Guanine estimation

Relevant kinetic information on two competitive reactions of guanine radical cations within double stranded DNA, namely hydration and hole transfer to another guanine residue, has been examined [13]. Thus, the pseudoorder rate for hydration of guanine radical cations 38 has been estimated to... 

One of the major problems has been to determine the site of attachment of the PAH to the base. Some information may be obtained directly from the nmr spectra eliminating certain points of attachment. As mentioned above, if the C-8 proton of guanine or adenine can be identified, then this cannot be the point of attachment of the carcinogen. Estimation of the pKa s of the adducts either by titration (108) or partition (110) has, however, provided additional valuable information. Mass spectral fragmentation patterns can be of help in determining the site of substitution as well as in determining which bases are involved in binding (108.111-113). Substantial advances have been made in recent years on the mass spectral analysis of involatile compounds and derivatization is not always essential (114-118). X-ray analysis of DNA adducts has, to date, only been applied to model systems (119-121). 

With aquated Pt(II) compounds, numerous studies have revealed the kinetic preference of the 6-oxopurine N7 site [15,35]. In addition to the favorable electrostatic potential mentioned above [23] also steric factors seem to favor coordination to the guanine N7 site, in particular [36]. Estimated relative steric parameters (in parenthesis) suggest that the guanine N7 (1.00) and hypoxanthine N7 (1.03) atoms are the least sterically hindered binding sites in alkylated nucleobases, followed by the adenine N7 (1.17) and deprotonated hypoxanthine N1 (1.17) sites and the deprotonated N3 atoms of the different pyrimidine bases (1.39 for U, 1.44 for T, and 1.56 for C), while the adenine N1 (1.58) and... 

Several conductive CT solids with nucleobase skeletons have been developed in the TTF systems having uracil moieties (crt = 10 -2 S cm ) [123-127]. Also several attempts have been undertaken to investigate the CT complexes in a variety of biochemical systems, especially using nucleobases (Scheme 9) [18, 104]. Estimation of 7p of the nucleobases, as potential components in CT complexes, indicate that they are reasonably effective Tt-donors particularly in the case of guanine (G) 7d = 7.64—7.85 eV vs adenine (A, 7.80-8.26 eV), cytosine (C, 8.45-8.74 eV), and thymine (T, 8.74-8.87 eV) [128-131]. 

Several recent papers have reported Density Functional Theory (DFT) calculations on the primary oxidation and reduction products observed in irradiated single crystals of the common nucleobases thymine [53], cytosine [54], guanine [55], and adenine [56]. The theoretical calculations include estimates of spin densities and isotropic and anisotropic hyperfine couplings which can be compared with experimental results (obtained from detailed EPR/ENDOR experiments). 

The transient absorption spectra of duplexes with [2AP]A4GGAs are depicted in Fig. 5. At a delay time of 100 ns, the transient absorption spectrum is attributed to the superposition of the spectra of the 2AP(-H) and G /G (-H) radical products and the hydrated electrons. The structureless tail of the eh absorption in the 350-600 nm region decays completely within At<500 ns. The formation of G VG(-H) radicals monitored by the rise of the 310-nm absorption band and associated with the decay of the 2AP V 2AP(-H) transient absorption bands at 365 and 510 nm (Fig. 5) occurs in at least three well-separated time domains (Fig. 6). The prompt (<100 ns) rise of the transient absorption at 312 nm due to guanine oxidation by 2AP was not resolved in our experiments. However, the ampHtude, A((=ioo), related to the prompt formation of the G /G(-H) radicals (Fig. 6a) can be estimated using the extinction coefficients of the radical species at 312 and 330 nm (isosbestic point) [11]. The kinetics of the G VG(-H) formation in the yits and ms time intervals were time-resolved and characterized by two well-defined components shown in Fig. 6a (0.5 /zs) and Fig. 6b (60 /zs). 

Deprotonation of the dG radical cations in double-stranded DNA is evident from the EPR spectra of guanine radicals recorded in neutral solutions at room temperature [80, 81]. The EPR signal assigned to the neutral G(-H) radical derived from the deprotonation of G shows the singlet with g 2.004 and half width at a half height -0.8 mT. However, the deprotonation rate of G cannot be estimated from the conventional EPR spectra, and further time-resolved EPR studies with laser pulse generation of the radicals are required to address this problem. 

In double-stranded DNA, electron abstraction from the guanine radical cation can be associated with an extremely fast shift of the N1 proton to its Watson-Crick partner cytosine (Scheme 2a) [9]. The equilibrium constant for the protonation of C (pfCa=4.3) with the concomitant deprotonation of G estimated from the pK values of the free nucleosides, is about 2.5 [49]. Within these constraints, the guanine radical should retain some radical cation character [82] and the complete deprotonation of G would require a base pair opening event occurring on a millisecond timescale [74]. An alternative mechanism of G deprotonation is the release of the N2 proton (Scheme 2b). This mechanism was experimentally established for 1-methyl-guanosine conductometric results showed that in neutral solutions, the radical cation of this nucleoside rapidly deprotonates with the formation of the neutral radical [48]. Although the exact mechanism of the G deprotonation in double-stranded DNA requires further clarification, electron abstraction... 

Figure 10 shows that the values of kag for the oxidation of G and GG are close to one another, and are smaller by a factor of only -1.7 than the value of kag for the oxidation of guanine in the GGG sequence. Estimates by Lewis et al. [89] have shown that even small differences in the rate constants can provide modest selectivities for alkali-labile strand cleavage observed in a number of experimental studies [83-90]. 

Little is known of the physical properties of guanine aminohydrolase. Elution of rabbit liver enzyme from Sephadex gave a skewed activity peak the estimated molecular weight of the main component was 170,000, a minor shoulder component, 525,000 (60). 

Five subsequent experiments performed for electrochemical detection of DNA at concentration level of dsDNA as 16 pg/mL immobilized onto PGE during 1 h gave reproducible results with a mean guanine signal of 885 nA and an RSD value of 6.92% (n — 5). The detection limit (DL) estimated from S/N = 3 corresponds to 400 ng/mL dsDNA concentration in the 150 pL samples. 

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