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Timeline
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- Experiment
- 1. Formic acid hydrolysis of DNA (10/21/1975 - 10/25/1975)
Purpose/Intro: To determine effect of hydrolysis temperature on release of bases from DNA.
Results: Cytosine and thymine hardly released; adenine and guanine had temperature-dependent release profiles.
Links:
PDF: See this in the notebook!
Video: The Early Meeting with George Büchi
Video: An Early Approach: X-Ray Crystallography
Notebook page 2
Notebook page 3
Notebook page 4
Notebook page 5
- 2. 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: 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
Notebook page 6
Notebook page 7
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Notebook page 10
Notebook page 11
Notebook page 12
Notebook page 14
- 3. Injection of DNA nucleosides resulting from enzymatic hydrolysis of AFB1-DNA (11/05/1975)
Purpose/Intro: To test enzymatic hydrolysis as an alternative to acidic hydrolysis for liberating the adduct from DNA.
Results: Not a very clean chromatogram.
Notes: Enzymatic DNA hydrolysis was an alternative to acidic hydrolysis and it was commonly used at the time. It was carried out in basic (pH ~ 8) conditions. Since aflatoxin is sensitive to base, John concentrated his efforts on acid hydrolysis—but tried enzymatic just in case.
Links:
PDF: See this in the notebook!
Notebook page 13
- 4. Attempts at extraction of aflatoxin adduct (11/13/1975)
Purpose/Intro: "The chromatographic behavior of the adduct indicates it may be a lipophilic yet somewhat polar molecule. I will try water saturated ethyl acetate and butanol to remove it from the aqueous phase."
Results: Butanol was effective at extracting the adduct; water saturated ethyl acetate was not.
Links:
PDF: See this in the notebook!
Notebook page 15
Notebook page 16
- 5. HPLC analysis of butanol extract (03/01/1976)
Purpose/Intro: "The previous pages describe an experiment in which it was demonstrated that radioactivity covalently bound to a base was extracted with butanol. The sample (11-3-1) still has ~200 µL of BuOH in the lower phase. This will be concentrated and analyzed by HPLC."
Results: "Nice chromatogram with about 3-4 large peaks of 364 nm-absorbing material. The radioactivity was primarily in 2 of the peaks. Two large 254-absorbing peaks were observed. Conclusions: Generally, the results are somewhat encouraging. Base(?)-AFB1 adducts were separated well from 254-nm absorbing material. The reason that 2 major adducts were seen could be that there was chemical degradation or that there are actually 2 present—analysis of nucleoside adducts under these conditions shows only a single peak with radioactivity. The rest of the 3H primarily elutes early in the chromatogram and may be unhydrolyzed adducts which are qualitatively different from the late-eluting compound."
Links:
PDF: See this in the notebook!
Notebook page 17
- 6. In vitro AFB1-DNA adduct formation (04/06/1976 - 04/09/1976)
Purpose/Intro: "Scale up the microsomal incubation system developed by W. Busby and P. B(?)ous this winter. The ultimate goal of this research is to provide enough DNA for adduct identification."
Notes: Sample JME-1 or JE-1
Links:
PDF: See this in the notebook!
Notebook page 18
Notebook page 19
- 7. In vitro AFB1-DNA adduct formation (04/13/1976)
Purpose/Intro: "The experiment done last week (JME-1) went well and we have decided to make more adduct, since there are microsomes left over."
Notes: Sample JME-2 or JE-2
Links:
PDF: See this in the notebook!
Notebook page 20
- 8. Isolation of a DNA adduct (04/16/1976)
Purpose/Intro: "Background: over the past 4 months, Bill Busby and Bob Croy have established an hydrolysis method and an HPLC separation for an "adduct" in several in vitro incubation samples. I am now going to isolate enough material for physico-chemical studies."
Results: All hydrolysates had a peak at tR = 20 min (Peak 1). This could be the adduct.
Notes: DNA was hydrolyzed enzymatically (DNAase I and nuclease P1).
Links:
PDF: See this in the notebook!
Notebook page 21
- 9. Isolation of Peak 1 for UV spectrum (04/19/1976)
Purpose/Intro: To determine the UV spectrum of the material in Peak 1.
Results: There was a disproportionately large absorbance around 260 nm for Peak 1 to be aflatoxin alone. This is evidence for a DNA adduct.
Links:
PDF: See this in the notebook!
Notebook page 23
- 10. Isolation of a large amount of Peak 1 (04/20/1976)
Purpose/Intro: To determine the UV spectrum of the material in Peak 1.
Results: UV spectrum obtained.
Notes: The adduct, which is lipophilic, was pumped onto a reversed phase precolumn. Since an RPC column is also lipophilic, the adduct accumulated on the column. The precolumn was then attached directly to another column, and the adduct eluted using a gradient. This avoids having to do 10 injections to isolate enough adduct for a UV spectrum.
Links:
PDF: See this in the notebook!
Notebook page 23
- 11. Perchloric acid hydrolysis of Peak 1 (04/20/1976)
Purpose/Intro: To determine what base(s) (adenine, guanine, cytosine, thymine) are present in the putative adduct, Peak 1.
Results: "A small peak was seen in the hydrolysate at the retention time of guanine. These results were reproducible and peaks were not seen at the tRs of cytosine and adenine. Conclusions: 1. There is a peak at the tR of guanine after HClO4 hydrolysis of the presumptive adduct in Peak 1; 2. Can't make any generalizations, but there was a large peak early in chromatogram—this usually occurs, however, and no thymine adduct has ever been identified. Cytosine and adenine were not observed."
Links:
PDF: See this in the notebook!
Notebook page 24
- 12. Hydrochloric acid hydrolysis of Peak 1 (04/21/1976)
Results: "Possibly these conditions weren't severe enough to liberate guanine."
Notes: The purpose here may have been to repeat or modify the Millers' experiment.
Links:
PDF: See this in the notebook!
Notebook page 25
Notebook page 26
- 13. Guanine chromatography under RPC conditions (04/21/1976)
Purpose/Intro: "To get additional information on the base part of the adduct. A method will be developed using the Micromeritics LC and a reversed phase column."
Results: The guanine peak is also detectable on a reversed phase column. The identities of the two earlier eluting peaks are uncertain.
