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Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection.
Electrophoresis. 1998 Oct; 19(13):2296-300.E

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).

Authors+Show Affiliations

Shimura K
Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan. shimurak@pharm.teikyo-u.ac.jp
Matsumoto H
No affiliation info available
Kasai K
No affiliation info available

MeSH

Amino Acid SequenceElectrophoresis, CapillaryFluorescenceFluorescent DyesHydrogen-Ion ConcentrationIsoelectric FocusingIsoelectric PointKineticsPeptidesSensitivity and SpecificitySubstrate SpecificityTrypsin

Pub Type(s)

Journal Article

Language

eng

PubMed ID

9788312

Citation

Shimura, K, et al. "Assay of Trypsin Activity By Capillary Isoelectric Focusing With Laser-induced Fluorescence Detection." Electrophoresis, vol. 19, no. 13, 1998, pp. 2296-300.
Shimura K, Matsumoto H, Kasai K. Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection. Electrophoresis. 1998;19(13):2296-300.
Shimura, K., Matsumoto, H., & Kasai, K. (1998). Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection. Electrophoresis, 19(13), 2296-300.
Shimura K, Matsumoto H, Kasai K. Assay of Trypsin Activity By Capillary Isoelectric Focusing With Laser-induced Fluorescence Detection. Electrophoresis. 1998;19(13):2296-300. PubMed PMID: 9788312.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection. AU - Shimura,K, AU - Matsumoto,H, AU - Kasai,K, PY - 1998/10/27/pubmed PY - 1998/10/27/medline PY - 1998/10/27/entrez SP - 2296 EP - 300 JF - Electrophoresis JO - Electrophoresis VL - 19 IS - 13 N2 - 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). SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/9788312/Assay_of_trypsin_activity_by_capillary_isoelectric_focusing_with_laser_induced_fluorescence_detection_ L2 - https://doi.org/10.1002/elps.1150191308 DB - PRIME DP - Unbound Medicine ER -