Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis |
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Authors: | Carmen Calinescu Rodolfo Federico Bruno Mondovi Mircea Alexandru Mateescu |
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Institution: | 1. Department of Chemistry and Centre BioMed, Université du Québec à Montréal, CP 8888, Succ. A, Montréal, QC, H3C 3P8, Canada 2. Department of Biology, 3rd University of Rome, 00146, Rome, Italy 3. Department of Biochemical Sciences “Rossi-Fanelli”, University of Rome “La Sapienza”, 00185, Rome, Italy
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Abstract: | A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior
at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish
peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product,
led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image
analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic
bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 °C), the DAO
from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin.
The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in
the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases.
After 10 h of incubation at 37 °C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin
and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in
the presence of pancreatin.
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