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Titolo:
REAL-TIME ENZYME-KINETICS MONITORED BY DUAL-COLOR FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY
Autore:
KETTLING U; KOLTERMANN A; SCHWILLE P; EIGEN M;
Indirizzi:
MAX PLANCK INST BIOPHYS CHEM,DEPT BIOCHEM KINET D-37077 GOTTINGEN GERMANY
Titolo Testata:
Proceedings of the National Academy of Sciences of the United Statesof America
fascicolo: 4, volume: 95, anno: 1998,
pagine: 1416 - 1420
SICI:
0027-8424(1998)95:4<1416:REMBDF>2.0.ZU;2-V
Fonte:
ISI
Lingua:
ENG
Soggetto:
ECORI RESTRICTION ENDONUCLEASE; SYNTHETIC OLIGODEOXYNUCLEOTIDES; EVOLUTIONARY BIOTECHNOLOGY; RECOGNITION SEQUENCE; RI ENDONUCLEASE; DNA; CLEAVAGE; ASSAY; HYBRIDIZATION; DIFFUSION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
43
Recensione:
Indirizzi per estratti:
Citazione:
U. Kettling et al., "REAL-TIME ENZYME-KINETICS MONITORED BY DUAL-COLOR FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY", Proceedings of the National Academy of Sciences of the United Statesof America, 95(4), 1998, pp. 1416-1420

Abstract

A method for sensitively monitoring enzyme kinetics and activities byusing dual color fluorescence crosscorrelation spectroscopy is described, This universal method enables the development of highly sensitiveand precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two fluorophores that can be discriminated spectrally, In this work a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing theGAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range(>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored online at various initial substrate concentrationsin the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a K-M of 14 +/- 1 nM and a k(cat) of 4.6 +/- 0.2 min(-1). In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-correlation spectroscopy will be very usefulfor high-throughput screening and evolutionary biotechnology.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 24/09/20 alle ore 04:59:48