Thermodynamic considerations of arteriovenous gradients of hydrogen ion concentration and carbon dioxide tension.

It is shown that, in a multicompartmental homeostatic system, the extent of interaction between any two compartments can be assessed by determination of the difference in free energy change of one particular reaction, or a series of coupled reactions, operative in both of the compartments under consideration. Hydrogen ion concentration and carbon dioxide tension have been used to determine free energy change difference relationships between the venous and arterial compartments (-deltadeltaG(a-v)) of the circulatory system. Data from the literature (from two studies of congestive heart failure and one study of experimentally induced cardiac arrest) are used to calculate -deltadeltaG(a-v). It was found that in control subjects -deltadeltaG(a-v) is close to zero, whereas in congestive heart failure or cardiac arrest, the value rises to 150 cal mol(-1) or more, whereas in blood, the approach towards equilibrium between hydrogen and bicarbonate ions and dissolved carbon dioxide (aqueous CO2) is known to be only moderately rapid. It is concluded that, in the system under study, and with respect to the reaction H+ + HCO3- = CO2 + H2O, a high value for the free energy change difference between the two compartments (high -deltadeltaG(a-v)) must be due to an insufficient blood circulation rate. Accordingly, -deltadeltaG(a-v) is probably a quantitative measure of cardiac insufficiency.

Reactivity of 6-phosphogluconolactone with hydroxylamine: the possible involvement of glucose-6-phosphate dehydrogenase in endogenous glycation reactions.

The reactivity of 6-phosphogluconolactone and of delta-gluconolactone with hydroxylamine (a model compound in electrophilicity determination studies) was examined and compared with the reactivity of several other electrophiles, such as acid anhydrides and esters, some of which exhibit adverse biological effects (e.g. carcinogenicity). At pH 7.6 and 30 degrees C, and with an excess of hydroxylamine concentration, most of the compounds tested disappear from the medium in a monoexponential reaction. On the other hand, the reaction of 6-phosphogluconolactone with hydroxylamine is biexponential. This finding indicates the existence of 6-phosphogluconolactone in two interconvertible, isomeric forms. The reactivity, towards hydroxylamine, of 6-phosphogluconolactone and, to a lesser extent of delta-gluconolactone, is on the upper scale of reactivity of the electrophiles tested. It is concluded that 6-phosphogluconolactone (and in particular, one of its isomeric forms) is a highly electrophilic compound, and may possibly react with sundry intracellular nucleophiles, thereby exerting untoward metabolic effects. In this connection, it is of interest that a positive correlation has been found to exist between glucose-6-phosphate dehydrogenase activity and cell proliferation.

Kinetic analysis of 6-phosphogluconolactone hydrolysis in hemolysates.

Hydrolysis of enzymatically generated 6-phosphogluconolactone in hemolysates has been found to be a first-order reaction, in all cases studied. It follows that the Michaelis constant of the reaction is higher than the highest 6-phosphogluconolactone concentration used in this study, viz., it is higher than 0.30 mmol l-1. The reaction first-order rate constant was found to decrease as the concentration, in the enzyme assay mixture, of the 6-phosphogluconolactone preparation was increased. It follows that this paradoxical effect may give rise to determinations involving spurious Michaelis constant and enzyme activity results. The possible causes of this effect are discussed.

Kinetic evidence for the existence of an unstable intermediate in the trinitrophenylation-induced rhodanese inactivation reaction.

1. Rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate, in the presence of n-butylamine in the reaction medium, has been studied by a kinetic analysis of the data, based on the assumption that enzyme inactivation is brought about by direct reaction of this with the modifying agent. 2. Initial reaction rates for rhodanese activity loss were determined by a mathematical analysis of the first three recorded values of rhodanese residual activity. 3. It was found that fractional rhodanese activity values, at infinite reaction time with 2,4,6-trinitrobenzenesulphonate (end-point values), were significantly lower than the values calculated on the assumption of rhodanese inactivation being entirely due to direct trinitrophenylation of enzyme protein. 4. Also, initial enzyme inactivation values were higher in the presence, rather than in the absence, of n-butylamine. 5. These results indicate that 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation, in the presence of n-butylamine in the reaction medium, is due to the generation of a highly reactive, unstable intermediate, probably a free radical species.

Utilization of the free energy of the reversible binding of protein and modifying agent towards the rate-enhancement of protein covalent modification.

