Ishii T, Iwahashi H, Sugata R, Kido R
Department of Biochemistry, Wakayama Medical College, Japan.
Arch Biochem Biophys. 1992 May 1;294(2):616-22. doi: 10.1016/0003-9861(92)90733-d.
Using ESR, a radical (g = 2.004) was detected in the reaction mixture of 3-hydroxykynurenine (3-HKY), H2O2, and horseradish peroxidase. The radical was stable and was detected even after 5 h. On HPLC analysis of the reaction mixture, two radical peaks (Peak-1 and Peak-2) were detected using ESR. The ESR spectra of Peak-1 and Peak-2 radicals were the same and identical with that of the original radical in the reaction mixture. The retention times of Peak-1 and Peak-2 corresponded to those of authentic xanthommatin (XA) and hydroxanthommatin (Hydro-XA), respectively, XA being formed in the oxidation of 3-HKY by potassium ferricyanide and Hydro-XA being formed in the reduction of XA by sodium metabisulfite. The absorbance spectra of Peak-1 and Peak-2 were nearly identical with those of authentic XA and Hydro-XA. The absorbance spectrum of Peak-2 changed from that of Hydro-XA to that of XA, indicating that Hydro-XA auto-oxidized to XA in the air. The ESR signal intensity of the Peak-2 radical developed in accordance with the progress of this auto-oxidation of Hydro-XA to XA. It was supposed that the Peak-2 radical was generated in the auto-oxidation of Hydro-XA after its elution from the HPLC column. Thus, the radical seemed to be the one-electron oxidized form of Hydro-XA. The Peak-1 radical appeared to be the true retention of the radical on the column and to be eluted with a much larger amount of XA. The separation of the radical from XA was impossible on the column. Hemoglobin (Hb) or hematin also induced the same radical in the reaction mixture of 3-KHY, H2O2, and Hb or hematin.
使用电子自旋共振（ESR）技术，在3-羟基犬尿氨酸（3-HKY）、过氧化氢（H₂O₂）和辣根过氧化物酶的反应混合物中检测到一种自由基（g = 2.004）。该自由基很稳定，即使在5小时后仍能被检测到。对反应混合物进行高效液相色谱（HPLC）分析时，使用ESR检测到两个自由基峰（峰1和峰2）。峰1和峰2自由基的ESR光谱相同，且与反应混合物中原始自由基的光谱一致。峰1和峰2的保留时间分别对应于纯品黄蝶呤（XA）和羟基黄蝶呤（Hydro-XA）的保留时间，XA是由铁氰化钾氧化3-HKY形成的，而Hydro-XA是由焦亚硫酸钠还原XA形成的。峰1和峰2的吸收光谱与纯品XA和Hydro-XA的吸收光谱几乎相同。峰2的吸收光谱从Hydro-XA的光谱变为XA的光谱，表明Hydro-XA在空气中自动氧化为XA。峰2自由基的ESR信号强度随着Hydro-XA自动氧化为XA的过程而增强。据推测，峰2自由基是在Hydro-XA从HPLC柱洗脱后自动氧化过程中产生的。因此，该自由基似乎是Hydro-XA的单电子氧化形式。峰1自由基似乎是自由基在柱上的真实保留形式，并与大量的XA一起洗脱。在柱上无法将该自由基与XA分离。血红蛋白（Hb）或血红素在3-KHY、H₂O₂和Hb或血红素的反应混合物中也能诱导产生相同的自由基。
