These reactions in which dyes are reduced and oxygen is consumed have been followed in vivo by Martin and Logsdon.183 Dyes were found to be reduced by E. coli, and then, after the living cells were separated form the dye solution by filtration, the dyes were found to reduce cytochrome c. Furthermore, drawing on the fact that normal respiration of oxygen is inhibited by cyanide, while the consumption of oxygen by dye reactions is not and is therefore known as cyanide-resistant respiration, it was shown that oxygen consumption by cells is increased by the addition of dyes in the presence of light. This cyanide resistant respiration in the presence of illumination and dyes may equal or even exceed the level of oxygen consumption seen in normal respiration by the cell (fig. 6) .
Figure 6. Cyanide -sensitive and cyanide-resistant respiration.
In this study, the reactions between specific dyes and NADH were followed in vitro. The oxidation of NADH in the presence of dyes was observed, showing whether NADH is capable of serving as an oxidizable substrate for each of the dyes, and the SOD-inhibitable reduction of ferricytochrome c was followed similarly in order to determine whether each of these reactions was capable of O2- production. These reactions were followed with four dyes in solution and with dyes that had been intercalated into a target molecule so that
they were potentially inaccessible to reductants and/or oxygen. The shifts in the
spectra of dyes as they became intercalated into
DNA
were followed in order to determine how much DNA was required to ensure that
most of the dye molecules were in the intercalated state, so that the dye reactions
could be studied under the condition of being bound to DNA, as the intercalating
dyes normally would be if present within a bacterial cell.
Absorption spectra of dyes The absorption spectra of the dyes azure c,
methylene blue, thionin, proflavin, and acridine orange were observed on an IBM
scanning spectrophotometer. DNA intercalation leads to a red shift and decrease in
absorbance of the spectrum of methylene
blue.
Spectra were recorded in the absence of DNA and then in the presence of
increasing amounts of purified calf thymus DNA at room temperature. When
changes in the spectra stopped, except for the lowering of amplitude due to the
dilution effect of adding the additional DNA, the dye was taken to be fully
intercalated. Ratios of DNA to dye were expressed in terms of moles of phosphates
in the DNA to moles of the dye. Dye concentrations were: methylene blue, 5 mm;
thionin, 40 mm; azure c, 7.5 mm; proflavin, 10 mm; acridine orange, 5 mm; fluorescein,
10 mm. Buffer was 100 mM potassium phosphate, 0.1 mM EDTA, pH 7.6.
The calf thymus DNA was purified by dissolving 50 mg in 10 mls proteinase K
buffer
(10 mM Tris, pH 7.8, 5 mM EDTA, 0.5% SDS) and incubating with 50 mg/ml proteinase K (Sigma) for one hour at 505C. The DNA was then extracted twice with 5 mls phenol, once with 10 mls chloropane (1:1 phenol:chloroform), twice with 10 mls chloroform (chloroform:isoamyl alcohol, 24:1 v/v), and twice with ether. Each extraction involved adding the solvent to the aqueous DNA layer, shaking, centrifuging 3 minutes at 1600 G, and transferring aqueous phase to a new centrifuge tube; ether was removed by pipetting it from the top of the aqueous phase followed by blowing nitrogen gas over the aqueous layer to remove remaining traces of ether. This DNA was devoid of associated proteins and should be similar in its dye-binding characteristics to bacterial DNA. The absorption of the DNA did not contribute to the spectra obtained for the dyes.
Cytochrome c reduction The reduction of cytochrome c by O2- was followed spectrophotometrically at 550 nm. Cuvettes were filled with a solution of dye, oxidizable substrate, and 20 mM cytochrome c, and illuminated by being placed in direct contact with a 24-inch GE Warm White fluorescent light bulb for the indicated time intervals. The dyes studied were methylene blue, rose bengal, fluorescein, acridine orange, proflavin, thionin, and azure c; all were at a concentration of 0.5 mm. The oxidizable substrate present was 0.125 mm NADH. The concentration of reduced cytochrome c at the beginning of each experiment was 20 mm, and all reactions took place in 0.05 m potassium phosphate buffer, 0.1 mm EDTA, pH 7.8. DNA concentrations were chosen to be at least twice the values at which most of the available dye molecules would be intercalated, in order to ascertain that the results obtained in the absence of DNA would still hold true for dyes that were intercalated into DNA. 40 units (as defined by McCord and Fridovich)104 of SOD were added to test for its ability to inhibit the reduction of cytochrome c.
