Assay of trypsin activity by capillary isoelectric focusing with laser‐induced fluorescence detection
- 1 October 1998
- journal article
- research article
- Published by Wiley in Electrophoresis
- Vol. 19 (13), 2296-2300
- https://doi.org/10.1002/elps.1150191308
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°C allowed the determination of 50–250 pg of trypsin, with a relative standard deviation of 5.3% (n = 5).Keywords
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