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Hydrolysis of 3H-AFB1-DNA (11/03/1975 - 11/07/1975)
Purpose/Intro: To test different conditions for formic acid hydrolysis of modified DNA and to experiment with HPLC detection of the adduct.
Results/Conclusion: Release of purines was observed even at the lower temperature. It took different conditions to wash the radioactivity off the column; a sharp peak was not obtained. Ion-exchange chromatography is probably not the way to go since the adduct is so nonpolar.
Notes: It had already been demonstrated that DNA hydrolysis with formic acid at 65 ºC for one hour could remove about 95% of the purines. The current experiment was a broad test of hydrolysis efficacy on aflatoxin-modified DNA. Though the chromatograms are not very clean, they nonetheless have several peaks—indicating that formic acid hydrolysis also works on modified DNA. The experiment also is useful in determining the chemical nature of what was later found to be the adduct. As is mentioned on the 7th page of the linked notebook section (see below), the "bulk of the radioactivity is washed off of the column with the lipophilic highly concentrated buffer," which suggests a lipophilic adduct. However, opposite of the previous page is an extraction experiment which shows that chloroform (which is largely nonpolar) is not at all efficient at removing the adduct from the aqueous phase. These two pieces of information are the basis of the comment, "...behavior of the adduct indicates it may be a lipophilic yet somewhat polar molecule" in the experiment performed on 11/3/1976.
Links:
PDF: See this in the notebook!
Video: Hydrolysis Methods
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