The ability of proline to directly react with ROS has been investigated by numerous laboratories (58, 114). Previous studies have shown that free and polypeptide-bound proline can react with H2O2 and OH• (pH 7–8) to form stable free radical adducts of proline and hydroxyproline derivatives as shown in Figure 3 (e.g., 4-hydroxyproline and 3-hydroxyproline) (38, 58, 102, 106, 127). Although Floyd and Nagy (38) observed that nitroxyl radicals accumulate during the incubation of proline with H2O2, the reaction is very slow relative to that of proline and OH• (5.4×108 M−1s−1) (3). Recently, the ability of proline to scavenge H2O2 was compared with pyruvate, a well-established scavenger of H2O2. At 30 min, H2O2 levels were diminished by >90% in cell medium supplemented with 1 mM pyruvate, whereas no significant decrease was observed with proline (5 mM) (70). This observation further indicates that a direct reaction between H2O2 and proline does not significantly contribute to the scavenging of cellular H2O2 (48). Proline has also been shown not to directly scavenge O2•− (58).