Identification of a radical formed in the reaction mixtures of ram seminal vesicle microsomes with arachidonic Acid using high performance liquid chromatography-electron spin resonance spectrometry and high performance liquid chromatography-electron spin resonance-mass spectrometry

Abstract

The reaction of ram seminal vesicle (RSV) microsomes with arachidonic acid (AA) was examined using electron spin resonance (ESR), high performance liquid chromatography-electron spin resonance spectrometry (HPLC-ESR), and high performance liquid chromatography-electron spin resonance-mass spectrometry (HPLC-ESR-MS) combined use of spin trapping technique. A prominent ESR spectrum (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT) was observed in the complete reaction mixture of ram seminal vesicle microsomes with arachidonic acid containing 2.0 mg protein/ml ram seminal vesicle (RSV) microsomal suspension, 0.8 mM arachidonic acid, 0.1 M 4-POBN, and 24 mM tris/HCl buffer (pH 7.4). The ESR spectrum was hardly observed for the complete reaction mixture without the RSV microsomes. The formation of the radical appears to be catalyzed by the microsomal components. In the absence of AA, the intensity of the ESR signal decreased to 16 +/- 15% of the complete reaction mixture, suggesting that the radical is derived from AA. For the complete reaction mixture with boiled microsomes, the intensity of the ESR signal decreased to 49 +/- 4% of the complete reaction mixture. The intensity of the ESR signal of the complete reaction mixture with indomethacin decreased to 74 +/- 20% of the complete reaction mixture, suggesting that cyclooxygenese partly participates in the reaction. A peak was detected on the elution profile of HPLC-ESR analysis of the complete reaction mixture. To determine the structure of the peak, an HPLC-ESR-MS analysis was performed. The HPLC-ESR-MS analysis of the peak showed two prominent ions, m/z 266 and m/z 179, suggesting that the peak is a 4-POBN/pentyl radical adduct. An HPLC-ESR analysis of the authentic 4-POBN/pentyl radical adduct comfirmed the identification.

Keywords: COX; ESR; arachidonic acid; peroxidation; radical.

Fig. 1

ESR spectra of the reaction mixtures of ram seminal vesicle microsomes with arachidonic acid. The reaction and ESR conditions were as described in Materials and Methods. Total volume of the reaction mixtures was 200 µl. A, a complete reaction mixture of sam seminal vesicle microsomes with arachidonic acid; B, same as in A except that microsomes were omitted; C, same as in A except that arachidonic acid was omitted; D, a complete reaction mixture with boiled microsomes; E, same as in A except that indomethacin (2 mM) was added.

Fig. 2

HPLC-ESR analyses of the reaction mixtures of ram seminal vesicle microsomes with arachidonic acid. The reaction and HPLC-ESR conditions were as described in Materials and Methods. Total volume of the complete reaction mixtures was 10 ml. A, 10 ml of complete reaction mixture; B, 10 µl of 4-POBN/pentyl radical adducts; C, 10 ml of complete reaction mixture mixed with 10 µl of 4-POBN/pentyl radical adducts diluted by one fifteenth.

Fig. 3

HPLC-ESR-MS analysis. The reaction and HPLC-ESR-MS conditions were as described in Materials and Methods. Total volume of the reaction mixtures was 10 ml.

Scheme 1

A possible reaction path for the formation of pentyl radical.