Gel diffusion assays for endo-beta-mannanase and pectin methylesterase can underestimate enzyme activity due to proteolytic degradation: a remedy.
The accuracy of the sensitive gel-diffusion assay for endo-beta-mannanase activity was improved when protein was added to fruit extracts or into the substrate-gel matrix in which the enzyme assays were conducted. Mixing of commercially available protease inhibitors with fruit enzyme extracts also resulted in increased assayable activity. These treatments were less effective when applied to extracts from tomato seeds, which contained over three times more endogenous protein than fruit extracts. Thus the presence of added or higher amounts of endogenous proteins served as the protectant for endo-beta-mannanase during the course of the gel-diffusion assay, which required an incubation at 32 degrees C for at least 18 h. There was no difference in assayable endo-beta-mannanase activity in the presence and absence of added protein when measured rapidly by viscometry. An effective modification was made to the galactomannan substrate gel assay for endo-beta-mannanase, which is the most efficient method for assaying large numbers of extracts, to improve its accuracy when the enzyme is obtained from tissues containing a low endogenous protein content. This involved incorporating an optimal concentration of gelatin into the galactomannan assay matrix gel. Much higher enzyme activities were recorded, with up to a 10-fold increase for tomato fruit extracts, compared to the same samples assayed on gels with no gelatin added. This increased activity was also obtained using extracts from the fruit of cantaloupe, peach, and nectarine. When incorporated into esterified pectin substrate gels, gelatin also increased the assayable activity of pectin methylesterase. Thus the incorporation of protein (gelatin) into substrate gels during the assay also should be widely more useful for other cell-wall-mobilizing enzymes and hydrolases.
Bourgault
Richard
R
Department of Botany, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Bewley
J Derek
JD
D001667:Binding, Competitive; D028463:Cucumis melo / Q000201:enzymology; D005638:Fruit / Q000201:enzymology; D005780:Gelatin / Q000378:metabolism; D005779:Immunodiffusion / Q000379:methods*; D018551:Solanum lycopersicum / Q000201:enzymology; D008361:Mannosidases / Q000032:analysis*; D010447:Peptide Hydrolases / Q000378:metabolism*; D053830:Plant Viral Movement Proteins; D027861:Prunus / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis*
D053830:Plant Viral Movement Proteins; D014764:Viral Proteins; D012710:Serum Albumin, Bovine; D005780:Gelatin; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase; D010447:Peptide Hydrolases; D012273:Ribulose-Bisphosphate Carboxylase