Mechanism for reduction of the dye

It is not known how the dye is reduced. It is not known why toxic chemicals inhibit the reduction. It is thought that tetrazolium dyes are reduced by cytochromes (Altman, 1975). But this has been questioned (Marshall et al., 1993). In eucaryotic cells, all the cytochromes are in the mitochondria. Marshall s group has found that the dyes are reduced in preparations from cells with the mitochondria removed. It has been found that a mutant of Escherichia coli lacking one of two major cytochromes found in this bacterium is unable to reduce MTT (Botsford, unpublished). But in E. coli, reduction of the dye is not inhibited by toxic chemicals. This may not be an analogous situation. The dye could be reduced by a different meeh-anism. 

meliloti, like most other bacteria, has many reductases. Some of these are membrane associated and damage to the membrane could affect the reductase. One of these reductases could be responsible for reduction of the dye. It has been found that the MTT is transported into the cell before it is reduced. The reduced dye is inside the cells. Cells with the dye can be concentrated by centrifugation, the dye appears in flie cell pellet. None of the dye is in the supernatant. Toxic chemicals could interfere with the transport of dye into the cell prior to reduetion. 

Transposon insertion mutants unable to reduce the dye have been obtained and five mutants have been isolated. All grow very slowly in minimal media supplement with 0.1% easamino acids and obviously all have lost a critical function. Wifli fliese mutants it should be possible to elone and then to sequence the function responsible for reduction of the dye. From the sequence, the nature of the function can be determined. 

This work shows that the Rhizobium test provides results comparable to other tests. Tests seem particularly comparable to work with Daphnia magna and with results from in vitro tests with animal cells. The test is simple, unskilled laboratory workers can master it quickly. The test is inexpensive, no specialized equipment is required, given cells, any laboratory able to carry out simple chemical analysis should be able to perform the assay. The test is rapid, a sample can be tested and analyzed in an hour, the test does not take several days. It offers an ideal first test for toxic chemicals (Blaise, 1991) 

Altman, F. P. 1976. TetrazoUnm salts and formazans. Progress is Histochemistry and Cytochemistry 9 6-52. Bitton, G., Dutka, B. 1. (1986) Toxicity testing using microorganisms. CRCPress Inc. Boca Raton, Florida 163 

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Other examples include acridine dyes (with absorption peaks aroimd 475 nm), xanthene dyes ( 500-550 nm), fluorone dyes ( 450-550 nm), coiunarin dyes (" 350-450 nm), cyanine dyes ( 400-750 nm), and carbazole dyes ( 400 nm) (12,19-21). The oxidation or reduction of the dye is dependent on the co-initiator for example, methylene blue can be photoreduced by accepting an electron from an amine (22) or photo-oxidized by transferring an electron to benzyltrimethyl-stannane (12). Either mechanism will result in the formation of a free-radical active center capable of initiating a growing polymer chain. For a more detailed discussion of the mechanisms, see Reference 12. 

The acceleration mechanism of redox mediators are presumed by van der Zee [15]. Redox mediators as reductase or coenzymes catalyze reactions by lowering the activation energy of the total reaction. Redox mediators, for example, artificial redox mediators such as AQDS, can accelerate both direct enzymatic reduction and mediated/indirect biological azo dye reduction (Fig. 3). In the case of direct enzymatic azo dye reduction, the accelerating effect of redox mediator will be due to redox mediator enzymatic reduction in addition to enzymatic reduction of the azo dye. Possibly, both reactions will be catalyzed by the same nonspecific periplasmic enzymes. In the case of azo dye reduction by reduced enzyme cofactors, the accelerating effect of redox mediator will either be due to an electron shuttle between the reduced enzyme cofactor and redox mediator or be due to redox mediator enzymatic reduction in addition to enzymatic reduction of the coenzymes. In the latter case, the addition of redox mediator simply increases the pool of electron carriers. 

Optimal conditions were found for analysis of the azo dye 2,6-dichloro-4-nitro-2,-(acetylamino)-4 -(diethylamino)azobenzene (257) by various polarographic reduction methods and a mechanism was proposed for the process531. 

A large part of the reduction of silver chloride by hydrazine evidently takes place by a different mechanism from that of the reduction by hydroxylamine. The effect of gelatin and dye on the process, together with the appearance of colloidal silver in the solution when gelatin is present to stabilize it, shows that the reaction involves dissolved silver chloride to a greater degree than the hydroxylamine reaction. Indeed, if the reaction rate is plotted against a silver ion concentration calculated on the assumption that a saturated solution of silver chloride is maintained, the same relation is obtained as is found for the reduction of silver ions from a solution of the sulfite ion complex. 

We have already described how nitration leads eventually to aromatic amines by reduction of the nitro group. In the next chapter you will meet the further development of these amines into diazoni-um salts as reagents for nucleophilic aromatic substitution by the S l mechanism with loss of nitrogen. In this chapter we need to address their potential for electrophilic aromatic substitution without the loss of nitrogen as this leads to the important azo dyes. Treatment of the amine with nitrous acid (H0N=0) at around 0°C gives the diazonium salt. 

The mechanism of spectral sensitisation has been shown to involve the direct transfer of an electron from the excited state of the dye into the AgX conduction band (see Fig. 11.8). In confirmation of this mechanism, excellent correlation has been demonstrated between the sensitisation capability of dyes and their electrochemical reduction potential. For the most efficient dyes, the quantum yield of the electron transfer step is close to 1.0. Using picosecond laser pulse excitation to measure the fluorescence decay rates of dyes adsorbed to AgX crystals, it has been shown that the electron transfer is very fast, with rate constants in the region of 10 s having been reported [24]. Once an electron has been transferred into the AgX conduction band, the latent image formation process ensues more or less as it does following direct absorption by AgX itself. The difference in the case of the dye-sensitised process is that the positive holes are trapped at the dye molecules. 

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