Notes: PCA stands for perchloric acid. In yesterday's experiment, the Peak 1 hydrolysate was injected onto an ion-exchange column. In this experiment, the column was reversed phase, just like the one used to isolate Peak 1 in the experiment performed on 4/16/1976.
Links:
PDF: See this in the notebook!
Notebook page 26
- 14. Reaction of aflatoxin B2α with guanosine (04/23/1976)
Purpose/Intro: "1. To attempt a chemical synthesis of an adduct; 2. To test the reactivity of B2α with a typical nucleic acid component. B2α may exist in the cell and is a possible ultimate carcinogen metabolite."
Results: Aflatoxin B2α seemed unstable—the B2α peak changed over time. There was no peak at the retention time of Peak 1, indicating that the adduct probably wasn't formed.
Links:
PDF: See this in the notebook!
Notebook page 27
Notebook page 28
- 15. (05/19/1976 - 05/20/1976)
Purpose/Intro: "To develop chemical hydrolysis methods for liberation of AFB1-DNA adducts."
Results: "Formic acid liberates a peak with exactly the same tR as the Peak 1 from enzymatic digestion of adducted DNA."
Notes: Relates to 4/16 Isolation of a DNA adduct (that experiment used enzymatic hydrolysis).
Links:
PDF: See this in the notebook!
Video: Hydrolysis Methods
Notebook page 30
Notebook page 31
Notebook page 32
Notebook page 33
Notebook page 34
- 16. Hydrolysis of dirty JE-2 DNA by formic acid (05/21/1976 - 05/24/1976)
Purpose/Intro: To isolate a large amount of adduct for structural characterization
Results: "A huge Peak 1 was collected at 24 min."
Notes: The sample is "dirty" because it accidentally tipped over onto the countertop during the experiment.
Links:
PDF: See this in the notebook!
Notebook page 35
Notebook page 36
Notebook page 37
- 17. Perchloric acid hydrolysis of adducts (05/25/1976)
Purpose/Intro: "A pilot experiment done on 4/20 gave some somewhat shaky evidence that the adduct involved guanine. This experiment will now be repeated with more material to be sure."
Results: Peak obtained at the tR of guanine.
Notes: Both JE-1 and JE-2 tested. JE-1 was sample 5-25-1 and JE-2 was sample 5-25-2.
Links:
PDF: See this in the notebook!
Notebook page 38
Notebook page 39
- 18. In vitro incubation of AFB1 and DNA (05/26/1976)
Purpose/Intro: "JE-2 was lost, for analytical purposes, because it tipped over in the cold room. A new incubation of AFB1, DNA, and rat liver microsomes will be made in order to provide adduct for identification."
Notes: Sample JE-3
Links:
PDF: See this in the notebook!
Notebook page 41
- 19. Collection of guanine from 5-25-2 (05/26/1976)
Purpose/Intro: To isolate the base (suspected guanine) from the adduct so it can be compared with known guanine.
Notes: Sample 5-26-1 is the pooled fractions of guanine from hydrolysis of 5-25-2.
Links:
PDF: See this in the notebook!
Notebook page 40
- 20. Isolation of a large quantity of Peak 1 (06/02/1976 - 06/03/1976)
Notes: Sample JE-1 used. Sample 6-2-1 is the material in Peak 1.
Links:
PDF: See this in the notebook!
Notebook page 44
- 21. Removal of salt from guanine isolated from perchloric acid hydrolysis of Peak 1 adduct (06/02/1976)
Purpose/Intro: To compare the base isolated from the adduct with guanine. The hypothesis is that guanine is that base, so there should be no distinguishable difference.
Results: UV spectra don't match.
Notes: Since this adduct was isolated from the "dirty" DNA, it's possible that it was contaminated.
Links:
PDF: See this in the notebook!
Notebook page 42
Notebook page 43
- 22. Further processing of guanine isolated from adduct (06/03/1976)
Purpose/Intro: "The UV spectrum obtained yesterday was not conclusive. The sample will be re-processed to see if a better spectrum can be obtained of the material eluting from the LC column directly into a 1 mL cuvette."
Links:
PDF: See this in the notebook!
Notebook page 46
- 23. UV spectrum of guanine from adduct (06/04/1976)
Purpose/Intro: "The experiment on p. 17y failed to produce an interpretable UV spectrum. The guanine peak was evaporated to dryness and dissolved in water as described above."
Results: "The adduct contains a base with an identical UV spectrum to guanine."
Links:
PDF: See this in the notebook!
Notebook page 45
Notebook page 46
- 24. UV spectrum of Peak 1 adduct (06/04/1976)
Purpose/Intro: "The Peak 1 adduct isolated from the enzymatic hydrolysis of JE-1 has been purified (sample 6-2-1). Before further purification and obtaining a mass spectrum, a UV spectrum will be obtained."
Results: "1. This UV spectrum easily could be published—get another of the HCOOH-hydrolyzed adduct for comparison; 2. The guanine part of the spectrum superimposes the actual UV spectrum of guanine; 3. This is exactly the same UV spectrum as the one obtained by the Millers of the dihydrodiol."
Links:
PDF: See this in the notebook!
Notebook page 47
- 25. Adduct chromatography (06/05/1976 - 06/07/1976)
Purpose/Intro: "To investigate alternative chromatographic procedures to the ones currently being used for adduct chromatography."
Results: Waters C18 reversed phase column is best.
Notes: Sample JE-1 used. Sample 6-5-1 is the concentrated ethanol phase from JE-1.
Links:
PDF: See this in the notebook!
Notebook page 48
Notebook page 49
- 26. Final purification of adduct Peak 1 (06/08/1976)
Results: "There are about 9.2 µg of base-aflatoxin adduct (more, if it is a nucleoside adduct). A good mass spectrum can be obtained on 2 µg of material, so it should be divided into about 4 portions."
Links:
PDF: See this in the notebook!
Notebook page 50
Notebook page 51
Notebook page 52
Notebook page 53
Notebook page 54
- 27. Hydrolysis of in vivo adducted DNA (06/09/1976)
Purpose/Intro: "RC and WFB injected 1 mg/kg AFB1-3H (50 (?)) into a rat and waited 2 hours before killing. Liver and kidney DNA are being prepared by the Kirby procedure. Since the HCOOH technique has been so successfully applied to efficient and facile adduct liberation, it will be used to see if in vivo adduct can be observed bound to DNA."
