The reduction of tetrazolium salts by plant mitochondria

Summary Data has been obtained concerning the reduction of tetrazolium salts by mitochondria isolated from Jerusalem artichoke tubers with succinate as the substrate using a direct recording spectrophotometric method of assay. ATP was found to increase the rate of reduction of the tetrazolium salts, this being independent of the effect ATP had on the rate of oxygen uptake. The magnitude of the stimulation by ATP depended on the concentration of tetrazolium salts present and under certain circumstances was suppressed by the addition of azide and cyanide. The sites at which the tetrazolium salts were reduced along the electron transport chain were investigated. The role of ATP has been discussed in relation to the mechanism of tetrazolium reduction.Abbreviations TTC 2,3,5-triphenyl-2,1,3,4-tetrazolium chloride - BT 5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-diphenylene)-ditetrazolium chloride - NT 2,2,5,5-tetraphenyl-3,3-(p-diphenylene)-ditetrazolium chloride - MTT 3-(4,5-dimethyl thiozolyl-2)-2,5-diphenyl tetrazolium bromide - INT 2-(p-iodophenyl)-3-p-dinitrophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride - NBT 2,2-dinitrophenyl-5,5-diphenyl-3,3-dimethoxy-4,4-diphenylene)-ditetrazolium chloride - TNBT 2,2-5,5-tetra-p-nitrophenyl-3,3-dimethoxy-4,4-diphenylene) ditetrazolium chloride     

Anti-Plasmodium activity of tetrazolium salts

We have previously reported that sulfated cyclodextrins inhibit the invasion of Plasmodium merozoites by interacting with receptors present on the surface of erythrocytes. The observation that tetrazolium salts formed stable complexes with the inhibitory sulfated cyclodextrins suggested that tetrazolium salts might have anti-Plasmodium activity as well. Evaluation of commercially available tetrazolium salts indicated that some were active in the low nanomolar range and showed specificity in their inhibition of Plasmodium. Synthesis of a further 54 structures allowed us to determine that activity results from an aromatic component attached to the tetrazolium carbon atom (R1) and its size is not critical to the activity of the compound. Nitro modifications of active compounds are poorly tolerated, however, the presence of halogen atoms on aromatic groups attached to the nitrogen atoms of the tetrazolium ring (R2 and R3) has little effect on activity. Methoxy groups are tolerated on R2 and R3 components; however, they are disruptive on the R1 component. The overall results suggest that the R1 component is interacting with a specific hydrophobic environment and the R2 and R3 components are less constrained. The activity of these compounds in several human and mouse Plasmodium cultures suggests that the compounds interact with a component of the parasite that is both essential and conserved.     

On the oxygen-sensitivity of various tetrazolium salts

Summary 1. Eight different tetrazolium salts have been chemically reduced with NADPH and PMS¹ under oxygenated and oxygen-free conditions. 2. PMS has been shown to be able to remove all of the hydrogen from NADPH very rapidly, and to transfer all of this hydrogen onto tetrazolium salts, under suitable atmospheric conditions. 3. MTT, INT, TNBT, and NBT¹ produced the same amount of formazan under both conditions; NT BT, TV, TT¹ produced formazan under oxygen-free conditions, but produced no formazan under oxygenated conditions. 4. These results are explained on the basis of competition for the NADP Hhydrogen between oxygen and the four tetrazolium salts NT, BT, TV and TT.I should like to thank The Arthritis and Rheumatism Council for financial support.     

Reduction of tetrazolium salts by sulfate-reducing bacteria

Abstract The reduction of tetrazolium salts by the sulfate-reducing bacteria, Desulfovibrio desulfuricans and Desulfotomaculum orientis , was examined. D. desulfuricans and D. orientis reduced triphenyltetrazolium chloride (TTC) and 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyltetrazolium chloride (INT) forming intracellular formazan deposits. The reduction rate of INT was higher than that of TTC. INT reduction was not inhibited by the addition of sulfate or molybdate, and sulfate uptake was inhibited by the addition of both INT and molybdate. The ratio of intracellular formazan forming cells to acridine orange direct counts in both strains decreased with culture age and starvation time.     

Effects of tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria

1. The effects of five different tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria have been investigated. 2. In all cases the mitochondria were uncoupled by very low concentrations of the tetrazolium salts. Further, the transition from a system just exhibiting respiratory control to one in which the mitochondria were totally uncoupled has been shown to occur over very small concentration ranges of the tetrazolium salts. 3. The effectiveness of the five tetrazolium salts as uncoupling agents is discussed in the light of their standard electrode potentials and effectiveness as electron acceptors in dehydrogenase-linked reactions.     

Studies on the reduction of nitroblue tetrazolium chloride mediated through the action of NADH and phenazine methosulphate.

The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic condition the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O2-.; this reaction is strongly inhibited by superoxide dismutase and by a number of O2-. scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ions inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence of O2-. is fraught with difficulties due to an equally rapid reduction of NBT by NADH and PMS under anaerobic conditions.     

Colorimetric microbial viability assay based on reduction of water-soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substances

The applicability of a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antimicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antimicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated.     

On the role of oxygen in dehydrogenase reactions using tetrazolium salts

Summary Fundamental aspects of the reduction fo tetrazolium salts were investigated and, in particular, the role of oxygen in the reduction. It was found that oxygen had a competitive inhibitory effect on the reduction of (Tetra)Nitro BT mediated by NADH and phenazine methosulphate. This competitive effect, under aerobic conditions, could be reversed by using tetrazolium concentrations of 5mm. Oxygen did not have a signIficant effect on BPST reduction, whereas the inhibitory effect of oxygen on the reduction of Neotetrazolium was not reversed by increasing the tetrazolium concentration. The oxygen effect on Nitro BT reduction was considerably less when macromolecular substances such as albumin or polyvinyl alcohol were added to the medium. This may be due to increased Nitro BT concentrations being built up at the surface of macromolecules due to the nonpolar components of the Nitro BT molecule. When demonstrating glucose-6-phosphate dehydrogenase activityin vitro or in tissue sections with the use of Nitro BT, oxygen also had a direct inhibitory effect, even when azide was added to the medium for the inhibition of flavoprotein-mediated electron transfer to oxygen. Again, this direct inhibition of Nitro BT reduction by oxygen could be excluded by using a high Nitro BT concentration. Macromolecules present in the incubation medium or in tissue sections counteracted the oxygen effect. It is concluded that the maximum reaction rate and optimum localization of dehydrogenases is obtained when histochemical media are used containing 5mm (Tetra)Nitro BT and 20% polyvinyl alcohol.