An-Najah Staff

BACKGROUND: Riboflavin finds ubiquitous occurrence in plants and animals and functions as a coenzyme participating in various oxidation-reduction reactions during the course of metabolism. Photosensitized riboflavin generates reactive oxygen species (ROS). Aminophylline is an antiasthmatic drug and a known phosphodiesterase inhibitor. In this study we examined the effect of photoilluminated riboflavin on aminophylline using trypsin as the target molecule.
MATERIAL/METHODS: The possible loss of trypsin activity due to autolysis was assayed after incubation in fluorescent light. Changes in trypsin activity caused by photoilluminated riboflavin alone and with aminophylline were monitored as functions of concentration and time. These effects were also analyzed by SDS-PAGE to visualize protein degradation. Spectra of riboflavin, alone and with aminophylline, under different conditions were taken to monitor the structural changes for elucidating the possible reaction mechanism involved. Free radical scavengers were also included in some experiments.
RESULTS: Aminophylline alone is not known to posses any photosensitizing characteristics. However, in the presence of riboflavin and fluorescent light, aminophylline caused inactivation and fragmentation of trypsin. This fragmentation was found to be concentration dependent and was mediated by ROS. In all cases, thiourea, a scavenger of hydroxyl radicals, was most effective in scavenging the damaging effect of the riboflavin-aminophylline combination.
CONCLUSIONS: Based on our results we suggest that photoilluminated riboflavin generates the singlet and triplet excited states that, upon energy transfer, generate (1)O(2). and (3)O(2). oxygen. These activated oxygen species probably attack aminophylline leading to its oxidation, generating hydroxyl radicals which presumably cause inactivation and fragmentation of trypsin.

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.