Analysis of the RNase T1 Mediated Cleavage of an Immobilized Gapped Heteroduplex via Fluorescence Correlation Spectroscopy
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Abstract
We report a new method for studying the activity of hydrolytic enzymes. Fluorescence correlation spectroscopy was used to observe online the hydrolyzation of a rhodamine Blabeled substrate by ribonuclease T1. A gapped heteroduplex substrate a hybrid of a ribooligonucleotide and two smaller complementary deoxyribooligonucleotides was immobilized via biotin to a streptavidincoated surface of a coverslip. The reported method opens the possibility to study the cleavage of small substrates differing only slightly in molecular weight from the enzyme reaction product. The use of fluorescence correlation spectroscopy allows the detection of very low enzyme concentrations (down to 10[-21] mol 0.05 f of RNase T1, corresponding to about 600 RNase T1 molecules in 0.02 ml).
Copyright © 2000 by Walter de Gruyter GmbH & Co. KG
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Artikel in diesem Heft
- Interaction of E. coli Single-Stranded DNA Binding Protein (SSB) with Exonuclease I. The Carboxy-Terminus of SSB Is the Recognition Site for the Nuclease
- p38/SAPK2-Dependent Gene Expression in Jurkat T Cells
- The First Laminin G-Type Domain in the SHBG-Like Region of Protein S Contains Residues Essential for Activation of the Receptor Tyrosine Kinase Sky
- Permutation of the Active Site Motif of Tryparedoxin 2
- Structural Investigations of the Highly Flexible Recombinant Ribosomal Protein L12 from Thermotoga maritima
- Polyhistidine-Tagged Hepatitis B Core Particles as Carriers of HIV-1/gp120 Epitopes of Different HIV-1 Subtypes
- In Vitro Evolution of Ligands for HCV-Specific Serum Antibodies
- Stability of Bacteriophage T4 Short Tail Fiber
- Analysis of the RNase T1 Mediated Cleavage of an Immobilized Gapped Heteroduplex via Fluorescence Correlation Spectroscopy
- Binding of Selenoprotein P to Heparin: Characterization with Surface Plasmon Resonance
- Biotin-Avidin Microplate Assay for the Quantitative Analysis of Enzymatic Methylation of DNA by DNA Methyltransferases