NADH oxidation The oxidation of NADH was studied similarly; the cuvettes contained dye, 0.05 m potassium phosphate buffer, 0.1 mm EDTA, pH 7.4 and 0.5 mm NADH. Thionin, rose bengal, methylene blue, and azure c were all studied at 1 mm. Bovine superoxide dismutase was added as an inhibitor of cytochrome c reduction, to determine the percentage of the reduction reaction that was dependent on O2-.
As the dyes exhibit a change upon intercalating into DNA, it is important to study their reactions in both the absence and presence of DNA. Experiments
Figure 7. Shift in the absorption spectrum of azure c with addition of calf thymus DNA. Top line is spectrum of 7.5 mM azure c in 50 mM phosphate buffer, pH 7.6. The second line from the top is the spectrum of the same solution with the addition of 71 mM DNA (DNA concentration expressed in phosphate residues); third, same with 142 mM DNA; fourth, same with 213 mM DNA; fifth, same with 284 mM DNA; sixth (occupying same position on graph as line number five), same with 355 mM DNA.
Figure 8. Shift in the absorption spectrum of methylene blue with addition of calf thymus DNA. Top line is spectrum of 5.0 mM methylene blue in 50 mM phosphate buffer, pH 7.6. The second line from the top is the spectrum of the same solution with the addition of 14 mM DNA; third, with 29 mM DNA; fourth, with 43 mM DNA; fifth, with 57 mM DNA; sixth, with 71 mM DNA; seventh, with 85 mM DNA; eighth, with 98 mM DNA; ninth, with 112 mM DNA; tenth, with 139 mM DNA.
Figure 9. Shift in the absorption spectrum of thionin with addition of calf thymus DNA. Top line is spectrum of 40 mM thionin in 50 mM phosphate buffer, pH 7.6. The second line from the top is the spectrum of the same solution with the addition of 36 mM DNA; third, same with 71 mM DNA; fourth, same with 107 mM DNA; fifth, same with 142 mM DNA; sixth , same with 178 mM DNA; seventh, same with 214 mM DNA; eighth, same with 249 mM DNA.
Figure 10. Shift in the absorption spectrum of acridine orange with addition of calf thymus DNA. Top line is spectrum of 5.0 mM acridine orange in 50 mM phosphate buffer, pH 7.6. The second line from the top is the spectrum of the same solution with the addition of 12 mM DNA; third, same with 23 mM DNA; fourth, same with 35 mM DNA; fifth, same with 46 mM DNA; sixth , same with 57 mM DNA; seventh, same with 68 mM DNA.
Figure 11. Shift in the absorption spectrum of proflavin with addition of calf thymus DNA. Top line is spectrum of 10.0 mM proflavin in 50 mM phosphate buffer, pH 7.6. The second line from the top is the spectrum of the same solution with the addition of 2.9 mM DNA; third, same with 14 mM DNA; fourth, same with 26 mM DNA; fifth, same with 37 mM DNA; sixth , same with 49 mM DNA; seventh, same with 71 mM DNA; eighth, same with 93 mM DNA; ninth, same with 115 mM DNA.
Figure 12. No shift in the absorption spectrum of fluorescein with addition of calf thymus DNA. Top line is spectrum of 10.0 mM fluorescein in 50 mM phosphate buffer, pH 7.6. The second line from the top is the spectrum of the same solution with the addition of 29 mM DNA; third, same with 139 mM DNA. The latter amount of DNA was added in a volume of 50 ml; this represents a dilution of the dye of 5%.
Table II. Ratio of moles of phosphate residues of DNA to moles of dyes at which dyes were fully intercalated.
Dye DNA/dye ratio
Azure c 38 Methyl ene blue 25 Thionin 5 Proflavin 7 Acridine Orange 11 Fluorescein 0
on reactions with NADH and cytochrome c were performed in both the absence and presence of concentrations of DNA greater than required for complete intercalation in order to make sure that the dyes are active as oxidants and reductants even when intercalated into the DNA.
NADH oxidation was followed as the decrease in absorbance at 340 nm. Even when dye was complexed with a saturating quantity of calf thymus DNA, it was capable, upon illumination, of oxidizing NADH (fig. 13 to 16.) The ability of thionin (fig. 13) to oxidize NADH was greatest in the absence of DNA, inhibited slightly by a small amount of DNA, and inhibited more by a saturating concentration of DNA; twice as much DNA as was required for saturation caused the solution to be fairly viscous, slowing the oxidation of NADH markedly, but not to zero. Rose bengal (fig. 14), which does not intercalate into DNA, showed very similar slopes for NADH oxidation curves for all four treatments; an amount of DNA equivalent to that needed to saturate methylene blue was used, since there is no such quantity for rose bengal. Methylene blue (fig. 15) and azure c (fig. 16), like thionin, showed a decreasing but not insignificant rate of NADH
oxidation as the DNA concentration increased. For methylene blue, the presence of the smallest ration amount of DNA appeared to increase reactivity to some extent.
