Linked Life Data

19610052

Source:http://linkedlifedata.com/resource/pubmed/id/19610052

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
2009-8-17
pubmed:abstractText
Single-molecule enzymology allows scientists to examine the distributions of kinetic rates among members of a population. We describe a simple method for the analysis of single-molecule enzymatic kinetics and provide comparisons to ensemble-averaged kinetics. To isolate our model enzyme, alpha-chymotrypsin, into single molecules, we use an array of cylindrical poly(dimethylsiloxane) wells 2 microm in diameter and 1.35 microm in height. Inside the wells, a protease assay with a profluorescent substrate detects alpha-chymotrypsin activity. We hold the concentration of alpha-chymotrypsin at 0.39 nM in a given well with an enzyme-to-substrate ratio of 1:6,666 molecules. Fluorescence emitted by the substrate is proportional to enzyme activity and detectable by a charge-coupled device. This method allows for the simultaneous real-time characterization of hundreds of individual enzymes. We analyze single-molecule kinetics by recording and observing their intensity trajectories over time. By testing our method with our current instruments, we confirm that our methodology is useful for the analysis of single enzymes for extracting static inhomogeneity.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
1520-6033
pubmed:author
pubmed:copyrightInfo
(c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009.
pubmed:issnType
Electronic
pubmed:volume
25
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
929-37
pubmed:meshHeading
pubmed:articleTitle
Microfabricated arrays of cylindrical wells facilitate single-molecule enzymology of alpha-chymotrypsin.
pubmed:affiliation
Department of Biomedical Engineering, Henry Samueli School of Engineering, University of California, Irvine, CA 92697, USA.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Evaluation Studies