GEL-DIFFUSION METHOD FOR THE ASSAY OF Α-Amylase

Abstract

α‐Amylase can be readily estimated by allowing a solution of the enzyme to diffuse from a filter paper disc into an agar gel containing starch or β‐limit dextrin. The starch is degraded in the zones into which the enzyme penetrates so that, when the gel‐surface is flooded with a solution of iodine, circular clear zones are obtained against the starch‐ or dextrin‐iodine background colour. When other conditions are constant, the diameter of the clear zone is proportional to the logarithm of enzyme concentration.

A Method for Quantitating Amylase and its Use in the Investigation of Various Body Fluids

“…Very short incubation times (down to 30 minutes) are adequate for qualitative studies, but 18-24 hours is convenient for quantitative work. When incubation time is prolonged to several days, the standard curve remains linear (Briggs, 1962). The decrease in zone diameter with increase in PRA level in the plate is a phenomenon also noted by Briggs (1962) for plates containing limit dextrin as amylase substrate.…”

“…When incubation time is prolonged to several days, the standard curve remains linear (Briggs, 1962). The decrease in zone diameter with increase in PRA level in the plate is a phenomenon also noted by Briggs (1962) for plates containing limit dextrin as amylase substrate. The effect of all these variables on plate diffusion assays has been reviewed by MoUonen (1970).…”

“…Previous workers have established that 24 hours incubation at 37°C is adequate for the development of a diffusion plate (Carter and Sykes, 1961;Briggs, 1962;Ceska, 1971). It has been demonstrated that higher temperatures may cause zone shrinkage due to loss of water from the gel.…”

Screening for α-amylase in cereals. Improved gel-diffusion assay using a dye-labelled starch substrate

“…Radial diffusion into a substrate-containing gel slab is a rapid technique for the determination of enzymes, and several methods using either [~-limit dextrin or starch as substrate for the assay of a-amylase have been described (2,4,6). As [~-amylase supplements the action of a-amylase both in the degradation of [~-limit dextrin and starch, interference by [~-amylase must be eliminated by heat-treatment of the sample extract before analysis (2).…”

“…As [~-amylase supplements the action of a-amylase both in the degradation of [~-limit dextrin and starch, interference by [~-amylase must be eliminated by heat-treatment of the sample extract before analysis (2). Several studies in recent years have shown that colorimetric measurement of the degradation of a commerci-0105-1938/79/0044/0021/$ 01.00 ally available cross-linked dye-labelled starch substrate presents a specific and quantitative alternative to the direct and indirect a-amylase methods normally used in cereal research (1,3,5,11).…”