Determination of the turnover number of the restriction endonuclease EcoRI using evanescent wave technology.
Binding and catalytic activity of the type II restriction endonuclease EcoRI on immobilized DNA has been observed in real time using three different evanescent wave biosensors and two different immobilization techniques. The method gives direct access to the turnover number (kcat) without the necessity for the determination of any concentration or activity. The combination of different evanescent wave techniques gives access to the catalytic mechanism and allows the determination of the rate limiting step.
Bier
Frank F
FF
Fraunhofer-Institute of Biomedical Engineering, Dept Molecular Bioanalytics and Bioelectronics, Bergholz-Rehbrucke, Germany.
Kleinjung
Frank
F
Schmidt
Peter M
PM
Scheller
Frieder W
FW
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D019921:Biotinylation; D002384:Catalysis; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry* / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding
D009841:Oligonucleotides; D015246:Deoxyribonuclease EcoRI