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Biomedical subjects

M Brunori

Publications and source records attributed to M Brunori.

At least 361 records · Page 20Linked to original sources
PubMed1975

Identification of chloride-binding sites in hemoglobin by nuclear-magnetic-resonance quadrupole-relaxation studies of hemoglobin digests.

35Cl minus-nuclear magnetic resonance (NMR) studies indicate that various digests of human hemoglobin with carboxypeptidase A and B, or a combination of the two, may be used for the identification of chloride binding sites. All the digestion products contain, like hemoglobin itself, at least two classes of binding sites, one of high, the others of low affinity. The pH dependence of the excess linewidth of the 35Cl minus NMR signal indicates that in the simple digests with either carboxypeptidase A or B, chloride is bound with high affinity at or near His-beta146-Asp-beta94 and at or near Val-alpha1-Arg-alpha141. The high-affinity sites show, in the case of the simple digests, a strong oxygen linkage which is lost in the forms digested with both carboxypeptidase A and B; this linkage may thus be correlated to the presence of conformational changes. Organic phosphates, like inositol hexaphosphate, show competition for some of the high-affinity chloride binding sites in hemoglobin and in the simple digests. This competition is likewise lost in the doubly digested hemoglobins.

Binding Sites↗
PubMed1975

Equilibrium and kinetics of the reaction of Aplysia myoglobin with azide.

The present paper reports a study on the equilibria and kinetics of the acid-alkaline transition and the azide binding reaction by ferric Aplysia myoglobin. A single completely reversible spectrophotometric titration curve is found over the pH range from similar to 5 to similar to 9, with an apparent pK equals to 7.5 for the acid-alkaline transition. The kinetics of the process, followed by the temperature-jump method, gives, at pH values close to the pK of the transition, one single, well-resolved, relaxation independent of protein concentration and of type of buffer used. The pattern accords to a simple pH dependent reaction, in buffered medium, between the two forms of the protein. The results of the azide binding reaction show that the process conforms to simple equilibrium as expected for a single site protein. The méasured association constant is reported as a function of pH. The kinetics of the reaction of Aplysia metMb with N3- minus shows, on the other hand, a complex behavior. The relaxation pattern is found to strongly depend on pH and ligand concentration in such a way to suggest a linkage between ligand binding and acid-alkaline transition. The system is discussed on the basis of two simplifying conditions, i.e., at low and higher pH with respect to the pK of the acid-alkaline transition. At acid pH the reaction corresponds to a single bimolecular process as expected for a simple binding reaction; at alkaline pH, the dependence of relaxation time on ligand concentration implies the existence of a rate-limiting monomolecular step. On the basis of a reaction scheme implying that binding of the ligand can only occur through the acid (aquomet) form of the protein via the displacement of the water molecule, the experimental data are quantitatively accounted for.

Azides↗
PubMed1975

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.

Animals↗
PubMed1975

Electron transfer between azurin and cytochrone c-551 from Pseudomonas aeruginosa.

The electron-transfer reaction between azurin and cytochrome c1 isolated from Pseudomonas aeruginosa was investigated by rapid-reaction techniques. Temperture-jump studies clearly reveal two chemical relaxations, the amplitudes of which have ikentical spectral distributions, but relaxation times show different dependencies on reactant concentrations. Stopped experiments also showed complex kinetics. A model is proposed which is consistent with the kinetic and equilibrium data obtained. The central feature of this model is the proposal that two intercenvertible forms of reduced azurin exist in solution, only one of which si able to participate directly in the electron-transfer reaction with cytochrome c-551. Support for the hypothesis that two forms of reduced azurin exist is derived from studies on the electron-transfer reaction between azurin and Pseudomonas cytochrome oxidase. The possible physiological significance of such a situation is discussed.

Azurin↗
PubMed1975

Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.

Copper↗
PubMed1975

A temperature-jump study of the reaction between azurin and cytochrome c oxidase from Pseudomonas aeruginosa.

The electron-transfer reaction between azurin and the cytochrome oxidase from Pseudomonas aeruginosa was investigated by temperature-jump relaxation in the absence of O2 and in the presence of CO. The results show that: (i) reduced azurin exists in two forms in equilibrium, only one of which is capable of exchanging electrons with the Pseudomonas cytochrome oxidase, in agreement with M. T. Wilson, C. Greenwood, M. Brunori & E. Antonini (1975) (Biochem. J. 145, 449-457); (ii) the electron transfer between azurin and Pseudomonas cytochrome oxidase occurs within a molecular complex of the two proteins; this internal transfer becomes rate-limiting at high reagent concentrations.

Azurin↗
PubMed1975

Kinetics of binding of carbon monoxide to lumbricus erythrocruorin: a possible model.

This paper represents a kinetic study of the binding of carbon monoxide by Lumbricus erythrocruorin. Observations on the quantum and the relaxation of the system both to equilibrium and to the steady state realized in the presence of constant illumination under various conditions are reported. The results, besides indicating the existence of at least two types of binding sites, give indications as to the behavior of a complex polyfunctional molecule, such as an enzyme, working under steady-state conditions.

Animals↗