ABSTRACT
Optimal conditions for the preparation of avidin-peroxidase conjugates by the periodate method were studied. A method based on hydrophobic interaction chromatography was developed for the isolation of active oligomers. The probable structure of oligomers with highest sensitivity is discussed.
Subject(s)
Avidin/chemistry , Horseradish Peroxidase/chemistry , Biotin , Chromatography, High Pressure Liquid , Horseradish Peroxidase/chemical synthesis , Oxidation-Reduction , Polymers/chemical synthesis , Polymers/isolation & purificationABSTRACT
Chemical modification of the elongation factor G (EF-G) with tetranitromethane and iodine has been studied. It has been shown by spectrophotometric titration that EF-G contains two exposed tyrosine residues, one of which has an unusually low pK value for a phenol hydroxyl group at pH 8.5. Modification of one tyrosine residue with either tetranitromethane or iodine results in a 70--80% loss of EF-G activity in all ribosome-dependent reactions. Modification of three or four residues inhibits 90--100% of activity. Binding of EF-G with the 70-S ribosome and 50-S subunit is equally effective for protection of tyrosine residues against modification. The rate of EF-G modification with tetranitromethane is considerably higher in the presence of guanyl nucleotides than for free EF-G. The modified residues are located in the C-terminal domain of EF-G and are presumably contained in one of the sites of EF-G interaction with the ribosome.
