Jerome C. Y. Wong

Single molecules of alkaline phosphatase are captured in a capillary filled with a fluorogenic substrate. During incubation, each enzyme molecule creates a pool of fluorescent product. After incubation, the product is swept through a high-sensitivity laser-induced fluorescence detector; the area of the peak provides a precise measure of the activity of each molecule. Three studies are performed on captured enzyme molecules. In the first study, replicate incubations are performed on the same molecule at constant temperature; the amount of product increases linearly with incubation time. Single enzyme molecules show a range of activity; the most active molecules have over a 10-fold higher activity than the least active molecules. In the second study, replicate incubations are performed on the same molecule at successively higher temperatures. The activation energy of the reaction catalyzed by a single molecule is determined with high precision. Single enzyme molecules show a range of activation energy; microheterogeneity extends to thermodynamic properties of catalysis. The average activation energy is within experimental error of the activation energy obtained from analysis of a bulk sample. These results are consistent with the first postulate of statistical thermodynamics: a thermodynamic property obtained from the time average of an individual molecule is identical to that produced by an ensemble average over a large number of molecules. In the third study, the activity of single enzyme molecules is measured after partial heat denaturation. The number of active molecules decreases in proportion to the extent of denaturation. However, the activity of the surviving molecules is experimentally indistinguishable from the activity of control enzyme. Thermal denaturation of alkaline phosphatase is a catastrophic process, wherein the molecule undergoes irreversible conversion to an inactive form.

Alkaline phosphatase can be assayed by monitoring the conversion of the fluorogenic substrate AttoPhos into the highly fluorescent product AttoFluor. We have used capillary electrophoresis with laser-induced fluorescence detection to monitor this reaction. The concentration limit of detection (3σ) of alkaline phosphatase is 1.5 × 10-17 M (2.1 fg/mL), which corresponds to a mass limit of detection of nine molecules (1.5 × 10-23 mol) contained within a 1-μL sample volume.

ADVERTISEMENT RETURN TO ISSUEPREVFeaturesNEXTPeer Reviewed: Life and Death of a Single Enzyme Molecule Details of enzyme behavior, previously hidden in the ensemble average of classic methods, are revealed by studying individual molecules. Douglas B. Craig, Edgar Arriaga, Jerome C. Y. Wong, Hui Lu, and and Norman J. DovichiCite this: Anal. Chemi. 1998, 70, 1, 39A–43APublication Date (Web):June 2, 2011Publication History Published online2 June 2011Published inissue 1 January 1998https://pubs.acs.org/doi/10.1021/ac9817164https://doi.org/10.1021/ac9817164research-articleACS PublicationsRequest reuse permissionsArticle Views321Altmetric-Citations26LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Molecules,Peptides and proteins Get e-Alerts