Use of fluorogenic substrates to visualize trypsin and chymotrypsin inhibitors after electrophoresis.
Electrophoresis
; 14(3): 220-6, 1993 Mar.
Article
em En
| MEDLINE
| ID: mdl-8486134
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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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Quimotripsina
/
Inibidores da Tripsina
/
Eletroforese em Gel de Poliacrilamida
/
Corantes Fluorescentes
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Limite:
Animals
Idioma:
En
Revista:
Electrophoresis
Ano de publicação:
1993
Tipo de documento:
Article