Cytochrome c reduction was observed in the presence and absence of DNA and SOD(fig. 17-23). Every treatment included 20 mM cytochrome c in buffer and 0.5 mM NADH to reduce the dyes. An important question concerned whether SOD would inhibit cytochrome c reduction when the dyes were intercalated into DNA, thus showing that the dyes were accessible to reductants and to O2 when intercalated and capable of producing O2- when intercalated. This O2- could diffuse away from the site of production to reduce cytochrome c. A concentration of DNA was used that was at least twice that sufficient to intercalate all of the dye. The reduction of cytochrome c was observed for methylene blue (fig. 17), rose bengal (fig. 18), fluorescein (fig. 19), acridine orange (fig. 20), proflavin (fig. 21), thionin (fig. 22), and azure c (fig. 23). For all dyes cytochrome c was reduced and in every case the rate of the cytochrome c reduction curve was diminished in the presence of SOD, showing that O2- was being produced by dye-mediated NADH oxidation. The non-intercalating xanthene dyes, rose bengal and fluorescein, showed a minor decrease in the rate of reduction of cytochrome c when DNA was present, presumably due to an increase in solution viscosity, although this effect could also be due to some interaction between the DNA and the cytochrome c or NADH. The reduction of cytochrome c in the presence of the acridine dyes, acridine orange and proflavin, both showed a somewhat greater decrease in reaction due to the presence of DNA, but less than 50%. The cytochrome c reduction mediated by thiazine dyes was greatly reduced by the addition of DNA to the reaction mixture, indicating that
Figure 17. Reduction of 20 mM cytochrome c mediated by 0.5 mM methylene blue in the presence of 0.125 mM NADH. Line 1, no additions; line 2, + 40 units SOD; line 3, + DNA; line 4. + 40 units SOD and DNA. DNA phosphate to dye molar ratio was 2800:1.
Figure 18. Reduction of 20 mM cytochrome c mediated by 0.5 mM rose bengal in the presence of 0.125 mM NADH. Line 1, no additions; line 2, + 40 units SOD; line 3, + DNA; line 4. + 40 units SOD and DNA. DNA phosphate to dye molar ratio was 200:1.
Figure 19. Reduction of 20 mM cytochrome c mediated by 0.5 mM fluorescein in the presence of 0.125 mM NADH. Line 1, no additions; line 2, + 40 units SOD; line 3, + DNA; line 4. + 40 units SOD and DNA. DNA phosphate to dye molar ratio was 200:1.
Figure 20. Reduction of 20 mM cytochrome c mediated by 0.5 mM acridine orange in the presence of 0.125 mM NADH. Line 1, no additions; line 2, + 40 units SOD; line 3, + DNA; line 4. + 40 units SOD and DNA. DNA phosphate to dye molar ratio was 200:1.
Figure 21. Reduction of 20 mM cytochrome c mediated by 0.5 mM proflavin in the presence of 0.125 mM NADH. Line 1, no additions; line 2, + 40 units SOD; line 3, + DNA; line 4. + 40 units SOD and DNA. DNA phosphate to dye molar ratio was 200:1.
Figure 22. Reduction of 20 mM cytochrome c mediated by 0.5 mM thionin in the presence of 0.125 mM NADH. Line 1, no additions; line 2, + 40 units SOD; line 3, + DNA; line 4. + 40 units SOD and DNA. DNA phosphate to dye molar ratio was 5:1.
Figure 23. Reduction of 20 mM cytochrome c mediated by 0.5 mM thionin in the presence of 0.125 mM NADH. Line 1, no additions; line 2, + 40 units SOD; line 3, + DNA; line 4. + 40 units SOD and DNA. DNA phosphate to dye molar ratio was 76:1.
intercalation of the dye into the DNA markedly reduces its accessibility to
cytochrome c, whether due to the interception of O2- by the DNA or due to different
chemistry of thiazines when existing in the hydrophobic environment inside the
DNA. Nonetheless, even when intercalated in DNA, the thiazines are capable of
superoxide dismutase-inhibitable reduction of cytochrome c. The observation that
some of the reduction of cytochrome c is not SOD-inhibitable is explanable by
mediation of cytochrome c reduction directly by reduced dye or by semi-oxidized
NADH.
