Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity.
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).
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
Tadashi
T
Hayama
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
Ena
E
Kiyokawa
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
Hideyuki
H
Yoshida
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
Osamu
O
Imakyure
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
Masatoshi
M
Yamaguchi
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan. Electronic address: nohta@fukuoka-u.ac.jp.
Hitoshi
H
Nohta
D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005290:Ferric Compounds / Q000737:chemistry*; D054879:Halogenation; D007098:Imino Acids / Q000737:chemistry*; D007202:Indicators and Reagents; D009842:Oligopeptides / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D012235:Rhodamines / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005290:Ferric Compounds; D007098:Imino Acids; D007202:Indicators and Reagents; D009842:Oligopeptides; D010748:Phosphopeptides; D012235:Rhodamines; C103931:iron(III) iminodiacetic acid; C037642:kemptide; D017868:Cyclic AMP-Dependent Protein Kinases
Fluorometry measurement; Fluorous; Metal chelate affinity; Phosphopeptide; Protein kinase