An analysis is presented of the catalytic factors responsible for the rate-enhancement that may be observed when a protein modification reaction is compared with a reaction of the same modifying agent with a model micromolecular compound exhibiting the same reactive group as the protein under study. It is seen that affinity-mediated rate-enhancement of protein modification is realized by the loss of activation entropy. On the assumption that attainment of maximal affinity-mediated rate-enhancement presents with an activation entropy of the protein modification reaction equal to zero, whereas the activation enthalpy of the reaction remains unchanged, it is shown that the value for maximal affinity-mediated rate-enhancement is equal to e-delta s++/R. Accordingly, protein modification reactions may be differentiated into (i) reactions the rate-enhancement of which (relative to the reaction of the same modifying agent with a model compound) is primarily entropy-controlled and (ii) reactions the rate-enhancement of which is primarily enthalpy-controlled. It is seen that modifying agents of low reactivity towards model compounds, but with a high, i.e. highly negative, activation entropy are better suited as prospective affinity-based protein-modifying agents, since the potential affinity-mediated rate-enhancement, and hence the selectivity, of these compounds is necessarily high. Kinetic and thermodynamic constants of the reaction of modifying agents with proteins, and with model compounds, and values of maximal affinity-mediated rate-enhancement, based on published data of the reaction of several modifying agents with model compounds, are presented and discussed.

Kinetic analysis of regeneration by dilution of a covalently modified protein.

An analysis of regeneration by dilution of a covalently modified protein is presented. It is shown that, when protein regeneration is realized through the intermediacy of a protein-modifying agent adsorptive complex, the reaction is described by a summation of two exponential functions of reaction time plus a constant-term equation. The conditions whereby this equation reduces to a single-exponential equation are delineated. It is shown that, when protein regeneration is described by a single-exponential function of reaction time, the first-order protein-regeneration rate constant is a function of modifying-agent concentration and also of the microscopic reaction rate constants. Accordingly, the protein-modifying agent dissociation constant (Ki), as well as the protein-covalent-modification and -regeneration, rate constants (k+2 and K-2), may be determined by an analysis of dilution-induced protein-regeneration (or enzyme-reactivation) data obtained at different dilutions of the covalently modified protein-modifying agent preparation.

Diethylbarbiturate potentiation of 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation.

The rate of rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate is increased in the presence of diethylbarbiturate in the reaction medium. A "rate saturation effect" indicates the formation of a rhodanese-diethylbarbiturate complex, prior to modification-induced enzyme inactivation. The dissociation constant of this complex is 19.0 mM. Diethylbarbiturate has no effect on the trinitrophenylation rate of the free amino groups of rhodanese. When rhodanese modification, in the presence of diethylbarbiturate in the reaction medium, is carried out by the use of a 2,4,6-trinitrobenzenesulphonate concentration much lower than the concentration of rhodanese modifiable amino groups, reaction stoichiometry indicates that 3 to 5 moles of rhodanese are rendered inactive for each mole of 2,4,6-trinitrobenzenesulphonate utilized. This finding indicates the existence of a chain-reaction type mechanism of rhodanese inactivation.

Kinetic analysis of protein modification reactions at equilibrium.

A kinetic analysis is presented of reactions of protein modification, and/or of modification-induced enzyme inactivation, which can formally be described by a single exponential function, or by a summation of two exponential functions, of reaction time plus a constant term. The reaction schemes compatible with the kinetic formalism of these cases are given, and a simple kinetic criterion is described whereby the identification of one of these cases, strong negative protein modification co-operativity, may be carried out. The treatment outlined in this paper is applied to a case from the literature, the inactivation of glyceraldehyde-3-phosphate dehydrogenase by butane-2,3-dione [Asriyants, Benkevich & Nagradova (1983) Biokhimiya (Engl. Transl.) 48, 164-171].

Inactivation of rhodanese from human gastric mucosa and stomach adenocarcinoma by 2,4, 6-trinitrobenzenesulphonate and by 4,4'-diisothiocyanatostilbene-2,2'-disulphonate.

Rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) was partially purified from normal human gastric mucosa and from gastric adenocarcinoma by DEAE-cellulose column chromatography. Rhodanese inactivation by 2, 4, 6-trinitrobenzenesulphonate and by 4,4'-diisothiocynatostilbene-2-2'-disulphonate was studied by an analysis of the time-dependence of rhodanese activity loss. Rhodanese inactivation by 2, 4, 6-trinitrobenzenesulphonate was, under all conditions tested, found to be first-order with regard to enzyme residual activity. In contrast to this, rhodanese inactivation by 4, 4'-diisothiocyanatostilbene-2, 2'-disulphonate was found to be biphasic, when log residual enzyme activity was plotted vs reaction time. The first-order rate constant, k, for rhodanese inactivation by 2, 4, 6-trinitrobenzenesulphonate was, at all pH values tested, higher with the gastric adenocarcinoma enzyme than with the normal mucosa enzyme: at pH 8.00, k is 27.0 per hour, for the normal mucosa enzyme, while for the adenocarcinoma enzyme k is 69.0 per hour. In contrast to this, no difference in the inactivation profile of the normal mucosa enzyme and the gastric adenocarcinoma enzyme was to be observed with 4,4'-diisothiocyanatostilbene-2,2'-disulphonate.