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Talanta. 2016 Aug 15;156-157:1-5. doi: 10.1016/j.talanta.2016.04.058. Epub 2016 Apr 27.

Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity.

Author information

1
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
2
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan. Electronic address: nohta@fukuoka-u.ac.jp.

Abstract

We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA).

KEYWORDS:

Fluorometry measurement; Fluorous; Metal chelate affinity; Phosphopeptide; Protein kinase

PMID:
27260427
DOI:
10.1016/j.talanta.2016.04.058
[Indexed for MEDLINE]

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