Tritiation of lysozyme by the free-radical interceptor method and determination of the tritium distribution among individual residues of the chromatographically homogeneous protein.
Lysozyme has been tritiated by the free-radical interceptor method, which involves reaction of the carbon free radicals, resulting from gamma-irradiation of the lyophilized protein in a vacuum, with tritiated hydrogen sulfide. This technique resulted in a high yield of tritiated enzyme which was chromatographically indistinguishable from the native protein. To characterize this material with respect to its tritium distribution, it was first reduced, carboxymethylated, and digested with chymotrypsin. The peptides were separated and purified by ion exchange chromatography and electrophoresis and then analyzed to determine the specific activities of 78 of the 129 residues in the protein chain. The tritium was found to be broadly distributed, with only 12 residues apparently devoid of label. The relative specific activity, defined as the ratio of the specific activity of a given residue to the average specific activity for all residues of the same kind within the protein, did not exceed the value of 3.73 for isoleucine at position 88. A distinct spatial relationship is seen among the more heavily labeled residues when these results are related to the x-ray diffraction model of lysozyme. Thus, residues 16, 18, 19, 21, 23, 29, 31, and 32 may be grouped with 105, 110, 113, and 115; these highly labeled residues are close to a strongly hydrophobic region. Another heavily labeled area involves residues 50, 54, 57. 83, 84, and 88. In addition, residues 1 and 6 at the amino end of the chain are heavily labeled, as are 118, 121, and possibly 119 at the carboxyl end. This distribution of tritium appears to reflect the secondary free-radical distribution in the irradiated protein. These observations confirm previous indications that the secondary distribution is influenced by the conformation of the protein molecule.
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National Heart Institute, Laboratory of Biochemistry, Section on Cellular Physiology, National Institutes of Health, Public Health Service, U.S. Department of Health, Education and Welfare, Bethesda, Maryland, USA.
D000465:Algorithms; D000596:Amino Acids / Q000737:chemistry / Q000528:radiation effects; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005609:Free Radicals / Q000737:chemistry; D005720:Gamma Rays; D009113:Muramidase / Q000737:chemistry* / Q000528:radiation effects*; D014316:Tritium / Q000737:chemistry* / Q000528:radiation effects*
D000596:Amino Acids; D005609:Free Radicals; D014316:Tritium; D009113:Muramidase