Continuous spectrophotometric assay for restriction endonucleases using synthetic oligodeoxynucleotides and based on the hyperchromic effect.
A continuous spectrophotometric assay for the EcoRV restriction endonuclease has been developed. The synthetic self-complementary oligonucleotide d(GACGATATCGTC) (which is double stranded under the assay conditions) is used as the substrate. The EcoRV endonuclease recognizes d(GATATC) sequences cutting between the central T and dA bases. Thus d(GACGATATCGTC) is converted to d(GACGAT) and d(pATCGTC) during catalysis. Both of the hexameric products are single stranded under the assay conditions. The conversion of the dodecameric substrate to the two hexameric products and the concomitant change from double- to single-stranded DNA is associated with an increase in absorbance at 254 nm due to the hyperchromic effect. This change can be used to monitor column effluents for endonuclease activity and also for Km and kcat determination under steady-state kinetic conditions.
Waters
T R
TR
Department of Biochemistry SERC Centre for Molecular Recognition, University of Southampton, Bassett Crescent East, United Kingdom.
Connolly
B A
BA
D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases