Specific Determination of α-Amylase Activity in Crude Plant Extracts Containing β-Amylase

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Abstract
The specific measurement of .alpha.-amylase activity in crude plant extracts is difficult because of the presence of .beta.-amylases which directly interfere with most assay methods. Methods compared in this study include head treatment at 70.degree. C for 20 min, HgCl2 treatment and the use of the .alpha.-amylase specific substrate starch azure. In comparing alfalfa (Medicago sativa L.), soybeans (Glycine max [L.] Merr.) and malted barley (Hordeum vulgare L.), the starch azure assay was the only satisfactory method for all tissues. While .beta.-amylase can liberate no color alone, over 10 international units [IU]/ml .beta.-amylase activity has a stimulatory effect on the rate of color release. This stimulation becomes constant (.apprx. 4-fold) at .beta.-amylase activities > 10,000 IU/ml. Two starch azure procedures were developed to eliminate .beta.-amylase interference: the dilution procedure, the serial dilution of samples until .beta.-amylase levels are below levels that interfere; and the .beta.-amylase saturation procedure, addition of exogenous .beta.-amylase to increase endogenous .beta.-amylase activity to saturating levels. Both procedures yield linear calibrations up to 0.3 IU/ml. These 2 procedures produced statistically identical results with most tissues, but not for all tissues. Differences between the 2 methods with some plant tissues was attributed to inaccuracy with the dilution procedure in tissues high in .beta.-amylase activity or inhibitory effects of the commercial .beta.-amylase. The .beta.-amylase saturation procedure was preferable with most species. The heat treatment was satisfactory only for malted barley, as .alpha.-amylases in alfalfa and soybeans are heat labile. Whereas HgCl2 proved to be a potent inhibitor of .beta.-amylase activity at concentrations of 10-100 .mu.m, these concentrations also partially inhibited .alpha.-amylase in barley malt. The reported .alpha.-amylase activities in crude enzyme extracts from a number of plant species are apparently the 1st specific measurements reported for any plant tissues other than germinating cereals.