Results: "Three peaks of A365 were observed in the adduct region. Fractions were collected. This may be very promising. This DNA wasn't pure, of course—it may have been contaminated with RNA, protein and AFB1. The aflatoxin possibly was degraded to B2α which may represent the peaks we observe."
Links:
PDF: See this in the notebook!
Notebook page 55
Notebook page 56
Notebook page 57
Notebook page 58
- 28. Mass spectrometry of adduct (06/17/1976)
Purpose/Intro: To determine the mass of the AFB1-DNA adduct to aid in structural identification
Results: "Material started coming off the probe at a relatively low temperature (~70 ºC)—a complex and clean mass spectrum was obtained…"
Notes: Probably about the first time mass spectroscopy of adduct was attempted.
Links:
PDF: See this in the notebook!
Notebook page 59
- 29. (06/18/1976)
Purpose/Intro: "The other sample was brought to K. Biemann's group for a high resolution MS."
Results: "This did not, however, have the same MS as the sample I obtained the low resolution MS of. The sample came off their probe at two different temperatures."
Links:
PDF: See this in the notebook!
Notebook page 60
- 30. Preparation of two purified Peak 1 samples (06/22/1976)
Purpose/Intro: To prepare pure adduct for use in the mass spectrometer.
Notes: Sample 6-2-1 used. Samples 6-22-1 and 6-22-2 are concentrated duplicates of sample 6-2-1.
Links:
PDF: See this in the notebook!
Notebook page 61
- 31. Adduct mass spectrometry (06/23/1976)
Purpose/Intro: To determine the mass of the AFB1-DNA adduct to aid in structural identification.
Links:
PDF: See this in the notebook!
Notebook page 61
- 32. Hydrolysis of 100 mg DNA for Peak 1 isolation (07/21/1976 - 07/23/1976)
Purpose/Intro: To liberate the adduct from modified DNA so that it can be isolated for structural determination.
Notes: Sample 7-21-1
Links:
PDF: See this in the notebook!
Notebook page 64
Notebook page 65
Notebook page 66
Notebook page 67
- 33. Peak 1 purification (07/26/1976)
Purpose/Intro: To isolate the adduct from the DNA hydrolysate 7-21-1.
Results: "1. Only 12% of the hydrolysate was recovered as semipurified Peak 1—this is much lower than expected—and I know that more than 70% of the hydrolysate radioactivity was present as Peak 1; 2. Peak 1 probably was there, but it didn't stick to the precolumn."
Notes: With regard to the precolumn: see the lower half of page 10w and the explanation given in the "Notes" section for the experiment entitled, "Isolation of a large amount of Peak 1" performed on 4/20/1976. As John mentions, the adduct does not "stick" to the precolumn as he hoped it would.
Links:
PDF: See this in the notebook!
Notebook page 68
Notebook page 69
Notebook page 70
Notebook page 71
- 34. Peak 1 isolation—2nd stage of purification (08/02/1976 - 08/04/1976)
Purpose/Intro: To isolate the adduct from the DNA hydrolysate 7-21-1
Results: "Total sample has 9.7 ug of adduct—which is ~1/4 what was expected (but ~what I started with this morning)."
Links:
PDF: See this in the notebook!
Notebook page 71
Notebook page 72
Notebook page 73
Notebook page 74
Notebook page 75
- 35. Sample preparation for mass spectroscopy (08/04/1976)
Notes: Sample 8-4-1 used. Samples 8-4-1-MS and 8-4-2-MS are duplicates taken from sample 8-4-1.
Links:
PDF: See this in the notebook!
Notebook page 76
- 36. Mass spectrometry [of adduct] (08/05/1976)
Purpose/Intro: "A sample of adduct was prepared (described previously) and divided into two portions for mass spectroscopy."
Results: "The high resolution mass spectrum initially showed nothing—which led (?) to think that the MS sample was a salt. I took back the 2 tubes (broken in half for the MS) and put 2 into scintillation vials—there was a lot of cpm (could probably account for the entire sample in this way). The third sample was dissolved in HPLC mobile phase and injected—a small but definite peak was observed. Finally, (?) resubmitted the last remaining sample to MS—and at very high temperature (for a long time) got a burst of ions."
Links:
PDF: See this in the notebook!
Notebook page 77
- 37. Adduct preparation (08/22/1976 - 08/25/1976)
Purpose/Intro: Continued attempted purification of adduct using a precolumn.
Results: "The huge adduct peak was obtained (Peak 1) though note that there is massive A254 contamination."
Links:
PDF: See this in the notebook!
Notebook page 78
Notebook page 79
Notebook page 80
Notebook page 81
Notebook page 82
- 38. Formic acid hydrolysis of 10 mg modified DNA (08/25/1976)
Purpose/Intro: "The adduct isolation from postcolumn effluent may not go well, so I will start another sample of adduct from one stick (i.e. 10 mg) of modified JE-3 DNA (should have ~30 µg adduct)."
Notes: Sample 8-25-2. "Go ahead to 9/23/76."
Links:
PDF: See this in the notebook!
Notebook page 83
- 39. Further processing of precolumn adduct (08/26/1976)
Purpose/Intro: Continued attempted purification of adduct using a precolumn. Peak 1 was re-treated in the same manner as in the previous experiment.
Results: A254 contamination is lower, but it still coelutes with the adduct. 123 µg adduct obtained.
Links:
PDF: See this in the notebook!
Notebook page 84
Notebook page 85
Notebook page 86
- 40. Preparation of adduct for HRMS (08/26/1976 - 08/27/1976)
Purpose/Intro: "To give Verne Reinhold enough sample for an unambiguous HRMS—we have had technical problems in the past."
Links:
PDF: See this in the notebook!
Notebook page 87
- 41. Attempted formation of a methoxy-derivative of adduct (09/16/1976 - 09/17/1976)
Purpose/Intro: "The adduct should have a 3-OH group on the aflatoxin moiety. I will try to form the —O-CH3 derivative."
Results: "Chromatogram showed a lot of 254-absorbing crud—and some early-eluting 365. A very sharp peak was observed ~0.4 min earlier than the adduct under these conditions—not known whether this compound is different from the adduct."
Notes: In order to get a mass spectrum of a compound, the sample must "fly," that is, be volatilized. Aflatoxin, since it is large and polar, is not readily volatile and therefore John attemped to reduce the polarity by converting a hydroxyl group to a methoxy group, which would be much less polar.
