Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis

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.FigureFigure Zymographic assay of DAO with peroxidase included in polyacrylamide gels

Carboxymethyl starch/alginate microspheres containing diamine oxidase for intestinal targeting

“…In order to further identify the protein profiles, samples of free DAO and of DAO extracted from microspheres were run in SDS‐PAGE under nonreducing conditions for protein staining and for zymography. Previous studies indicated the presence of a single band at 72 kDa in denaturating SDS‐PAGE for vegetal DAO extracted from L. sativus and a band at 95 kDa for DAO extracted from Pisum sativum L . The molecular mass of pea seedling amine oxidase was also investigated using the method of gel filtration, indicating a value of 184.0 ± 2.6 kDa .…”

“…Our uncoated microspheres have been investigated in SGF and SIF in order to evaluate the physicochemical properties of the CaCMS/alginate complex. Previous studies showed that free DAO is sensitive to gastric acidity . It is also known that alginate microspheres (with porous structure) allow the diffusion of gastric acidity into microspheres inducing a rapid inactivation of enzyme.…”

“…Enzymatic detection of DAO by zymography was carried out on peroxidase entrapped in PAGE as described by Calinescu et al [40]. For the entrapment, 1 mL of horseradish peroxidase (1 mg/mL) was incorporated in the solution of polyacrylamide (final volume of 5 mL), just prior to trigger the polymerization.…”

“…a single band at 72 kDa in denaturating SDS-PAGE [40,43] for vegetal DAO extracted from L. sativus and a band at 95 kDa for DAO extracted from Pisum sativum L [44]. The molecular mass of pea seedling amine oxidase was also investigated using the method of gel filtration, indicating a value of 184.0 ± 2.6 kDa [45].…”

In vitro determination of diamine oxidase activity in food matrices by an enzymatic assay coupled to UHPLC-FL

“…In humans, DAO is mainly located in the intestines, placenta and kidneys [6,11]. Intestinal DAO acts as a protective barrier against exogenous histamine, especially of food origin [12][13][14]. A deficiency of DAO enzyme may thus lead to excess the normal plasmatic levels of histamine (0.3 -1.0 ng/mL) and the subsequent appearance of histamine intolerance symptoms [15,16].…”