An ESR study of gamma-irradiated haemoglobin and DNA.

The first half of this thesis is concerned with studying the mechanism of autoxidation of haemoglobin and myoglobin. The paramagnetic Y-irradiated forms were used in this ESR study as the native protein is diamagnetic. On irradiation of haemoglobin distinct radical centres are seen representing the ? and ? subunits formed by electron addition to the FeO2 unit. The radical yields are found to be both pH and solvent dependent. A similar centre is formed on irradiation of myoglobin which is pH but not solvent dependent. Warming these centres above 77 K results in conversion into new centres with g-values approaching those of low-spin Fe(III). This conversion is interpreted mechanistically in terms of proton transfer from the distal histidine to the (FeO2) unit [assuming that the (FeO2)- unit is hydrogen-bonded to the distal histidine]. Further anneal results in the loss of signal in the g = 2 region and the growth of the high-spin Fe(III) signal at g = 6 as a result of loss of HO2-. To substantiate this theory experiments were done using haemoglobin Glycera which has a distal leucine instead of histidine (i.e. there are no hydrogen bonding sites available) . The primary centre formed in this case has a g-value which is close to that of superoxide. This indicates that the centres formed in haemoglobin and myoglobin have more spin-density on iron which would result if the centres were hydrogen bonded. The second half of the thesis concerns the mechanism of Y-irradiation damage in DNA. In irradiated frozen samples of DNA two ESR signals are found at 77 K. One has been shown to be the guanine cation, the other the thymine anion. Annealing these samples, in the absence of oxygen, results in the protonation of the anion and loss of the cation. Further anneal results in loss of signal altogether. As y-irradiation produces strand breaks in DNA it is surprising that no sugar radicals are detected. By incorporation of different additives I found that this mechanism of radical interconversion can be altered. Addition of electron scavengers such as CuCl2 prevented the formation of the thymine radicals without affecting the guanine cation yield. Addition of I had the reverse effect reducing the formation of guanine cations. However, addition of soft heavy metal compounds such as HgCl2 and cis Pt (NH3)2Cl2 resulted in the formation of a new radical which has identical parameters with radicals previously found in the sugar moiety of dCMP and in the thymine base moiety of dTMP.