Links:
PDF: See this in the notebook!
Notebook page 88
- 42. Preparation of samples for low resolution MS (09/19/1976 - 09/22/1976)
Purpose/Intro: "The HRMS data are not as clear as I would have hoped. I will go back to LRMS, because it's faster and easier to troubleshoot."
Results: "The instrument has been running well lately, and Pete Wishnok (?) said that the sensitivity has been very high. Unfortunately, the MS blew its filament when I put my sample in—and it wouldn't be possible to replace it for some time."
Links:
PDF: See this in the notebook!
Notebook page 89
Notebook page 90
Notebook page 91
- 43. Further processing of 8-25-2 (HCOOH-hydrolyzed DNA) (09/23/1976)
Purpose/Intro: "Sample 8-25-2 was prepared from 10 mg of adducted DNA—it should contain up to 30 ug of adduct—I need adduct for identification studies, so this sample will be processed further."
Results: "HPLC: A peak appeared at ~tR of the adduct, but it was very broad—column problems developed. Explicitly, the pressure went up and it took 3 days to repair the damage. Radioactivity: There should be 2.2e6 counts in 10 mg! Therefore I have ~1/4 the amount of adduct that I want."
Links:
PDF: See this in the notebook!
Notebook page 92
Notebook page 93
- 44. Further processing of 8-25-2 cont. (09/27/1976)
Results: "The column finally was restored to more or less normal behavior. The 365 adduct peak was ~2 min early! There was a lot of 254 contamination which would complicate using this method for adduct isolation. Note the phthalate contamination—this is a result of omitting the charcoal column in the clean up of the water in the mobile phase.
Links:
PDF: See this in the notebook!
Notebook page 94
Notebook page 95
- 45. Preparation of an adduct standard (09/27/1976)
Links:
PDF: See this in the notebook!
Notebook page 95
- 46. Further processing of 8-25-2 cont. (09/29/1976)
Results: "There are a lot of peaks in the region of the bases—possibly these peaks represent di, tri nucleotides, etc."
Notes: SN-1 and SN-2 (supernatants).
Links:
PDF: See this in the notebook!
Notebook page 97
- 47. Processing of adduct in SN-1 (10/04/1976)
Purpose/Intro: "This sample originally was obtained by dissolving the non-MeOH soluble material in water and centrifuging down the pellet."
Results: 5 ug adduct obtained.
Notes: SN-1 = supernatant 1
Links:
PDF: See this in the notebook!
Notebook page 98
Notebook page 99
- 48. Further processing of 8/25 modified DNA adduct (10/05/1976)
Purpose/Intro: "The SN-1 sample didn't have an overwhelming amount of adduct, so I will pool it with other samples in hopes of getting a higher concentration. SN-1 was combined with the MeOH extract (which contained ~same amount of adduct and two samples of Bob Croy, which he knew contained substantial quantities of adduct (subsequently, however, we didn't find too much)."
Results: 18 µg adduct obtained. "There was a fair amount of 254 interference—I probably should reprocess this sample through the Waters column."
Notes: Sample 10-5-1
Links:
PDF: See this in the notebook!
Notebook page 99
Notebook page 100
Notebook page 101
- 49. JE-4 DNA (10/06/1976 - 10/11/1976)
Purpose/Intro: "We now have the capability to produce modification levels in DNA of 1:18 to 1:30, depending on exact conditions. We will attempt in this experiment to achieve ~1:30 modifications on a large quantity of DNA in order to produce enough DNA adduct to identify by NMR and MS."
Results: 9.45 mg adduct in DNA.
Notes: Sample 10-9-1.
Links:
PDF: See this in the notebook!
Notebook page 102
Notebook page 103
Notebook page 104
Notebook page 105
Notebook page 106
- 50. Experiments on removing or neutralizing HCOOH (10/10/1976)
Purpose/Intro: "Removing HCOOH by lyophilization was a slow and risky business last time. I will investigate alternative neutralization and lyophilization methods."
Results: 1. Neutralization: none of these conditions are appropriate. 2. Lyophilization: 1 mL HCOOH + 4 mL H2O worked well—the apparatus described on the facing page was used and lyophilization was ~2 hr."
Links:
PDF: See this in the notebook!
Notebook page 107
Notebook page 108
- 51. JE-4 DNA: 10-9-1 processing (10/11/1976)
Purpose/Intro: To isolate more of the adduct to determine its structure
Results: "Publishable chromatogram."
Notes: This is when John forgot to set the gradient before going to lunch. As a result, the system kept running at 10% methanol for several hours. Despite running for so long, the adduct remained on the column—after the 254 nm-absorbing material had been washed off. When John ran the gradient, the adduct eluted at the same retention time, indicating that it had stayed at the start of the column for all that time.
Links:
PDF: See this in the notebook!
Video: The Two Liter Problem Part I
Video: The Two Liter Problem Part II
Notebook page 109
Notebook page 114
- 52. Preparative column for adduct purification (10/18/1976 - 10/19/1976)
Purpose/Intro: "A preparative reversed phase column was made to purify the adduct from other DNA components."
Results: "The adduct had a tR of 32 min and gave a symmetrical peak. These should be good conditions to try for its isolation."
Notes: Two ideas resulted from the previous experiment. The first is that multiple injections could be done before running the gradient, thus accumulating material on the column. The second is that the column could be hooked up directly to a pump, thus getting around the 1 mL injection limit of the HPLC system. Both of these solutions allow a lot of adduct to build up at the start of the column, thus achieving a high concentration in the collected peak and getting around the problem of evaporating 2 L of water.
Links:
PDF: See this in the notebook!
Notebook page 115
- 53. Hydrolysis of JE-4 DNA (10/19/1976 - 10/21/1976)
Purpose/Intro: "To produce several mgs of adduct."
Notes: The large preparative column was made on 10/18/1976. The next day, a lot more DNA was hydrolyzed, producing a large amount of impure adduct which could be used to test the preparative column as a method for purifying large quantities of adduct.
Links:
PDF: See this in the notebook!
Notebook page 116
- 54. Use of preparative column to isolate adduct (10/19/1976)
Purpose/Intro: "To evaluate the potential of the preparative column for making large quantities of adduct."
Results: The adduct elutes as a separable peak.
