A rapid method for monitoring the activity of certain carbohydrases

ANALYTICAL

BIOCHEMISTRY

67, 66 l-663 ( 1975)

SHORT

A Rapid

COMMUNICATIONS

Method

for Monitoring

of Certain

the Activity

Carbohydrases

A method for localizing activity peaks in eluates from chromatographic columns is described for hydrolases normally assayed by reducing-sugar determinations. The technique combines rapidity and small amounts of reagents. Results obtained with yeast exo-,B-( 1 + 3)-glucanase are shown.

The localization of activity peaks in fractions collected from chromatographic columns during the purification of enzymes is often very timeconsuming and costly. During a study of yeast exo-p-( 1 + 3)-glucanase, a technique was developed for achieving such localization rapidly and with very small amounts of reagents. The usual assay for this enzyme is to measure the release of reducing sugar (glucose) during the incubation of enzyme solutions in the presence of its substrate laminarin (I). The technique proposed here can be applied to most hydrolases measured by reducing-sugar determinations, for example amylases and many glycosidases. MATERIALS

AND

METHODS

Five-microliter aliquots of each fraction were applied with a 50+1 Hamilton microsyringe to form droplets on a sheet of powder paper (Eli Lilly, Indianapolis, IN; 14.5 X 11 cm) in an orderly pattern. Equal volumes of the substrate, in this case 1% laminarin (Nutritional Biochemicals, Cleveland, OH) in 0.05 M sodium succinate buffer, pH 5.5, were added to each drop, and the piece of paper was held for a suitable period of time at an appropriate temperature, e.g., 30 min at 30°C. The paper was then dried in an oven at 90°C. Visualization was initially done by a method for paper chromatography, using the alkaline silver nitrate reaction (2). However, varying concentrations of salts in fractions obtained from ionic strength gradient elutions caused interference with the action of the reagents. It was found that the following modifications gave clearer results: The paper is first immersed in 0.5 N sodium hydroxide in 95% ethanol (ca. 20 ml) for 1 min. with mild agitation, after which it is allowed to drip and dry in air. A short immersion of the paper in the silver nitrate solution in acetone (2) is followed by air drying and an additional immersion in the ethanolic 661 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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NaOH. The intensity of the reaction can be increased by repeating these steps one or more times. The dried paper is then fixed in Rapid Fixer (Eastman Kodak, Rochester, NY) and washed gently in water before a final rinse in acetone is applied. Acetone allows the paper to dry quickly if pressed between sheets of absorbent paper. When glucose was the reaction product, as in the example given, the dark background could be completely cleared by rinsing the paper in a 1.O% solution of aqueous ammonia before the fixation step. Application of this step in the visualization of the products of an endoglucanase resulted in the flaking off of the spots. RESULTS

Figure 1 shows an example of the localization of an activity peak in fractions collected from a Sephadex G- 100 column. The corresponding enzyme activity as determined by the conventional reducing-sugar method is indicated for two spots (1 unit = 1 pmole of glucose released/min at 30°C and pH 5.5, with a total substrate concentration of 0.5% in the reaction mixture). When incubation at 30°C is continued

FIG. 1. Localization of an exo-p-(1 + 3)-glucanase activity peak in 70 fractions collected from a Sephadex G-100 column. Fractions were spotted from left to right, top to bottom. Numerals indicate glucanase activity (unit/ml).

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until the drops have dried, the lower limit of sensitivity 0.00.5-0.010 unit/ml.

of this method is

DISCUSSION

This technique is obviously not intended to replace the traditional assays, since it does not give quantitative information such as asymmetry and other irregularities of the peaks representing activity. Moreover, it does not discriminate between products of enzyme activity and reducing sugars already present in the fractions containing the enzyme. Used within its range of applications, it can save much time, as well as glassware and reagents. The rapidity of the test is such that every fraction of a large eluate can be routinely assayed, eliminating the chance of missing small but potentially important secondary peaks of activity. Incubation of the drops containing the reaction mixtures in an enclosure with high relative humidity should allow indefinite prolongation of the incubation period and, consequently, this would increase the sensitivity sufficiently to detect very low concentrations of enzymes. ACKNOWLEDGMENT This work was made possible Research Council of Canada.

by a scholarship

awarded

to M.-A.

L. by the National

REFERENCES 1. Abd-El-Al, A. T. H.. and Phti, H. J. (I 968) Biochern. J. 109, 347. 2. Green. S. R., and Stone, I. (1953) Wailerstein Lab. Commun. 15, 347 MARC-ANDRE HERMAN Department of Food Science md Technology University of California Davis. California 95616 Receiized January 20, 1975: accepted March

IO, I975

LACHANCE

J.

PHAFF