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Plant Physiol. (1983) 71, 229-234 0032-0889/83/71/0229/06/$00.50/0

Specific Deteriination of a-Amylase Activity in Crude Plant Extracts Containing fl-Amylasel
Received for publication August 26, 1982 and in revised form October 4, 1982

DOUGLAS C. DOEHLERT2 AND STANLEY H. DUKE3

Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706
ABSTRACT
The specific measurementof a-amylase activity in crude plant extracts is difficult because of the presence of f8-amylases which directly interfere with most assay methods. Methods compared in this study include heat treatment at 70° C for 20 min, HgCl2 treatment, and the use of the aamylase specific substrate starch azure. In comparing alfalfa (Medicago sativa L.), soybeans (Glyjine max IL.l Merr.), and malted barley(Hodum vsdgare L.), the starch azure assay was the only satisfactory method for all tissues. While famylase can Uberate no color alone, over 10 International units per mililiter f8-amylase activity has a stimulatory effect on the rate of color release. This stimulation becomes constant (about 4-fold) at fiamylase activities over 1,000 International units per millilter. Two starch azure procedures weredeveloped to eliminate .8-amylase interference: (a) the dilution procedure, the serial dilution of samples until -amylase levels are below levels that interfere; (b) the ,B-amylase saturation procedure, addition of exogenous f8-amylase to increase endogenous f8-amylase activity to saturating levels. Both procedures yield linear calibrations up to 0.3 International units per milliliter. These twoprocedures produced statisticaliy identical results with most tissues, but not for ali tissues. Differences between the two methods with some plant tissues was attributed to inaccuracy with the dilution procedure in tissues high in ,-amylase activity or inhibitory effects of the commercial fi-amylase. The f8-amylase saturation procedure was found to be preferable with most species. The heattreatment was satisfactory only for malted barley, as a-amylases in alfalfa and soybeans are heat labile. Whereas HgCI2 proved to be a potent inhibitor of f8-amylase activity at concentrations of 10 to 100 micromolar, these concentrations also partially inhibited a-amylase in barley malt. The reported a-amylase activities in crude enzyme extracts from a number of plant species are apparently the firstspecific measurements reported for any plant tissues other than germinating cereals.

The specific determination of a-amylase activity in crude plant extracts is difficult because of the presence of 8-amylase activity in these tissues that directly interferes with most assay methods. The most commonly used procedure involves the selective inactivation of ,B-amylase by heating. This procedure isdescribed by Briggs (4) and is based on the original observations of Schwarzer (23) which were elaborated on by Ohlsson (16) and Olson et aL (19). The procedure was developed for the determination of aamylase activity in barley malt. f8-Amylases are selectively inac' Supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison. 2Present address: United StatesDepartment of Agriculture/Agricultural Research Service, Plant Science Research, 3127 Ligon Road, North Carolina State University, Raleigh, NC 27650. 3 To whom reprint requests should be addressed.
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assayed by reducing power production or starch-iodine color disappearance. Whereas this procedure is used routinely in the malting industry, it has also been applied to tissues and plant species wherethe heat stabilities of the constitutive a- and ,Bamylases are unknown (1, 5, 12, 24). In fact, several investigators have shown that in stveral plant species, a-amylases are heat labile under these conditions (1, 17, 18). Another procedure involves the putative selective inactivation of fi-amylases by the addition of low concentrations of HgC12, (10-12). This method is clearly dependent on the...