the isolated chains of human hemoglobin has made it possible to prepare frozen intermediates in which one type of chain is in the ligand-bound (ferric) state, and the other is free to react with oxygen or carbon monoxide [l-4] . However, it has not been possible to isolate intermediates in which either one or three sites in the tetramer are frozen in the ligand-bound state. With human hemoglobin the inability to isolate such intermediates has been interpreted on the basis of the fast dimer-exchange process occurring in the ligand bound form [S] . It has recently been reported that one of the hemoglobin components from trout’s blood, the so-called Hb trout I, is a very stable tetramer, the value of the dissociation equilibrium constant being lo100 times smaller than that of human hemoglobin [6]. Therefore, it seemed feasible in the case of Hb trout I to attempt the preparation and isolation of all the species which should exist upon partial oxidation of Hb CO. As reported below, it was indeed possible to isolate the five species expected on theoretical grounds, i.e.: (as&)(CO), , W-W W%, (MW2 (W2, W2T3 (CO), and

Studies on cobalt-reconstituted trout hemoglobins

1. Introduction Thermodynamic and kinetic studies of the oxygen and carbonmonoxide binding to trout Hb IV, the main hemoglobin component of trout’s blood, have recently contributed to the understanding of the molecular mechanism and the physiological role of the Root effect, i.e., the drop in oxygen affinity and cooperativity observed at low pH. This effect has been interpreted in the framework of a classical two states model [ 13, as a stabilization by protons or organic phosphate of the low affinity form of the ligand-bound derivative [2]. On the other hand, in the last few years recon- stitution of myoglobin and hemoglobin containing cobalt in the place of iron have been successfully employed to elucidate structure-function relation- ships in hemeproteins [3,4]. The reaction of these cobalt-reconstituted hemeproteins with oxygen has been shown to be cooperative and reversible. More- over, the paramagnetic character of the Co(I1) allowed a detailed EPR study of the deoxy- and oxy-state of the reconstituted proteins [4]. The availability of globins from Hb I and Hb IV from trout blood [5] and the purpose of gaining new relevant information on the effect of protons on the liganded and the un~ganded derivative of these proteins, prompted to prepare cobalt containing trout hemoglobins and to study their functional and EPR properties. 2. Materials and methods Cobalt-meso-Hb IV and cobaltlproto- and meso-

INFRARED STRETCHING FREQUENCIES OF CO IN CARBOMONOXY- HEMOGLOBIN FROM TROUT

The infrared spectra of the carbomonoxy derivatives of the hemoglobin components I and IV from trout have been measured in the CO stretching frequency region using a high resolution infrared spectrometer. The CO stretching frequency of Hb I CO is very close to that of carbomonoxy human hemoglobin and is pH-independent. In contrast, the CO stretching frequency of Hb IV CO is higher and shows a small but significant pH dependence in the range 6.2-7.8. These results point to a decreased strength of the iron-CO bond in Hb IV CO at low pH, in agreement with the conclusions drawn from the reported difference spectra of Hb IV CO as a function of pH.

Hemoglobin in fishes: Structural and functional properties of trout hemoglobins

Abstract Homogeneous components of trout hemoglobin (Salmo irideus), have been investigated from the structural and functional point of view. Only one (Hb trout IV) of the 2 major components found in trout hemoglobin is characterized by a Root effect; the other components (Hb trout I and II) are not affected by pH at all. No change was observed in the CD spectrum going from ligand bound to ligand free Hb trout I; instead ligand binding by Hb trout IV has an effect on the CD which is very similar to that observed for human hemoglobin. Kinetic experiments on the O2 and CO reactions show a similarity in behaviour of trout hemoglobin components at pH > 7.5. Hb trout I, as well as Hb trout II, is pH independent, while the kinetic behaviour of Hb trout IV is strongly pH dependent. Where the Root effect is operative, the dissociation velocity of oxygen increases largely, thus suggesting that the kinetic basis of the Root effect resides, at least in part, in the off constants.