Notes: Sample 10-9-1 used. Samples 10-19-9 and 10-19-10 are the fractions collected from the adduct peak (hypothetically pure adduct). The procedure here was to load the 7 mL sample onto the preparative column using a pump, then disconnect the pump and connect the column to the Micromeritics pump (which could pump gradients), wash the column with 10% methanol until 254 nm-absorbing material is washed off, then finally elute the adduct using a gradient to 80% methanol. This differs from the precolumn method in two ways: in the earlier experiment, no Micromeritics system is used, and in the earlier experiment, no methanol wash is used.
Links:
PDF: See this in the notebook!
Notebook page 117
Notebook page 118
- 55. Quality control check of 10-9-1 adduct prepared on 10/19/1976 (10/20/1976)
Purpose/Intro: To determine whether the preparative column is effective at purifying the adduct.
Results: "1. There is a lot of adduct here; 2. It appears to be quite pure—more work should be done to evaluate this, however."
Notes: Sample 10-19-9 used.
Links:
PDF: See this in the notebook!
Notebook page 119
Notebook page 120
- 56. Further processing of JE-4 adduct (10/21/1976)
Notes: Sample 10-21-1.
Links:
PDF: See this in the notebook!
Notebook page 120
- 57. Further preparative chromatography on 10-9-1 (10/21/1976)
Results: Again, the adduct elutes as a separable peak.
Notes: "This is the 2nd half of 10-9-1, which was a hydrolysate of JE-4 DNA (one rod). Sample vol = 7 mL."
Links:
PDF: See this in the notebook!
Notebook page 121
Notebook page 122
- 58. Preparative run on 25 mL of 10-21-1 (JE-4 DNA) (10/21/1976)
Purpose/Intro: "The prep run on the adduct done earlier in the day worked well, so 25 mL of the new sample will be processed now (this is < 25% of entire 10-21-1 sample). In terms of the amount of DNA represented, this is probably in excess of 20 mg."
Results: "1. 254 was off-scale much of the time; 2. the middle peak is building up on the prep column."
Notes: "Oligon pk" means "oligonucleotide peak" and is the large peak immediately before the 32 min adduct peak. "254" refers to "254 nm-absorbing material," in other words, the four DNA bases.
Links:
PDF: See this in the notebook!
Video: The "Go for it all" Story
Notebook page 123
Notebook page 124
- 59. Preparative run on 35 mL of 10-21-1 done by Paul Donahue (10/22/1976)
Notes: Sample 10-22-1 is the adduct fraction.
Links:
PDF: See this in the notebook!
Notebook page 125
Notebook page 126
- 60. Final processing of 10-21-1 (10/25/1976)
Purpose/Intro: "All but 50 mL of 10-21-1 (from JE-4 DNA) has been processed. The remainder will be put through the column in lots of 25 and 25 mL."
Notes: Samples 10-25-1 and 10-25-2 are the adduct fractions collected from each preparative run of 25 mL of 10-21-1.
Links:
PDF: See this in the notebook!
Notebook page 127
Notebook page 128
- 61. Hydrolysis of nine rods of JE-4 DNA and lyophilization of DNA hydrolysate (10/26/1976 - 10/28/1976)
Purpose/Intro: To prepare more adduct for structural determination
Notes: Sample 10-28-1
Links:
PDF: See this in the notebook!
Notebook page 133
- 62. Further purification of adduct (10/26/1976 - 10/27/1976)
Purpose/Intro: "I will take the fractions checked above and concentrate them down to remove most of the MeOH. Then, if all goes well, put the concentrate through the preparative column."
Results: "The adduct probably is in the precipitate. It consists of white crystals that are needle-like in shape."
Notes: Sample 10-26-1 is the pooled fractions. Solid materials precipitated during rotary evaporation. The supernatant is 10-26-2; the insoluble material is 10-26-3.
Links:
PDF: See this in the notebook!
Notebook page 129
Notebook page 130
Notebook page 131
Notebook page 132
- 63. Dissolving of pellet of ? adduct (10/27/1976)
Purpose/Intro: To try and dissolve the white crystals to determine they are pure adduct.
Results: Dimethyl formamide was effective at dissolving the crystals. HPLC and scintillation counter showed that the crystals are in fact crystals of the adduct.
Links:
PDF: See this in the notebook!
Notebook page 134
Notebook page 135
Notebook page 136
- 64. Adduct purification (10/27/1976)
Notes: Sample 10-26-2 used.
Links:
PDF: See this in the notebook!
Notebook page 134
- 65. Adduct preparation: attempt to recover more adduct from DNA pellet by DMF extraction (10/28/1976)
Purpose/Intro: "The adduct has low solubility in most aqueous or organic solvents tested. This may be a limiting factor in determining the efficiency with which the adduct is solubilized by 10% MeOH after lyophilization. If there is pure adduct at that stage that doesn't go into solution, it may be solubilized by hot DMF, which was found yesterday to be a good solvent for the adduct."
Results: The DMF was unable to extract significant quantity of adduct from the pellet.
Links:
PDF: See this in the notebook!
Notebook page 137
- 66. Solubility of adduct in DMSO (10/28/1976)
Purpose/Intro: To determine whether the adduct is soluble in DMSO. If it is, deuterated DMSO can be used to dissolve the pure adduct crystals to get a good NMR spectrum.
Results: "[Adduct] went in [to solution] within 30 sec. at 90 ºC and did not reprecipitate at refrigeration temperatures."
Links:
PDF: See this in the notebook!
Notebook page 137
- 67. Preparative chromatography of 10-28-1 (10/29/1976)
Notes: "Excellent chromatogram to publish."
Links:
PDF: See this in the notebook!
Notebook page 140
- 68. Transfer of adduct into PS vial (10/29/1976)
Purpose/Intro: "This was accomplished to quantitate adduct."
Results: 3.86 mg adduct
Notes: Sample may not have been completely dry.
Links:
PDF: See this in the notebook!
Notebook page 138
Notebook page 139
- 69. Preparation of samples for mass spectrometry (11/01/1976)
Notes: Sample 11-1-1
Links:
PDF: See this in the notebook!
Video: Mass Spectrometry of the Adduct
Notebook page 141
Notebook page 142
- 70. Preparation of adduct for MS and NMR (11/03/1976 - 11/04/1976)
Results: "The [NMR] spectrum was generally interpretable in that most of the expected protons were present, and fortunately, the sample's purity probably was good because of the integral relationship in the protons observed. The sample appeared to have H2O, however."
