Enzyme coupling method on calibrated nylon spheres: application to the selective trypsinization of histones in chromatin.
A new method consisting of a two-step activation was developed in order to covalently immobilize enzymes on calibrate nylon 66 spheres. This efficient method associates for the first time peptide bond cleavage and O-alkylation of the support. Optimal conditions for activation and protein coupling were defined, and immobilized trypsin was used to investigate the histone accessibility on chromatin. This approach, which allows us to degrade first progressively H1, indicates that H4 seems inaccessible both in relaxed and condensed chromatin.
Michalon
P
P
Laboratoire de Physico-Chimie Biologique, LBTM-CNRS UM 24, Université Lyon, France.
Couturier
R
R
Hacques
M F
MF
Favre-Bonvin
G
G
Ville
A
A
Marion
C
C
D055598:Chemical Phenomena; D002621:Chemistry; D002843:Chromatin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006868:Hydrolysis; D009757:Nylons; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*
D002843:Chromatin; D004800:Enzymes, Immobilized; D006657:Histones; D009757:Nylons; D014357:Trypsin