Fig. 1. Optimizations of the preparation of protein extracts. Protein extracts were prepared under various conditions and assayed for OGG activity. (A) PBMC frozen in hypotonic buffer were quickly thawed in a water bath at 30 °C followed by extraction using either shearing by passage (column 1), additional 0–3 freeze-thaw cycles (columns 2–5, respectively), sonication (column 6), or detergent treatment (columns 7–15). Columns 7–9, NP40 at final concentrations of 0.12, 0.06, and 0.03%, respectively; columns 10–12, triton X-100 at final concentrations of 0.12, 0.06, and 0.03%, respectively; columns 13–15, CHAPS at final concentrations of 0.17, 0.09, and 0.04%, respectively. (B) Extracts were prepared from PBMC samples each frozen in a hypotonic solution with one of the indicated buffers at a concentration of 50 mM, and the indicated pH. (C) PBMC thawed at 30 °C were incubated with KCl at the indicated concentrations and time periods. (D) PBMC at concentrations of 10,000–50,000 cells/μl were suspended in hypotonic buffer to a final number of 2–10 million cells, after which extracts were prepared and assayed for protein concentration (black diamonds) and OGG activity (grey columns). The details are presented under Section 2.

Fig. 2. Effects of salt and oligonucleotide substrate on OGG activity. (A) Effect of KCl concentration on OGG activity, using substrate T5. (B) Effect of concentration of substrate T5. (C) Effect of substrate length and sequence. Nine different oligonucleotides (35 nM each) were used as indicated. OGG specific activities with the various substrates are presented relative to substrate T5, which was set to 100%. Reactions were performed at 37 °C for 1 h. See Section 2 for details.

Fig. 3. Protein extract titration and time course of the OGG activity reaction. Reactions were performed with a protein extract prepared from PBMC and substrate T5. The upper panels show gel images of ³²P-labeled reaction products. The 32-mer oligonucleotide band corresponds to the non-cleaved substrate, and the 17-mer oligonucleotide band corresponds to the OGG reaction product. The lower panels present quantification of gel images similar to those presented in the upper panels. (A) The effect on OGG activity of increasing protein extract amount. Reactions were performed with a control substrate having a G instead of 8-oxoG (lanes 1–5, marked G), and with substrate T5 (lanes 6–10, marked GO). Protein extract amounts of 0, 9.4, 14.1, 18.8, and 23.5 μg were used in each group of lanes 1–5, and 6–10, respectively. Reactions were performed with 25 nM substrate at 37 °C for 1 h. (B) Time course of OGG activity. Lanes 1–7 represent reaction times of 0, 15, 30, 45, 60, 75, and 90 min, respectively, for a reaction with 8 μg extract, and lanes 8–14 represent the respective times of a reaction performed with 11 μg extract. The lower panel shows the kinetics of reactions performed with various protein concentrations, derived from the gel image in the upper panel for 8 μg (squares) and 11 μg (circles) extract, and similar gels obtained with 2 μg (×) and 4 μg (triangles) extract/reaction.

Fig. 4. Analysis of the specificity of the OGG reaction by competition experiments. OGG reactions were performed under standard reaction conditions, except that the reaction mixture contained 2 pmol radiolabeled T5 substrate and increasing amounts of unlabeled competitor, as indicated. The reactions were incubated at 37 °C for 1 h. Hx, a duplex oligonucleotide similar to T5, except that it had a hypoxanthine instead of 8-oxoG; G, the control oligonucleotide with a G instead of 8-oxoG; GO, unlabeled substrate T5. (A) Gel image of the competition experiments. Lanes marked 0 contained no extract, and lanes marked 1 contained no competitor. Each group of lanes marked 2–5 contained reactions with 2, 10, 20, and 50 pmol of a competitor, respectively. (B) Quantification of the gel image shown in A performed using phosphorimaging.

Fig. 5. Distribution of OGG activity in a group of 120 healthy individuals. OGG activity was measured in protein extracts prepared from 10 ml blood samples obtained from 120 healthy subjects. (A) Distribution of OGG specific activity values of all 120 healthy subjects. (B) Sub-group comparison of OGG activity in 68 female subjects (white circles) and 52 male subjects (black circles). (C) Sub-group comparison of OGG activity in 85 non-smokers (black triangles) and 35 smokers (white triangle).

Fig. 6. Age sub-group analysis of OGG activity in females and males. OGG activity values of the 120 individuals were plotted for females 55 years old or younger (N = 37), compared to older females (N = 31) (panel A), and for males 55 years old or younger (N = 26), compared to older males (N = 26) (panel B).

Fig. 7. The relationship between OGG1 mRNA expression and enzymatic activity in human PBMC. Relative expression of OGG1 mRNA in PBMC from 11 individuals (8 healthy, 3 lung cancer patients) was determined by quantitative real-time RT-PCR, and plotted against their OGG activity. The detailed procedure is described under Section 2, and the numerical data are presented in Table 5.

Analysis of the reproducibility of protein extraction

PBMC isolated from 120 ml blood obtained from each of two blood bank donor were divided to 12 identical portions each containing 4 million cells. For each donor extracts were prepared from three to four samples in parallel, on 3 different days.

Reproducibility of the OGG activity assay

Stability of stored frozen PBMC and protein extracts

Distribution of selected characteristics and OGG activity values among healthy subjects

OGG activity was determined using 10 ml blood samples obtained from 120 healthy subjects.

Relationship between OGG enzymatic activity and expression of OGG1 mRNA

OGG enzymatic activity and OGG1 mRNA were determined in PBMC obtained from 11 individuals. Relative mRNA expression levels are given based on the indicated reference genes, or their combinations. See Section 2 for details.