Links:
PDF: See this in the notebook!
Video: NMR: The Backstory
Video: Analyzing the NMR Data to Obtain the Preliminary Structure
Audio: The Truth Table Explanation
Image: The Truth Table
Notebook page 145
Notebook page 146
- 71. Harvest crystals of adduct (11/03/1976)
Purpose/Intro: "It has been at least 24 hr since the last prep run was completed. Crystals are present in all 4 fractions."
Notes: Sample 11-3-1 is the supernatant and washings; sample 11-3-2 is the pellet containing the adduct.
Links:
PDF: See this in the notebook!
Notebook page 144
- 72. Purity check on 11-1-1 (11/03/1976)
Purpose/Intro: "To make sure that this sample, which previously was dissolved in DMF, is still stable."
Results: "It looked as though there was some early eluting impurities—there also appeared to be some minor contaminants in adduct region, but it was difficult to tell. REDO THIS ANALYSIS WITH CLEAN COLUMN AND SAME SAMPLE—FIX LC VALVE!"
Links:
PDF: See this in the notebook!
Notebook page 144
- 73. Acetylation of adduct (11/06/1976)
Purpose/Intro: To prove that the aflatoxin moiety was present by restoring its fluorescence.
Links:
PDF: See this in the notebook!
Video: Where Did the Fluorescence Go?
Notebook page 147
- 74. Preparative runs on samples 10-26-2 and 11-7-4-J (11/08/1976 - 11/09/1976)
Links:
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Notebook page 152
- 75. Preparation of field desorption MS sample (11/08/1976)
Notes: Sample 11-3-2 used; became sample 11-7-2-J which was submitted for MS.
Links:
PDF: See this in the notebook!
Notebook page 149
- 76. Analysis of acetylated reaction mixture (11/08/1976)
Purpose/Intro: "This sample was described on the previous page."
Results: "This was repeatable—and the adduct was confirmed to be absent from the chromatogram."
Notes: Sample 11-5-2 used.
Links:
PDF: See this in the notebook!
Notebook page 149
- 77. Preparation of crystalline adduct (11/08/1976)
Notes: Sample 11-3-1 used; sample 11-7-4-J is the supernatant and sample 11-7-3-J is the adduct crystals.
Links:
PDF: See this in the notebook!
Notebook page 151
- 78. Preparation of more adduct crystals (11/08/1976)
Purpose/Intro: "This sample will be used in the acetylation series to be run tomorrow."
Notes: Sample 10-26-2 used; became sample 11-7-5-J.
Links:
PDF: See this in the notebook!
Notebook page 151
- 79. Acetylation of adduct II (11/09/1976 - 11/10/1976)
Notes: Sample 11-7-5-J was acetylated to become sample 11-9-1. Sample 11-9-2 is 100 µL of sample 11-9-1.
Links:
PDF: See this in the notebook!
Video: Where Did the Fluorescence Go?
Notebook page 153
Notebook page 154
- 80. Chromatography of synthesized AFB1 diol (11/09/1976)
Results: "There are quite a few distinct components to this system." Perhaps the diol has cis and trans isomers.
Links:
PDF: See this in the notebook!
Notebook page 155
- 81. Preparation of acetylation samples for MS (11/11/1976)
Purpose/Intro: To determine whether introduction of acetyl groups in the molecule could make it more able to "fly" in the mass spectrometer.
Notes: Sample 11-11-1a is ~250 µL of 11-9-1; sample 11-11-1b is 100 µL of 11-9-1.
Links:
PDF: See this in the notebook!
Notebook page 155
- 82. Preparation of an acetylation product sample (11/11/1976)
Purpose/Intro: "The last eluting peak(s) from the acetylation mixture have been collected from the 3 injections made to this point."
Notes: Sample 11-11-2
Links:
PDF: See this in the notebook!
Notebook page 155
- 83. Mass spectrometry of adduct-related compounds (11/12/1976)
Results: "1. Acetylated adduct mixture: the probe turned black—probably got charring of compound; 2. Adduct: no spectrum—looked like nothing there, probe was clean; 3. 7-MethylGuanine: got proper spectrum, but it was weak. I think that I haven't been submitting enough sample to MS."
Links:
PDF: See this in the notebook!
Notebook page 156
- 84. Preparation of a pure, dry adduct sample (11/28/1976)
Purpose/Intro: "Sample 11-3-2 is good-quality adduct and some small portions have been used in HCl-stability studies by Paul Donahue. I want some dry adduct as a backup sample for the NMR to be run in D2O/DCl."
Notes: Sample 11-28-1
Links:
PDF: See this in the notebook!
Notebook page 157
Notebook page 158
- 85. Preparation of UV sample (12/01/1976)
Purpose/Intro: To obtain the UV spectrum of the adduct; to test for a bathochromic shift.
Results: "Sample was ~2x too concentrated, to I diluted 1:1 with 0.1 N HCl. New spectrum: perfect—looked exactly like MeOH/H2O spectrum. Add 2 drops 10 N NaOH—got very substantial drop in all absorbances (~factor of 2)—but no bathochromic shift."
Links:
PDF: See this in the notebook!
Video: Early Clues to the Adduct Structure
Notebook page 157
Notebook page 158
- 86. Quantitative study on the release of adduct with formic acid (12/07/1976 - 12/13/1976)
Purpose/Intro: "We believe that >90% of the radioactivity incorporated into DNA can be released as adduct with room temperature HCOOH hydrolysis. This experiment was designed to test that figure and its precision."
Links:
PDF: See this in the notebook!
Notebook page 159
Notebook page 160
Notebook page 161
Notebook page 162
- 87. JE-5 DNA modification (12/09/1976 - 12/15/1976)
Notes: Sample 12-15-1
Links:
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Notebook page 163
Notebook page 164
Notebook page 165
Notebook page 166
Notebook page 167
- 88. Fluorescence profile of 12-7-5 (modified DNA hydrolysate) (12/14/1976)
Results: "4 fluorescent peaks, one eluting close to the tR of adduct."
Links:
PDF: See this in the notebook!
Notebook page 168
Notebook page 169
- 89. Preparative run on adduct used for NMR work (12/14/1976)
Purpose/Intro: To isolate a large amount of adduct for structural characterization.
Links:
PDF: See this in the notebook!
