2014 Volume 60 Issue 4 Pages 319-326
In this review, I focus on three major subjects of my research life in Juntendo. The first subject, the work on which was carried out at Narashino campus, was purification and crystallization of iron-containing superoxide dismutase (Fe-SOD). Since we succeeded in the crystallization of Fe-SOD, we determined the three-dimensional structure of the enzyme by X-ray crystallographic analysis in collaboration with Dr. Mitsui (University of Tokyo), and then Prof. Gregory A. Petsko (MIT, USA). Finally, we succeeded in determining the three-dimensional structure of Fe-SOD for the first time. The second subject was determination of nitrated amino acids (Tyr34) in human recombinant manganese-containing superoxide dismutase (Mn-SOD). This modification caused inactivation of Mn-SOD and has been found in many disease states. Therefore, this modification has an important role for the process of oxidative injury in these diseases. The last subject was the establishment of a new oxidative stress marker, which was caused by reactive nitrogen species (RNS). We identified a major nitrated product of tryptophan residues in proteins reacted with RNS, 6-nitrotryptophan, and developed its specific monoclonal antibody. We successfully identified 6-nitrotryptophan-containing proteins in pathophysiological and physiological states of animals and also humans by the combination of the specific antibody and LC-MS/MS analysis. We believe that this technique could be a useful tool as a new oxidative stress marker and also a new tool to clarify new mechanisms of nitrative stress in vivo.