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Use of fluorogenic substrates to visualize trypsin and chymotrypsin inhibitors after electrophoresis.
Electrophoresis. 1993 Mar; 14(3):220-6.E

Abstract

Fluorogenic substrates were tested as a means of increasing both the sensitivity and the selectivity of trypsin and chymotrypsin inhibitor detection after electrophoretic separation. Out of six substrates applied to cellulose acetate membranes, N alpha-benzyloxycarbonyl-L-arginine-4-methylcoumarinyl-7-amide (Z-Arg-MCA) and benzyloxycarbonyl-glycyl-glycyl-L-arginine-4-trifluoromethylcoumariny l-7-amide (Z-Gly-Gly-Arg-TFMCA) were found to be suitable for trypsin, and L-alanyl-L-alanyl-L-phenylalanine-4-methylcoumarinyl-7-amide (Ala-Ala-Phe-MCA) was suitable for chymotrypsin. A procedure to detect trypsin and chymotrypsin inhibitors, and to discriminate between them, was developed. After electrophoresis, slab gels were first incubated with the enzyme (bovine trypsin, bovine chymotrypsin, or human duodenal juice) at 37 degrees C, and then covered with the respective substrate membrane and incubated at room temperature while being observed under UV light. Dark blue inhibitor bands on a light-blue-fluorescent background were obtained with Z-Arg-MCA/trypsin and Ala-Ala-Phe-MCA/chymotrypsin, whereas Z-Gly-Gly-Arg-TFMCA/trypsin resulted in dark inhibitor bands on a fluorescent green background. The "inhibitor overlay membrane technique" (IOM technique) was used after polyacrylamide gel isoelectric focusing with carrier ampholytes and immobilized pH gradients, pore-gradient polyacrylamide gel electrophoresis, and sodium dodecyl sulfate pore-gradient polyacrylamide gel electrophoresis.

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Authors+Show Affiliations

Weder JK
Institut für Lebensmittelchemie, Technische Universität München, Garching, Germany.
Haussner K
No affiliation info available
Bokor MV
No affiliation info available

MeSH

Amino Acid SequenceAnimalsCattleChymotrypsinElectrophoresis, Polyacrylamide GelEvaluation Studies as TopicFluorescent DyesIsoelectric FocusingMolecular Sequence DataOligopeptidesSensitivity and SpecificitySubstrate SpecificityTrypsin Inhibitors

Pub Type(s)

Journal Article

Language

eng

PubMed ID

8486134

Citation

Weder, J K., et al. "Use of Fluorogenic Substrates to Visualize Trypsin and Chymotrypsin Inhibitors After Electrophoresis." Electrophoresis, vol. 14, no. 3, 1993, pp. 220-6.
Weder JK, Haussner K, Bokor MV. Use of fluorogenic substrates to visualize trypsin and chymotrypsin inhibitors after electrophoresis. Electrophoresis. 1993;14(3):220-6.
Weder, J. K., Haussner, K., & Bokor, M. V. (1993). Use of fluorogenic substrates to visualize trypsin and chymotrypsin inhibitors after electrophoresis. Electrophoresis, 14(3), 220-6.
Weder JK, Haussner K, Bokor MV. Use of Fluorogenic Substrates to Visualize Trypsin and Chymotrypsin Inhibitors After Electrophoresis. Electrophoresis. 1993;14(3):220-6. PubMed PMID: 8486134.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Use of fluorogenic substrates to visualize trypsin and chymotrypsin inhibitors after electrophoresis. AU - Weder,J K, AU - Haussner,K, AU - Bokor,M V, PY - 1993/3/1/pubmed PY - 1993/3/1/medline PY - 1993/3/1/entrez SP - 220 EP - 6 JF - Electrophoresis JO - Electrophoresis VL - 14 IS - 3 N2 - Fluorogenic substrates were tested as a means of increasing both the sensitivity and the selectivity of trypsin and chymotrypsin inhibitor detection after electrophoretic separation. Out of six substrates applied to cellulose acetate membranes, N alpha-benzyloxycarbonyl-L-arginine-4-methylcoumarinyl-7-amide (Z-Arg-MCA) and benzyloxycarbonyl-glycyl-glycyl-L-arginine-4-trifluoromethylcoumariny l-7-amide (Z-Gly-Gly-Arg-TFMCA) were found to be suitable for trypsin, and L-alanyl-L-alanyl-L-phenylalanine-4-methylcoumarinyl-7-amide (Ala-Ala-Phe-MCA) was suitable for chymotrypsin. A procedure to detect trypsin and chymotrypsin inhibitors, and to discriminate between them, was developed. After electrophoresis, slab gels were first incubated with the enzyme (bovine trypsin, bovine chymotrypsin, or human duodenal juice) at 37 degrees C, and then covered with the respective substrate membrane and incubated at room temperature while being observed under UV light. Dark blue inhibitor bands on a light-blue-fluorescent background were obtained with Z-Arg-MCA/trypsin and Ala-Ala-Phe-MCA/chymotrypsin, whereas Z-Gly-Gly-Arg-TFMCA/trypsin resulted in dark inhibitor bands on a fluorescent green background. The "inhibitor overlay membrane technique" (IOM technique) was used after polyacrylamide gel isoelectric focusing with carrier ampholytes and immobilized pH gradients, pore-gradient polyacrylamide gel electrophoresis, and sodium dodecyl sulfate pore-gradient polyacrylamide gel electrophoresis. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/8486134/Use_of_fluorogenic_substrates_to_visualize_trypsin_and_chymotrypsin_inhibitors_after_electrophoresis_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0173-0835&date=1993&volume=14&issue=3&spage=220 DB - PRIME DP - Unbound Medicine ER -