Notebook page 168
Notebook page 169
- 90. Preparative chromatography (12/15/1976)
Purpose/Intro: To isolate more of the adduct to determine its structure.
Results: 5.2 mg obtained.
Links:
PDF: See this in the notebook!
Notebook page 170
Notebook page 173
- 91. Determination of the % of radioactivity in hydrolysate attributable to adduct (12/20/1976)
Links:
PDF: See this in the notebook!
Notebook page 174
Notebook page 175
- 92. Development of conditions for adduct methylation (12/22/1976)
Purpose/Intro: "We have good MS and NMR data on the adduct, and it is all consistent with substitution either on the N3, N9 or most likely the N7 of guanine."
Results: Unclear data obtained. Methylation of guanosine and subsequent hydrolysis, which should have resulted in 7-methyl guanine, showed no peak at that compound's retention time. "It is possible that the imidazole ring opened when I made the samples alkaline."
Notes: John's hypothesis was correct: a competing lab using enzymatic (and thus alkaline) hydrolysis identified a minor aflatoxin adduct (FAPY) that is formed when the guanine imidazole ring opens in basic conditions.
Links:
PDF: See this in the notebook!
Video: Closing the Deal
Video: What is the Guanine Site of Attachment?
Notebook page 176
Notebook page 177
- 93. Methylation of guanosine II (12/28/1976)
Purpose/Intro: To determine appropriate conditions to methylate the adduct without destroying it.
Results: "The guanosine probably is degrading under these conditions! The DMS probably is destroying the ribose—leading to production of colored products. Since the AFB1-Gua adduct has a ribose-like group, these conditions probably would not be satisfactory for its methylation."
Links:
PDF: See this in the notebook!
Notebook page 178
- 94. Guanosine methylation III (12/29/1976 - 12/30/1976)
Purpose/Intro: To determine appropriate conditions to methylate the adduct without destroying it.
Results: "7-MeGuanosine eluted at ~42 min under these conditions. The reaction mixture was diluted ~100 fold in buffer prior to injection. Results: a huge 7-MeGuanosine peak was obtained."
Links:
PDF: See this in the notebook!
Notebook page 179
Notebook page 180
- 95. Hydrolysis of 7-MeGuanosine (12/30/1976)
Purpose/Intro: To hydrolyze the 7-MeGuanosine sample. Theoretically, 7-MeGuanine should be obtained.
Results: No 7-MeGuanosine or 9-MeGua obtained. Large 7-MeGua peak.
Links:
PDF: See this in the notebook!
Notebook page 181
- 96. Analysis of guanine methylation mixture (12/30/1976)
Purpose/Intro: To determine what compounds methylation of guanine results in.
Results: Several unknowns produced, but the key point is that they can be separated and distinguished from each other.
Notes: "The sample of 'methylated' guanine from 12/22/76" was used.
Links:
PDF: See this in the notebook!
Notebook page 181
- 97. Adduct methylation I (12/31/1976)
Purpose/Intro: Now that appropriate conditions have been found using smaller test molecules, methylation of the adduct will be attempted. Methylation of guanine is the control sample.
Results: Possible methylated product obtained. "EXCELLENT CHROMATOGRAM."
Notes: There was no 9-MeGua in the lab at this point, so it had to be synthesized by methylating guanine. However, methylation of guanine produces a number of other methylated derivatives. As was showed, they are distinguishable by HPLC.
Links:
PDF: See this in the notebook!
Notebook page 182
Notebook page 183
- 98. Analysis of methylated adduct hydrolysis product (01/01/1977)
Purpose/Intro: To compare the methylation products of guanine (one of which is 9-MeGua) to the methylation and hydrolysis products of the adduct.
Results: Sharp peak at 6 minutes. Broad "hump" at 40 minutes. These data suggest 9-MeGua is present, indicating that the adduct is linked to the N7 of guanine, as suspected.
Links:
PDF: See this in the notebook!
Notebook page 186
- 99. Isolation [and hydrolysis] of putative methylated adduct (01/01/1977)
Purpose/Intro: To isolate and hydrolyze the putative methylated adduct.
Links:
PDF: See this in the notebook!
Notebook page 184
- 100. Analysis of Gua methylation mixture (the control) (01/01/1977)
Purpose/Intro: To compare the methylation products of guanine (one of which is 9-MeGua) to the methylation and hydrolysis products of the adduct.
Results: Six major peaks observed. 9-MeGua hypothesized to elute at approximately 6 minutes.
Notes: The early parts of chromatograms are notoriously unreliable. Peaks may be caused by air bubbles, material that was previously on the column, etc. An experienced investigator can (usually) tell what is an artefact and what is real data. In this case, the shaded peak is an artefact.
Links:
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Notebook page 186
- 101. Isolation of methylated guanine (9-MeGua?) for UV spectrum (Bob Croy) (01/02/1977)
Purpose/Intro: To get the UV-Visible spectrum of 9-MeGua so it can be compared to the compound produced by hydrolysis of methylated adduct.
Results: "The UV in base doesn't look like published spectrum, but 1. base came from plastic bottle and 2. the concentration is very low, and Al Nadzan says that spectra often are concentration dependent."
Notes: This experiment aims to back up evidence by the previous HPLC experiment showing that the adduct was an N7 adduct.
Links:
PDF: See this in the notebook!
Notebook page 187
- 102. Isolation of more methylated adduct (01/02/1977)
Links:
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Notebook page 187
- 103. Co-elution of 9-MeGua with methylated adduct hydrolysis product (01/03/1977)
Purpose/Intro: "The authentic sample of 9-MeGua arrived today. Its spectrum will be obtained and I will get information on its chromatographic behavior in the tRNA system."
Results: "Retention time: 8+ minutes. Retention time of methylated product: same. When spiked with 9-MeGua, the peak from the methylation system grew and no second peak or shoulder was observed. THERE ARE SOME VERY NICE CHROMATOGRAMS HERE. Data shown to G. Büchi."
Links:
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Notebook page 189
- 104. Adduct methylation II (01/03/1977 - 01/06/1977)
Purpose/Intro: "The previous experiment was an apparent success. It will be repeated with more material to characterize the product as 9-MeGua."
Results: Both HPLC and UV-Visible spectroscopy confirm the identity of the compound to be 9-methyl guanine.
