A fluorogenic, mixed-disulfide reagent for thiol assay

The design and synthesis of the mixed disulfide (ortho-nitroaniline-N-ethyldithio) 2-[pyridyl-5-thioureido-N'-(5-fluorescein)] are described and the chemical and spectroscopic properties of this thiol-specific fluorogenic reagent are presented. The high reactivity and sensitivity of this reagent in thiol assay are demonstrated with low-molecular-weight thiols and with human carbonmonoxyhemoglobin and its subunits. Comparison with conventional absorption methods shows that at least 100 times less material is needed; moreover, high background absorbance or turbidity do not interfere with the assay.     

Dimethylmaleic anhydride, a specific reagent for protein amino groups

The reagent dimethylmaleic anhydride does not cause a stable modification of thiol compounds under the conditions used for modification of protein amino groups, in contrast to maleic and monomethylmaleic anhydrides, which produce an irreversible modification of sulfhydryl groups. This behavior and the low reactivity toward hydroxyamino acid residues, shown in a previous work, make dimethylmaleic anhydride a specific reagent for protein amino groups.     

Production of thiol groups and retention of calcium ions by cardiac mitochondria.

Efflux of Ca2+ from previously Ca2+-loaded heart mitochondria was measured after inhibiting respiratory activity. The efflux was increased by p-chloromercuribenzoate, methylmercuric chloride, Cu2+, Fe2+, 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (uncoupler). 1,1,1-trifluoro-3-(2-thienylacetone and indomethacin; after such increase it could be diminished by dithiothreitol. The induced loss of the Ca2+ was accompanied by a loss of endogenous adenine nucleotide. Methylmercuric chloride was particularly effective, since it was active at ratios of about 1 nmol/mg of mitochondrial protein. The non-respiring mitochondria were found to regenerate bound thiol groups after their original complement had reacted with thiol-blocking reagent. This regeneration was diminished by the Ca2+-efflux stimulatig agents that were not themselves thiol-blocking reagents, such as thyroxine, uncoupler, trifluorothienylacetone and indomethacin. The external exposure of thiol groups was also diminished by thyroxine, uncoupler and trifluorothienylacetone. The results support the proposal made previously that the membrane is maintained in a state of low permeability by adenine nucleotide and Mg2+ being bound to thiol-dependent sites.     

The role of thiol groups in the structure and mechanism of action of arginine kinase

1. A detailed study of the reaction of iodoacetamide with arginine kinase has been carried out. 2. The enzyme contains five reactive thiol groups per 37000g. of protein, all of which can be alkylated. 3. Below pH8.5 loss of activity is substantially independent of pH and can be correlated with the alkylation of a single pH-independent thiol. 4. One catalytic site per enzyme molecule is inferred. 5. The progress curves of the alkylation reaction are polyphasic and reveal a pH-and time-dependent sequential release of thiols which is dependent upon the alkylation of the first pH-independent thiol. This is supported by electrophoretic investigations. 6. Comparison of alkylation rate and rate of loss of activity suggests that two thiol groups are not essential for catalytic activity. Variability in enzyme preparations with respect to alkylation rate appears to be associated with these two groups. 7. A complex protection pattern is revealed by the effects of various substrate combinations on rates of alkylation and of loss of activity. It is inferred that two thiol groups participate in conformational changes and nucleotide interactions. 8. Comparison with creatine kinase suggests a fundamentally similar catalytic mechanism, although for arginine kinase certain additional restrictions are necessary because of the protection observed with nucleotide substrates.     

The effect of a cross-bridging thiol reagent on the catecholamine fluxes of adrenal medulla vesicles

The thiol groups of the vesicular protein of bovine adrenal medulla were allowed to react with the bifunctional thiol reagent bis-(N-maleimidomethyl) ether and with the monofunctional thiol reagent N-ethylmaleimide, and the ATP-dependent and -independent catecholamine fluxes of the modified preparations were studied. 1. During the initial phase of the reaction bis-(N-maleimidomethyl) ether blocks twice as many thiol groups as does N-ethylmaleimide at equimolar concentrations. 2. Labelling of the bis-(N-maleimidomethyl) ether-protein compound with [(14)C]-cysteine shows that 70-80% of the blocked thiol groups are interconnected by the bifunctional thiol reagent. 3. At a low extent of reaction (1.5mol of thiol groups/10(6)g of protein) the catecholamine efflux is diminished. If more than 2mol of thiol groups/10(6)g of protein are blocked, the efflux is enhanced whichever thiol reagent is applied. 4. If 2-4mol of thiol groups/10(6)g of protein are blocked the inhibition of the catecholamine influx increases linearly with the proportion of the thiol groups blocked. 5. ATP protects the catecholamine influx and the adenosine triphosphatase activity against bis-(N-maleimidomethyl) ether poisoning somewhat less effectively than against N-ethylmaleimide poisoning.     

Stimulation of septation in mitochondria by diamide,a thiol oxidising agent

Summary Diamide (10^–4M), a thiol oxidizing agent, rapidly promotes septation in isolated frog liver mitochondria and also in situ in liver slices. The effect is partially inhibited by dithioerythritol. DNP does not have this effect, and it is concluded that diamide does not promote septation via an uncoupling action. The septate mitochondria have a different appearance from typical dividing mitochondria previously described; it is suggested that (1) diamide may act by favouring the fusion of the internal membranes, and (2) -SH oxidation is of importance in mitochondria in ageing and in various pathological conditions. The ways in which septa may develop in mitochondria in the orthodox and condensed configuration are discussed.     

Stabilising action of carnitine on energy linked processes in rat liver mitochondria

Rat liver mitochondria exposed to stressing conditions - ageing at room temperature, incubation in the presence of t-butyl hydroperoxide or damaging concentrations of Ca2+ and phosphate- undergo a rapid fall in their membrane potential (delta psi) with a concomitant release of endogenous Mg2+ and accumulated Ca2+. Addition of L-carnitine to the incubation medium considerably delays mitochondrial deenergization. A similar, though lower, protection has also been observed in L-carnitine pretreated and subsequently washed rat liver mitochondria. Furthermore mitochondria isolated from livers of starved rats, treated with L-carnitine 30 minutes before death and exposed to the same stressing conditions show similar delay in the decrease of delta psi and concurrent energy linked processes as compared with untreated animals. Both the in vitro and in vivo results strongly indicate that the stabilising action of L-carnitine on liver mitochondria is due to the removal of membrane bound long chain acyl CoA.     

On the mechanism of action of alkylguanidines on oxidative phosphorylation in mitochondria.

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.