Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection |
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Authors: | K Shimura H Matsumoto K Kasai |
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Affiliation: | Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan. shimurak@pharm.teikyo-u.ac.jp |
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Abstract: | Capillary isoelectric focusing is a highly effective method for the separation of proteins due to focusing as a function of their pI values in the separation process. This technique is also effective for certain types of peptides that focus well. Fluorescence labeling and subsequent detection by laser-induced fluorescence farther enhance the sensitivity of this technique. This paper demonstrates the utility of this technique in an enzyme assay. A synthetic nona peptide, H-Gly-Cys-His-Glu-Ala-Arg-Ala-Glu-Glu-OH, was labeled with an iodoacetyl derivative of Lissamine rhodamine B at the thiol group of the cysteine residue as a substrate for trypsin. Trypsin catalyzed the cleavage of the Arg-Ala bond of the labeled substrate, which focused at pH 4.8, and liberated a shortened, labeled product, H-Gly-*Cys-His-Glu-Ala-Arg-OH that focused at pH 6.9 (* indicates the label). The product peptide at 3-300 pM was determined with a relative standard deviation of 5.5% (n = 5) by fluorescence detection at 590 nm with excitation by a green line of He-Ne laser. Incubation of trypsin with the substrate for 10 min at 37 degrees C allowed the determination of 50-250 pg of trypsin, with a relative standard deviation of 5.3% (n = 5). |
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