Quantitation of the singlet oxygen produced by UVA irradiation of human lens proteins

Ultraviolet irradiation of aged human lens proteins in vitro causes extensive photolytic damage of His and Trp residues. Protection by sodium azide argues for a process mediated by singlet oxygen (1O2). In the work described here, the synthesis of 1O2 was measured by the bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), the oxidation of added histidine and the oxidation of furfuryl alcohol. To obtain a more accurate value for 1O2 generation, a known quantity of 1O2 was generated by the thermal dissociation of 3-(4-methyl-naphthyl)propionic acid endoperoxide, and the efficiency of each assay method to report on the 1O2 generated was determined. The values obtained were 0.003 mol of RNO bleached/mol of 1O2 generated, 0.55 mol of furfuryl alcohol oxidized/mol 1O2 and 0.5 mol of His oxidized/mol 1O2 generated. Irradiation of the human lens proteins with UVA light produced from 2.1 to 2.4 mM of 1O2 by RNO bleaching, 2.6-2.8 mM 1O2 by furfuryl alcohol oxidation and up to 1.9 mM of 1O2 by histidine oxidation during a 1 h irradiation period. The average value (2.2 mM of 1O2) corresponds to the theoretical production of 30 nmol of singlet oxygen at UVA light intensities equivalent to a 1 h exposure to sunlight at noon in the northern hemisphere.