Effects of chemical carcinogens and physicochemical factors on the UV spectrophotometric determination of DNA.
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.
Kim
Hyung Sik
HS
Laboratory of Molecular Toxicology, College of Pharmacy, Pusan National University, Jangjun-dong, Gumjung-ku, Pusan, South Korea.
Byun
Soo Hyun
SH
Lee
Byung Mu
BM
D016604:Aflatoxin B1; D001482:Base Composition; D001564:Benzo(a)pyrene; D001580:Benzopyrenes; D002273:Carcinogens; D002725:Chloroform; D004247:DNA / Q000032:analysis*; D000431:Ethanol; D006863:Hydrogen-Ion Concentration; D009705:Nucleosides; D009711:Nucleotides; D000439:Pentanols; D019800:Phenol; D011506:Proteins; D012313:RNA; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D001580:Benzopyrenes; D002273:Carcinogens; D009705:Nucleosides; D009711:Nucleotides; D000439:Pentanols; D011506:Proteins; D019800:Phenol; D001564:Benzo(a)pyrene; D000431:Ethanol; C072019:benzo(a)pyrene diolepoxide I; D012313:RNA; D002725:Chloroform; D004247:DNA; D016604:Aflatoxin B1; C029683:isopentyl alcohol