Notes: Sample 1-4-1 is the methylated adduct hydrolysate.
Links:
PDF: See this in the notebook!
Notebook page 190
Notebook page 191
Notebook page 192
Notebook page 193
Notebook page 194
Notebook page 195
- 105. Generation of publishable 9-MeGua chromatogram (01/14/1977)
Notes: Sample 1-4-1 used.
Links:
PDF: See this in the notebook!
Notebook page 196
- 106. Analysis of guanine methylation sample (01/14/1977)
Notes: Sample 12-31-2 used.
Links:
PDF: See this in the notebook!
Notebook page 196
- 107. Methylated base chromatogram for publication (01/14/1977)
Notes: "The 1-MeGua had to be made by hydrolysis of 1-methyl guanosine."
Links:
PDF: See this in the notebook!
Notebook page 196
- 108. Preparation of 9-MeGua sample for MS (02/01/1977)
Purpose/Intro: To use yet another method to ascertain that the product of adduct methylation and hydrolysis is in fact 9-MeGua.
Links:
PDF: See this in the notebook!
Notebook page 197
- 109. Perchloric acid hydrolysis of "Diol" peak from adduct methylation experiment (02/01/1977)
Notes: Sample 1-21-2P (from 1/3/77 adduct methylation) used.
Links:
PDF: See this in the notebook!
Notebook page 197
- 110. Methylated base analysis: on "Diol" hydrolysate from "Adduct Methylation II" [previous page] (02/03/1977)
Links:
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Notebook page 198
- 111. Obtain UV spectrum of "Diol" peak from adduct methylation experiment (02/05/1977)
Purpose/Intro: "The data reported on previous page imply that 3-MeGua may have been present in the "diol" peak. If this peak is a diol, it should undergo a bathochromic shift in alkali, like B2α."
Results: "In alkaline solution, no bathochromic shift was observed. The UV at pH 6 looked similar to adduct (high A264/A360). The data indicate that the "diol" peak probably is a derivative of the adduct and not a breakdown product."
Notes: Note the control sample; the authentic diol.
Links:
PDF: See this in the notebook!
Notebook page 199
Notebook page 200
- 112. Comparison of 3-Me-I and 9-Me-I peaks in adduct methylation mixture (02/06/1977)
Purpose/Intro: "To determine how much of each methylated adduct is in methylation mixture."
Results: The mixture has about twice as much 9-Me-I as 3-Me-I.
Notes: The "I" is the adduct.
Links:
PDF: See this in the notebook!
Notebook page 201
Notebook page 202
- Note
- 1. (04/16/1976)
Notes: "Bill Busby has finished the hydrolysis of JME-1 AFB1-DNA adduct. The hydrolysate was deproteinated with 2 volumes of ethanol (final volume ~ 300 mL) and centrifuged. The supernatant is stored in the refrigerator."
Links:
PDF: See this in the notebook!
Notebook page 20
- 2. (04/16/1976)
Notes: "Proposed method of operation: 1. remove the bulk of the ethanol by rotary evaporation; 2. attempt to extract the adduct with polar organic solvents from small portions of the hydrolysate; 3. concentrate extract and purify by RPC (reversed phase chromatography)."
Links:
PDF: See this in the notebook!
Notebook page 20
- 3. (06/14/1976 - 06/16/1976)
Notes: "A mass spectrum will be taken of the purified adduct Peak 1 (p21) samples which have been dried. Much of the week was spent in refamiliarizing myself with the MS and in obtaining spectral data on modified bases and nucleosides."
Links:
PDF: See this in the notebook!
Video: Mass Spectrometry of the Adduct
Notebook page 59
- 4. Quantitation of DNA from JE-3 (07/20/1976)
Notes: "There are 18 rods left from in vitro experiment JE-3. The DNA quantity per rod will be determined in order to get an idea on how much adduct is bound per unit weight of DNA. The DNA variability per rod probably is less than 10-15%."
Links:
PDF: See this in the notebook!
Notebook page 62
Notebook page 63
- 5. Search for missing counts (08/04/1976)
Notes: "The sample lost ~3/4 of the counts between the time it was at the 8.7 mL level and this morning—possibly lost in evaporation? Or not soluble in the 10% MeOH used to dissolve the sample after rotary evaporation (supported by the fact that a residue was observed after removing the liquid from the flask). I then added 400 µL of MeOH (pure) to the flask, and 4.6e4 counts were there (~10% of the original sample); therefore not enough to account for the large loss. Loss could be attributed to precipitate out of adduct as an insoluble salt, or it could have co-precipitated with another contaminating salt."
Links:
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Notebook page 76
- 6. Chromatography of aflatoxin G2 (10/18/1976)
Notes: "G2 was chosen because it should have chromatographic properties similar to the adduct. On e a 10 to 80% gradient (40 min), G2 had a tR of 36 min—a somewhat broad peak was obtained, but understandable under these conditions."
Links:
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Notebook page 115
- 7. Discussion with D. Trafficanti (10/28/1976)
Notes: "We have ~2+ mg of adduct in crystalline form, so we discussed the possibility of getting a Fourier-transform NMR with D. Trafficanti—he will run it himself next Thursday."
Links:
PDF: See this in the notebook!
Notebook page 137
- General
- 1. (10/25/1975)
Purpose/Intro: "Introduction: The research done on this project involves the binding of carcinogens to macromolecules, specifically DNA. This research has been under way for about six months now and detailed accounts can be found in notebooks 3 and 5. Specific references to experiments are listed below."
Notes: Notebooks 3 and 5 are not online. Most of the relevant information is contained in the videos or explanations.
Links:
PDF: See this in the notebook!
Notebook page 1
- 2. Meeting with George Büchi. Also present: Gerry Wogan, Bob Croy, Vern Reinhold, and Al Nadzen. (01/03/1976)
Notebook page
- 3. 9-Methyl Guanine arrives from Heterocyclic Chemical Corp. (01/03/1977)
Links:
PDF: See this in the notebook!
Notebook page 188
- 4. Mail manuscript to Proceedings of the National Academy of Sciences (PNAS) (02/08/1977)
Video: Publication
Notebook page
- 5. PNAS accepts manuscript (02/25/1977)
Notebook page
- 6. American Association for Cancer Research annual meeting (05/18/1977 - 05/22/1977)
Audio: The Scientific Summit Story
Image: The Scientific Summit
Notebook page
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