Electron Spin Resonance Studies of spin-Trapped Free Radicals Produced by Reaction of Metmyoglobins with Hydrogen Peroxide = $H_2O_2$에 의해 생성된 myoglobin 자유라디칼에 대한 ESR 연구

국문 초록 (Abstract)

Metmyoglobin을 spin trap 5,5-dimethyl pyrroline-N-oxide (DMPO) 존재 아래 $H_2O_2$반응시켰을 때, 폭이 넓은 다섯 가닥의 비대칭 ESR spectrum을 얻었는데, 기대했던 DMPO/OH adduct의 흔적은 찾을 수 없었다. 또 이 metmyoglobin을 DMPO 존재 아래서 Fenton 시약과 반응시켰을 때도 동일한 결과를 얻었을 뿐 아니라, Fenton 시약과 DMPO를 반응시켜 DMPO-OH adduct 생성을 ESR로 확인한 뒤에 그 용액에 metmyoglobin을 첨가했을 때, 기존의 DMPO-OH signal은 완전히 사라지고 위에서 얻었던 비대칭 다섯가닥 spectrum으로 대치되는 것을 확인했다. 이 결과는 myoglobin 분자의 heme crevice에서 생성된 1 차 및 2 차 라디칼 adduct는 물론, 단백질 분자 표면에서 Fenton 시약에 의해 생성된 DMPO/OH까지도 heme pocket 안에 굳게 고정되는 때문인 것으로 해석했다. 한편 DMPO 대신에 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO)를 사용했더니 DMPO의 경우와는 아주 다르게, 두 종류의 라디칼로 혼성된 ESR spectrum을 얻었는데. 그 중에서 폭넓은 특정 구조가 없는 signal은 TMPO가 단백질 backbone에 있는 ${\alpha}-$ 탄소 라디칼과, 그리고 선명한 세가닥 signal은 단백질의 곁가닥 라디칼과 이룩한 adduct를 각각 나타내는 것으로 해석된다. 그런데 TMPO는 DMPO보다 메틸기를 두 개 더 가지고 있어서 보다 큰 입체적 장애를 받아 pocket에는 들어갈 수가 없고, 단백질 분자의 표면에 생성된 라디칼만을 포착하는 것으로 이해된다.

다국어 초록 (Multilingual Abstract)

Metmyoglobin when incubated with hydrogen peroxide in the presence of the spin trap, 5,5-dimethyl pyrroline-N-oxide (DMPO) has produced an asymmetric ESR spectrum, consisting of five broad lines of different intensities, in which no evidence of DMPO/OH radical adduct was detected. This result was found to be consistent to other incubation in which metmyoglobin was mixed with Fenton's reagent in the presence of DMPO. Furthermore, DMPO/OH radical adducts validated by the characteristic ESR quartet while incubating Fenton's reagent with DMPO, disappeared completely and was displaced by the asymmetric broad five line signal immediately after addition of metmyoglobin to this incubation solution. These results strongly suggest that not only both of the primary and secondary radical species formed in the vicinity of the heme active site and hence trapped by DMPO, but also additional DMPO/OH radical adducts formed on the surface of the protein molecule by Fenton's reagent/DMPO incubation are together strongly immobilized in the heme pocket of the myoglobin molecule. A similar incubation in which metmyoglobin was mixed with hydrogen peroxide in the presence of the spin trap 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO), however, gave a quite different ESR spectrum with two different signal species, a sharp triplet of equal intensities with a parameter $a_N$ 6.8 G, and a broad, structureless signal covering about 40 G. This indicates that the TMPO, having two more substituents, traps radicals formed on the surface of the protein molecule, in contrast to DMPO, due to the increased steric hindrance and hence can hardly enter into the heme pocket. The broad signal species was ascribed to the TMPO radical adduct formed with ${\alpha}$-carbon atom in backbone and the triplet to that formed with side chains.