The ultraviolet (UV) absorbance ratio of 260/280 nm has been used as an
indicator of
DNA purity. However, the A260/A280 ratio may be beyond the normal range (1.8-1.9) due to physicochemical alterations produced by pH and temperature, and carcinogenic chemical modification. When the pH of the
DNA solution buffer increased from 3 to 11, the A260/A280 ratio changed significantly from 1.5 to 2.2 in mixtures of
DNA bases [A:T:C:G = 28.5:28.5: 21.5:21.5, i.e., (A +
T)/(all four bases) = 57%, expressed as mole percent], of
deoxyribonucleosides (
adenosine:
thymidine:
cytidine:
guanosine= 28.5:28.5:21.5:21.5, as mole percent), or of
deoxyribonucleotides (dAMP:
dTMP:
dGMP:
dCMP = 28.5:28.5:21.5:21.5, as mole percent) examined. The A260/A280 ratio increased with
RNA contamination and exceeded 1.9 when
RNA concentration was >30%, as mole percent. In contrast, the A260/A280 ratio was linearly reduced by increasing the
protein concentration.
Phenol (>0.02%) contamination also reduced the A260/A280 ratio to below 1.8.
Benzo[a]pyrene diol
epoxide (
BPDE), a reactive
carcinogen metabolite of
benzo[a]pyrene (BaP), decreased the A260/A280 ratio correlated with the degree to which it modified the
DNA. These results suggest that the UV A260/A280 ratio is significantly affected by pH and the presence of contaminating species of macromolecules and chemicals.