Chemical synthesis of biotinylated histones and analysis by sodium dodecylsulfate-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 stud y, we sought to biotinylate histones in vitro for later use as standards in the quantitation of histones biotinylated in vivo. We also sought to devel op a procedure for electrophoretic separation and streptavidin-peroxidase d etection of the various classes of biotinylated histones. Histones H1, H2a, H2b, H3, and H4 from calf thymus were biotinylated using sulfosuccinimidob iotin at pH 7.5. Stoichiometries of biotin/histone were determined either b y 4'-hydroxyazobenzene-2-carboxylic acid/avidin assay or by avidin-binding assay. The stoichiometries of biotinylation (mol biotin/mol histone) were a s follows: H1, 3.9 +/- 0.17; H2a, 1.7 +/- 0.11; H2b, 1.8 +/- 0.11; H3, 0.02 9 +/- 0.0012; H4, 0.006 +/- 0.0002. When two synthetic polypeptides were us ed 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-argin ine was less than 0.3 x 10(-3) mol biotin/mol. These data suggest that prim ary amino groups of histones biotinylated by sulfosuccinimidobiotin were ly sine rather than arginine. Detection and identification of biotinylated his tones 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 sub strate. We conclude that sulfosuccinimidobiotin does biotinylate each of th e five classes of histones and that the stoichiometry of biotinylation is s ufficient for detection on nitrocellulose transblots by streptavidin-peroxi dase. (C) 1999 Academic Press.