Detection of free radicals generated from hydrogen peroxide, gallic acid and haemoprotein chemiluminescence system by electron spin resonance spectroscopy.

Abstract

Low-level chemiluminescence is produced in a hydrogen peroxide (H(2)O(2))/gallic acid/haemoprotein system with single broad peaks around 520 nm, regardless of the biological role of the haemoprotein. The free haem iron systems (haemin and haematin systems) gave a higher photon intensity (1.5 x 10(4) and 2.0 x 10(4) cps) than that of the H(2)O(2)/gallic acid/haematoporphyrin system. These results indicated that haem iron plays a significant role in the photon emission of haemoprotein systems. A free radical with a g value of 2. 0058 was detected through a direct electron spin resonance (ESR) method. The photon intensity of the H(2)O(2)/gallic acid/haemoprotein system decreased in the order: HRP > cytochrome c > myoglobin > haemoglobin, and this corresponded to the decrease in radical intensity. These results indicated that the formation of the free radical with a g value of 2.0058 may be the key step for chemiluminescence in the H(2)O(2)/gallic acid/haemoprotein system. A quartet line similar to DMPO-OH adducts and uncomplexed free radicals (g = 2.0058) was detected using the ESR spin-trapping method in the H(2)O(2)/gallic acid/cytochrome c system.