Chemical synthesis of biotinylated histones and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/streptavidin-peroxidase.
Recently, Hymes and co-workers demonstrated that human biotinidase (EC 3.5.1.12) specifically biotinylates histones, suggesting that biotin may have a specific role in transcription and replication of DNA. In the present study, we sought to biotinylate histones in vitro for later use as standards in the quantitation of histones biotinylated in vivo. We also sought to develop a procedure for electrophoretic separation and streptavidin-peroxidase detection of the various classes of biotinylated histones. Histones H1, H2a, H2b, H3, and H4 from calf thymus were biotinylated using sulfosuccinimidobiotin at pH 7.5. Stoichiometries of biotin/histone were determined either by 4'-hydroxyazobenzene-2-carboxylic acid/avidin assay or by avidin-binding assay. The stoichiometries of biotinylation (mol biotin/mol histone) were as follows: H1, 3.9 +/- 0.17; H2a, 1.7 +/- 0.11; H2b, 1.8 +/- 0.11; H3, 0.029 +/- 0.0012; H4, 0.006 +/- 0.0002. When two synthetic polypeptides were used as substrates for biotinylation, the stoichiometry of poly-l-lysine was 2.8 +/- 0.14 mol biotin/mol; in contrast, the stoichiometry of poly-l-arginine was less than 0.3 x 10(-3) mol biotin/mol. These data suggest that primary amino groups of histones biotinylated by sulfosuccinimidobiotin were lysine rather than arginine. Detection and identification of biotinylated histones were accomplished by electrophoretic separation on 16% polyacrylamide gels; the separated histones on nitrocellulose transblots of the gels were detected using streptavidin-peroxidase with 4-chloro-1-naphthol as the substrate. We conclude that sulfosuccinimidobiotin does biotinylate each of the five classes of histones and that the stoichiometry of biotinylation is sufficient for detection on nitrocellulose transblots by streptavidin-peroxidase.
Zempleni
J
J
Division of Gastroenterology, Hepatology, and Nutrition, University of Arkansas for Medical Sciences and Arkansas Children's Hospital Research Institute, Little Rock, Arkansas 72202, USA.
Mock
D M
DM
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D043523:Biotinidase; D019921:Biotinylation; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006657:Histones / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D010544:Peroxidases; D011107:Polylysine / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate; D019809:Streptavidin; D013950:Thymus Gland
D006657:Histones; D010455:Peptides; D011107:Polylysine; C015462:polyarginine; D012967:Sodium Dodecyl Sulfate; D019809:Streptavidin; D010544:Peroxidases; D000581:Amidohydrolases; D043523:Biotinidase