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Photodynamic Inactivation of Trypsin by the Aminophylline-Riboflavin System: Involvement of Hydroxyl Radical

Journal Title, Volume, Page: 
Med Sci Monit.;12(8):BR283-9
Year of Publication: 
Iyad Ali
Department of Biochemistry, Faculty of Pharmacy, University of Aman, Aman, Jordan
Current Affiliation: 
Faculty of Medicine & Health Sciences, Department of Biomedical Sciences, An-Najah National University, Nablus, Palestine
Nazarul Hasan
Department of Biochemistry, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh, India
Imrana Naseem
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India
Preferred Abstract (Original): 

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.

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