Kinetics of the Reaction of Hemoglobin with Ethylisocyanide: INTERPRETATION OF THE RESULTS WITHIN A DIMER SCHEME

Abstract The kinetics of the reaction of human deoxyhemoglobin with ethylisocyanide has been studied, by rapid mixing, over a 50- to 100-fold range of ligand concentration, both as a function of protein concentration (from 3 to 30 x 10-6 m) and ionic strength (from 0.2 to 2.2 m). The results show that the progress curve, which is autocatalytic at high ligand concentration, tends to change shape as the ethylisocyanide concentration is decreased, and finally becomes markedly diphasic. The experimental results can be fitted satisfactorily with a simple dimer scheme, with only two combination and two dissociation velocity constants. Consideration of these results, in conjunction with other data, allows us to arrive at important conclusions concerning the kinetic origin of co-operativity as observed at equilibrium. The most significant of these is that, to a major degree, cooperative ligand binding finds its kinetic justification in a large decrease of the dissociation velocity constant as the reaction proceeds

Kinetics of the reaction with oxygen of mixtures of oxy- and carbon monoxide hemoglobin.

Abstract The paper reports rapid mixing and relaxation experiments performed on mixtures of oxy- (HbO2) and carbon monoxide (HbCO) human hemoglobin. On the (well justified) assumption that the two ligands will distribute at random between the available sites, intermediates containing different proportions of O2 and CO will be formed. In the stopped flow experiments mixtures containing different proportions of the two ligands have been mixed with sodium dithionite (Na2S2O4), which rapidly reduces to zero the O2 concentration in the system. The apparent dissociation velocity constant for O2 (koff) measured under these conditions decreases progressively as the fraction of HbCO in the mixture increases, in agreement with previous observations on sheep hemoglobin. Temperature jump experiments performed on mixtures of HbCO and HbO2 show that the amplitude of the faster relaxation time (τf) relative to that of the slower one (τf) increases as the percentage of HbCO in the mixture is progressively increased. At high enough percentage of HbCO (≥70%), the amplitude of the faster relaxation time becomes dominant. The reciprocal relaxation time (τf-1), measured under these conditions, is linearly dependent on oxygen concentration, while it is independent of protein concentration (so long as O2 is buffered). The apparent second order velocity constant is kon = 4.8 x 107 m-1 s-1 at 25°. Simple considerations indicate that the kinetics of the reaction with oxygen of mixtures containing high enough percentages of HbCO should represent the combination and dissociation velocity constants of high affinity forms of hemoglobin

Observations on rapidly reacting hemoglobin.

Abstract The time course of appearance of quickly reacting hemoglobin at pH 7 in dilute solutions (l10 µm in heme) was studied by means of a stopped flow-flash apparatus. When the flash is fired as soon as possible after the reaction of human deoxyhemoglobin with carbon monoxide or immediately after dilution of a concentrated human carbon monoxide hemoglobin solution, little of the quickly reacting form is present; the amount of quickly reacting material increases with time and reaches its final value in a few seconds. The results may be due to dissociation of ligand bound hemoglobin

THE EFFECT OF OXYGENATION ON THE RATE OF DIGESTION OF HUMAN HEMOGLOBINS BY CARBOXYPEPTIDASES.

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Studies on the Relations between Molecular and Functional Properties of Hemoglobin: VII. KINETIC EFFECTS OF THE REVERSIBLE DISSOCIATION OF HEMOGLOBIN INTO SINGLE CHAIN MOLECULES

Abstract The kinetics of the reactions of human hemoglobin with carbon monoxide and oxygen has been studied in photochemical and rapid mixing experiments over a large range of hemoglobin concentration. When the reaction is initiated by rapid removal of the ligand from ligand-bound hemoglobin, the kinetics of the combination of hemoglobin with CO shows a marked concentration dependence in both the photochemical and the rapid mixing experiments. In dilute hemoglobin solutions (below 10-5 m in heme), dissociation of the ligand from oxyhemoglobin or carbonmonoxyhemoglobin is followed by slow changes (half-time of the order of seconds) in the properties of the system. These results lead to the following picture, which is also consistent with other as yet unexplained aspects of hemoglobin kinetics. (a) Ligand-bound hemoglobin dissociates reversibly into single chain molecules at concentrations below 10-5 m. (b) Deoxygenated hemoglobin has a much lower tendency to dissociate into single chain molecules, and there is no appreciable dissociation even at concentrations of the order of 10-6 to 10-7 m. (c) The association of deoxygenated α and β chains is a relatively slow process. Therefore, after sudden dissociation of the ligand from dilute hemoglobin solutions, the properties of the system, for a brief time, are those of a mixture of deoxygenated hemoglobin and deoxygenated α and β chains. (d) The properties of the single chain molecules obtained by dilution of ligand-bound hemoglobin are the same as those of isolated α and β hemoglobin chains as obtained by preparative procedures

Light scattering and sedimentation of native human adult and fetal globin.

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Properties of modified cytochromes. I. Equilibrium and kinetics of the pH-dependent transition in carboxymethylated horse heart cytochrome c.

Reduced (Fe+2) carboxymethylated cytochrome c, Cm-cyt. c, undergoes a reversible pH-dependent transition with a pK of 7.16 at 20°. This pK is found to be nearly temperature-independent indicating that the over-all enthalpy for the transition is close to zero. The kinetics of this transition have been investigated by the temperature jump technique. A single well resolved relaxation process (in the millisecond time range) is observed over the pH region of the static titrations. The amplitude of this relaxation at different wave lengths fits the statically derived difference spectrum between the alkaline and acid forms of the protein. Both the amplitude and the relaxation time τ are pH dependent; the over-all enthalpy of the process is estimated to be ∼ +1 Cals per mole. The observed behavior may be accounted for by a model in which a proton-linked conformational change in the protein is responsible for the spectral changes. It is suggested that the deprotonation of an e-amino group of lysine (possibly that of lysine 79) is followed by the binding of this group to the ferrous iron to fill the vacant sixth coordination position. The observed spectral changes are attributed to the binding of a nitrogen atom to the iron. The thermodynamic parameters governing the conformational part of the reaction are calculated on the basis of the above model and values of ΔH = -10 Cals per mole and ΔS = -20 e.u. are found. These values are discussed in the context of the binding of protein residue to the iron and the consequent changes in the crevice structure

Properties of the Product of Partial Photodissociation of Carbon Monoxide Hemoglobin

Abstract Some properties of the quickly reacting hemoglobin generated by partial photodissociation of human CO hemoglobin have been studied using flash photolysis methods. The absorption spectrum of the rapidly reacting photoproduct is different from that of slowly reacting "normal" hemoglobin and, similarly to other quickly reacting forms, corresponds to that of isolated deoxyhemoglobin chains. The fraction of the fast reacting material increases linearly with the fraction of nonphotodissociated hemoglobin. On the basis of these results and of other information on hemoglobin kinetics it is suggested that this fast reacting species represents partially saturated intermediate which appears transiently when the ligand is suddenly removed from ligand-bound hemoglobin

Studies on the relations between molecular and functional properties of hemoglobin. I. The effect of salts on the molecular weight of human hemoglobin.

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THE OXYGEN EQUILIBRIUM OF CYSTINE-TREATED HUMAN HEMOGLOBIN WITHOUT FREE SULFHYDRYL GROUPS.

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