A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity.
A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent Km of 34.7 +/- 2.4 mM was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, alpha-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.
Department of Chemistry, Baylor University, Waco, Texas 76798.
M L
ML
Fink
Y Y
YY
Shao
G J
GJ
Kersh
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005470:Fluorometry / Q000379:methods*; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D011503:Transglutaminases / Q000032:analysis*
D011503:Transglutaminases