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| Type |
Poster Presentation |
| Area |
Electrochemistry |
| Room No. |
Event Hall |
| Time |
4월 20일 (금요일) 11:00~12:30 |
| Code |
ELEC.P-578 |
| Subject |
One label-based fluorescence detection of an endopeptidase |
| Authors |
Haesik Yang*, JeongHwa Shin
Department of Chemistry, Pusan National University, Korea
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| Abstract |
In order to detect a protease that cleaves the peptide bond between two specific amino acids via fluorescence, a synthetic peptide modified with two labels (a donor and an acceptor) for fluorescence resonance energy transfer(FRET) is commonly used. However, the preparation and optimization of a peptide for the sensitive and selective detection of a target protease are time-consuming. We report a homogeneous fluorescence assay that allows the simple and sensitive detection of BoNT/E-LC using (i) a peptide substrate modified with one fluorescence label [7-amino-4-methylcoumarin(AMC)] and (ii) a two-step proteolytic cleavage by BoNT/E-LC and L-leucine-aminopeptidase(LAP). BoNT/E-LC cleaves the specific peptide bond between arginine and isoleucine within C-terminally 7-amino-4-methylcoumarin(AMC)-labeled oligopeptide, leaving fragmented isoleucine-AMC. Subsequently, LAP cleaves the peptide bond between isoleucine and AMC, liberating fluorescent AMC. This method does not require two label-modified peptides. Capping the oligopeptide with the D-form of tyrosine does not result in better performance in terms of detection limit, although a higher concentration of LAP can be used. The detection limit for BoNT/L-EC in both phosphate-buffered saline and commercial bottled water is 2 ng mL-1 for incubation period of 1h. The fluorescence detection is selective for BoNT/E-LC among the four tested BoNTs. Fluorescence detection using one label can be readily applied to any type of proteases without using FRET. |
| E-mail |
chewjd30@gmail.com |
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121st General Meeting of the KCS