ABSTRACT
Kinetics of photolysis of the antibiotic tetracycline (TC) during irradiation at 365 nm was studied in three buffer solutions usually used for studies on TC binding to its main cell targets--a transcriptional repressor protein TetR and to the ribosome. These buffer solutions contain magnesium ions and an antioxidant--mercaptoethanol or dithiothreitol. The rate of TC photolysis was maximal in medium which contained 14 mM mercaptoethanol and 5 mM magnesium ions. In the absence of mercaptoethanol the photolysis rate was more than twofold decreased. The rate constants and quantum yields of the photolysis were determined under various conditions.
Subject(s)
Tetracycline/chemistry , Buffers , Chromatography, High Pressure Liquid , Kinetics , Magnesium/pharmacology , Mercaptoethanol/pharmacology , Photolysis , Quantum Theory , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Spectrophotometry, UltravioletABSTRACT
Results of a first successful application of a direct photo-induced affinity modification of Tet repressor (TetR(D)) protein with tetracycline within a complex of known three-dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR(D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59-Glu73 and Ala173-Glu183, which form a binding tunnel for the drug according to the X-ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.
