Dinitrophenol

Abstract: Publisher Summary High-performance liquid chromatography (HPLC) provides a rapid, sensitive, and reproducible means of separating and quantifying simultaneously a variety of sulfur-containing amino acids and related derivatives. The HPLC method described in this chapter is modified and is based on the initial formation of S-carboxy-methyl derivatives of free thiols followed by the conversion of free amino groups to 2,4-dinitrophenyl (DNP) derivatives. Following derivatization, nanomole levels of individual sulfur-containing amino acids are measured using UV detection at 365 nm after separation by reverse-phase ion-exchange HPLC. Because of the versatility of this HPLC method, biological specimen preparation as well as derivatization and HPLC analysis procedures are discussed. DNP derivatives of acidic amino acids (including thiol-containing compounds) are separated on a 3-aminopropyl column by reversed-phase ion-exchange HPLC. In the mobile phase, methanol is used to elute rapidly the excess 2,4-dinitrophenol and the DNP derivatives of basic and neutral amino acids. Acetic acid is present in the mobile phase to maintain the bonded-phase amino groups in the protonated form. By increasing the sodium acetate concentration of the mobile phase, selective elution of acidic DNP derivatives is accomplished. The eluted DNP derivatives are measured by detection at 365 nm.

Abstract: The ATP/ADP-antiporter inhibitors and the substrate ADP suppress the uncoupling effect induced by low (10-20 microM) concentrations of palmitate in mitochondria from skeletal muscle and liver. The inhibitors and ADP are found to (a) inhibit the palmitate-stimulated respiration in the controlled state and (b) increase the membrane potential lowered by palmitate. The degree of efficiency decreases in the order: carboxyatractylate (CAtr) greater than ADP greater than bongkrekic acid, atractylate. GDP is ineffective, Mg.ADP is of much smaller effect, whereas ATP is effective at much higher concentration than is ADP. Inhibitor concentrations, which maximally suppress the palmitate-stimulated respiration, correspond to those needed for arresting the state 3 respiration. The extent of the CAtr-sensitive stimulation of respiration by palmitate has been found to decrease with an increase in palmitate concentration. Stimulation of the controlled respiration by p-trifluoromethoxycarbonylcyanide phenylhydrozone (FCCP) and gramicidin D at any concentrations of these uncouplers is CAtr-insensitive, whereas that caused by a low concentrations of 2,4-dinitrophenol and dodecyl sulfate is inhibited by CAtr. The above effect of palmitate develops immediately after addition of the fatty acid. It is resistant to EGTA as well as to inhibitors of phospholipase (nupercain) and of lipid peroxidation (ionol). Moreover, palmitate accelerates spontaneous release of the respiratory control, developing in rat liver mitochondria under certain conditions. This effect takes several minutes, being sensitive to EGTA, nupercain and ionol. Like the fast uncoupling, this slow effect is inhibited by ADP but CAtr and atractylate are stimulatory rather than inhibitory. In artificial planar phospholipid membrane, palmitate does not increase the membrane conductance, FCCP increases it strongly and dinitrophenol only slightly. In cytochrome oxidase proteoliposomes, FCCP, gramicidin and dinitrophenol (less effectively) lower, whereas palmitate enhances the cytochrome-oxidase-generated membrane potential. In this system, monensin substitutes for palmitate. It is concluded that the ATP/ADP antiporter is somehow involved in the uncoupling effect caused by low concentrations of palmitate and, partially, of dinitrophenol, whereas uncoupling produced by FCCP and gramicidin is due to their action on the phospholipid part of the mitochondrial membrane. A possible mechanism of this effect is discussed.

Abstract: Publisher Summary This chapter discusses the application of inhibitors and uncouplers that are useful in the study of oxidative phosphorylation. The various inhibitors and uncouplers may be classified based on the chemical-coupling theory of oxidative phosphorylation, which can be formulated. The chapter discusses the non-site-specific inhibitors (oligomycin and aurovertin), site I-specific inhibitors (Amytal and alkylguanidines), site II-specific inhibitors (n-Heptylquirwline N-oxide and phenylethylbiguanide), site III inhibitors (synthalin), and inhibitor of phosphorylafion of added ADP. Oligomycin is used to inhibit the synthesis of ATP by respiratory-chain oxidative phosphorylation without inhibiting the initial conservation of energy. Aurovertin is used in ethanolie solution, and 0.4 micromole aurovertin per gram protein inhibits oxidative phosphorylation at all sites. As inhibition by Amytal is partially relieved by dinitrophenol, this compound may be classed as an inhibitor of oxidative phosphorylation. The most commonly used uncoupler is 2, 4-dinitrophenol, which affects all phosphorylation sites in the respiratory chain, but has no effect on the substrate-linked phosphorylation. 2,6 -Dinitrophenol acts very similarly to 2, 4-dinitrophenol. The concentrations of 2, 6-dinitrophenol, at different pH's, inducing the maximal ATPase and maximal stimulation of respiration in the absence of ADP and phosphate. Dicoumarol [3, 3 ' -methylenebis (4-hydroxycoumarin)] is active on all phosphorylating sites of the respiratory chain, and has no effect on the substrate-linked phosphorylation. Arsenate readily uncouples substrate-linked phosphorylations but is relatively ineffective against respiratory-chain phosphorylation. Other uncouplers gramicidin and valinomycin are also discussed.

Abstract: Sodium salicylate, aspirin and methyl salicylate uncouple oxidative phosphorylation in liver, kidney and brain mitochondrial preparations. Liver and kidney are more sensitive to these agents than is brain. The uncoupling action is qualitatively similar to that seen with the classical uncoupling agent, 2,4-dinitro-phenol. The concentrations of salicylates used in these studies are comparable to toxic levels in the intact animal. These compounds stimulate the oxygen consumption of brain mitochondria in an ortho phosphate and acceptor-saturated system, with pyruvate and malate as substrate. This effect is not abolished by incubation of brain mitochondria with ethylenediaminetetraacetate. Salicylates stimulate the respiratory rate of an acceptor-deficient system and inhibit fatty acid oxidation of rat liver mitochondria. Like dinitrophenol, these agents enhance the liberation of inorganic phosphate from ATP. Sodium salicylate, aspirin, methyl salicylate, and o-pyrocatechuate effectively depress oxidative phosphorylation while sodium benzoate, gentisate, β-resorcylate, γ-resorcylate, para-aminosalicylate, salicylamide, antipyrine and acetophenetidin are weak or ineffective uncoupling agents. The oxygen consumption of liver and muscle slices from salicylate treated-rats is increased. It is suggested that the above actions are responsible for many of the manifestations of salicylate poisoning.