Escherichia coli β-galactosidase is heterogeneous with respect to the activity of individual molecules

1998 ◽  
Vol 76 (5) ◽  
pp. 623-626 ◽  
Author(s):  
Douglas B Craig ◽  
Norman J Dovichi
Keyword(s):  
Escherichia Coli ◽  
Capillary Electrophoresis ◽  
Single Molecule ◽  
Enzyme Assay ◽  
Laser Induced Fluorescence ◽  
Enzyme Molecule ◽  
Fluorogenic Substrate ◽  
Individual Enzyme

Escherichia coli β-galactosidase molecules were incubated with fluorogenic substrate in a capillary. Upon flushing the reactor contents past the detector, individual randomly distributed peaks of product were observed, each representing the activity of an individual enzyme molecule. Individual molecules were found to differ with respect to their activities. Molecules showed a 23-fold distribution of activities with the majority of molecules within a 4-fold distribution.Key words: single-molecule chemistry, β-galactosidase, capillary electrophoresis, laser-induced fluorescence, enzyme assay.

High sensitivity detection of Escherichia coli based on the measurement of β-galactosidase activity by microchip capillary electrophoresis combined with field-amplified sample injection

2019 ◽  
Vol 11 (11) ◽  
pp. 1558-1565 ◽  
Author(s):  
Yan Zhang ◽  
Yating Zhang ◽  
Luqi Zhu ◽  
Pingang He ◽  
Qingjiang Wang
Keyword(s):  
Escherichia Coli ◽  
Capillary Electrophoresis ◽  
High Sensitivity ◽  
Laser Induced Fluorescence ◽  
Galactosidase Activity ◽  
Microchip Capillary Electrophoresis ◽  
Sample Injection ◽  
E Coli ◽  
High Sensitivity Detection ◽  
Sensitivity Detection

A sensitive strategy developed for the detection of Escherichia coli (E. coli) by microchip capillary electrophoresis (MCE) combined with laser-induced fluorescence (LIF) is described in this paper.

scholarly journals A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate

The Analyst ◽  
2015 ◽  
Vol 140 (15) ◽  
pp. 5065-5073 ◽  
Author(s):  
Yusuke Obayashi ◽  
Ryota Iino ◽  
Hiroyuki Noji
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Enzyme Assay ◽  
Fluorogenic Substrate ◽  
Enzymatic Assays ◽  
Array Technology

Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity.

Analyzing reactions of single mechanoenzyme molecules by light microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 426-427
Author(s):  
Jeff Gelles
Keyword(s):  
Image Processing ◽  
Reaction Mechanisms ◽  
Transient State ◽  
Nanometer Scale ◽  
Reaction Intermediates ◽  
Enzyme Molecule ◽  
Directed Movement ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Single Reaction

Mechanoenzymes are enzymes which use a chemical reaction to power directed movement along biological polymer. Such enzymes include the cytoskeletal motors (e.g., myosins, dyneins, and kinesins) as well as nucleic acid polymerases and helicases. A single catalytic turnover of a mechanoenzyme moves the enzyme molecule along the polymer a distance on the order of 10−9 m We have developed light microscope and digital image processing methods to detect and measure nanometer-scale motions driven by single mechanoenzyme molecules. These techniques enable one to monitor the occurrence of single reaction steps and to measure the lifetimes of reaction intermediates in individual enzyme molecules. This information can be used to elucidate reaction mechanisms and determine microscopic rate constants. Such an approach circumvents difficulties encountered in the use of traditional transient-state kinetics techniques to examine mechanoenzyme reaction mechanisms.

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