Adducts HPLC elution profiles

Figure 8. HPLC elution profiles for DMBA-deoxyribonucleoside adducts from female NIH Swiss mouse skin treated with 14 nmoles [3h]-DMBA 24 h (0-0) and from mouse embryo cells exposed to [ C]-DMBA (0.14 yg/ml) 24 h ( - ). The arrow shows the position of elution of an added uv-absorbing marker, toluene.

The relative amounts of syn and anti adducts produced in mouse embryo cells did not vary substantially with DMBA concentration (20). However, we found a dramatic difference in the relative amounts of these adducts when the dose of DMBA applied to mouse skin was varied (jl). Figure 9 shows the HPLC elution profiles for adducts formed at a low dose of 14 nmol [ HJ-DMBA. Peaks A,C and D are present in approximately equal amounts, i.e. 29, 21 and 22% of total radioactivity, respectively. However, at a 100-fold higher dose of 1400 nmol, peak C has increased to 39% while A and D have decreased to 13% and 9%. These results indicate that the formation of syn-bay region dihydrodiol epoxide adducts is favored at high doses. Due to this, the total binding to deoxyadenosine (peaks C and D) also increases with dose and ranges from 27% to 48% of the total DNA binding. 

The extent of HPLC separation between derivatized and underivatized peptides depends on the length of the polypeptide chain, the type of stationary phase used for separation (e.g. C4-C18), and the elution conditions. When using C18 columns the difference in retention time between the two species varies from about 10 min for peptides of 40-60 residues to about 5 min in the case of larger sequences. A typical elution profile for a 100-residue polypeptide is shown in Figure 1. As the length of the chain increases, a progressive reduction in the separation between labelled and unlabelled chains is observed. Unpublished results showed that beyond about 150 residues, the retention times of a probe-peptide adduct and underivatized chains are very similar. However, for sequences up to 120-130 residues, the method is sequence independent and can therefore be standardized. 

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