Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection.
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).
Shimura
K
K
Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan. shimurak@pharm.teikyo-u.ac.jp
Matsumoto
H
H
Kasai
K
K
D000595:Amino Acid Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D007700:Kinetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014357:Trypsin / Q000032:analysis* / Q000378:metabolism*
D005456:Fluorescent Dyes; D010455:Peptides; D014357:Trypsin