Oxine concentration quenching

It remained to be shown whether the toxic action of oxine was due to the withdrawal of essential metals, as had been suggested (Zentmyer, 1944), or whether it actually increased the toxic action of metals normally present in the medium. The latter proved to be the case for both the bacteriostatic and the bactericidal actions (Rubbo etal., 1950 Albert etaL, 1953), as was indicated in the outline of this work presented in Section 2.3 (p. 37). The first clue came from concentration quenching. 

Table 11.6 The effect of increasing concentration on the bactericidal action of oxine in broth ( concentration quenching ). Staph, aureus in meat broth at pH 7.0-7.3 (20°C)...

It is reasonable to assume that the toxic species of oxine is the 1 1 ferrous complex because it is unsaturated in other words it has the unused combining power that is necessary in a catalyst. The 2 1 complex 11.33), on the other hand, is saturated and unlikely to be catalytic. (In aerobic systems, both mous and ferreV species are present in equilibrium it follows that the 1 1 and 2 1 ferr V complexes are unsaturated and presumably catalytically active, whereas the 3 1 complex is saturated and likely to be inert.) This concept reinforces a similar conclusion made on the basis of concentration quenching (p. 470). 

The question was then posed does oxine act on bacteria by removing metals essential to bacterial welfare, or does it cause traces of metals to become more toxic to the bacteria The latter proved to be the case, as first indicated by the following example of concentration quenching . Staphy lococci were completely killed in an hour by 0.01 mM oxine but were unharmed by 0.70 mM oxine in fact even a saturated (5.0 mM) solution would not kill them (Albert, Gibson, and Rubbo, 1953). Streptococci behaved similarly. The meaning of this phenomenon became clear when it was found to occur only in media containing traces of iron or copper. The viability of staphylococci for 24 hours in distilled water permitted the decisive experiments, summarized in Table 2.3, to be made. 

Other chelating antimicrobials have been found that, while having a totally different structure, mimic the action of oxine by being active only in the presence of a variable-valence metal, and hence show concentration quenching. Such a substance is i-hydroxypyridine-2-thione (pyrithione), (2.2j) (Albert, Rees, and Tomlinson, 1956), which is much used in the dermatology of the scalp. Another example is dimethyldithiocarbamic acid (2.2 ), whose salts are widely used as selective fungicides in agriculture. 

Concentration quenching. It is highly unusual for the effect of a biologically active substance to decrease as the concentration is increased. However, oxine shows this phenomenon to an unprecedented degree. As will be seen from Table ii.6, staphylococci which are killed in an hour by M/100 000 oxine are not killed (even in 3 hours) by M/1600 oxine (in fact, even a saturated solution, which is M/200, will not kill them). There was, however, a degree of toxicity after 24 hours (Albert et al.y 1953). Streptococci behaved similarly. The meaning of this concentration quenching became evident when it was found that it occurred in broth, but not in distilled water. 

Experiments with oxine in broth. When broth is used instead of water, no added iron is necessary (Table 2.3), because a sufficient amount has been introduced from the meat. When the concentration of oxine was increased to M/800, the bactericidal action disappeared (concentration quenching). It seemed evident to us that the toxic action was caused by the i i-complex 2.22) (if ferroM5) but not by the saturated 2 i-complex ii.2j) which must be the only form present when oxine is in excess. Therefore we added iron in sufficient amount (M/800) to equal that of the oxine, and thus restore the unsaturated i i-complex. As expected, this combination proved just as highly bactericidal as the earlier one (see Table 2.3). It should be noted that M/800 iron is not toxic on its own, but the oxine has made it so. 

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