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ACKNOWLEDGMENTS

This investigation was supported by Biomedical Sciences Support Grant FR-7006 from the General Research Support Branch, Division of Research Resources, Bureau of Health Professions and Manpower Training, National Institutes of Health, and in part by U. S. Public Service Fellowship 1 F2 Al39965 from the National Institute of Allergy and Infectious Diseases.

REFERENCES

1. JACOBSEN, M., O’BRIEN, J. F., AND HEDGCOTH, C., Anal. Biochem. 25, 363 (1963). 2. SCHRECKER, A. W., JACOBSEN, D. W., AND KIRCHNER, J., Anal. Biochem. 26, 474

(1963). 3. MARMUR, J., AND DOTY, P., J. Mol. Biol. 5, 109 (1962). 4. CHARGAFF, E., LIPSHITZ, R., GREEN, C., AND HODES, M. E., J. Bid. Chem. 192,

223 (1951). 5. CHARGAFF, E., VISCHER, E., D~NIGIER, R., GREEN, C., AND MISANI, F., J. Biol. Chem.

177, 405 (1949). 6. DALY, M. M., ALLFREY, V. G., AND MIRSKY, A. E., J. Gen. Phys-iol. 33, 497 (1950).

G. H. BUTEAU, JR. J. E. SIMMONS

Department of Zoology University of Californti Berkeley, California 947.60

Received April 8, 870

Action of Amylolytic Enzymes on a

Chromogenic Substrate

The use of several colored derivatives of starch for the assay of cr- amylase has recently been described (1,2). The method of assay involves shaking the enzyme solution with a suspension of dyed substrate, followed by centrifugation and measurement of the color released into solution. One of these chromogenic substrates, Cibachron Blue F3GA-amylose (2) has been reported to be attacked by amyloglucosidase (glucoamylase, EC 3.2.1.3) and it has been suggested as a substrate for assay of this enzyme.

In this communication, evidence is presented showing that amyloglu- cosidase is not able to release soluble colored products from Cibachron Blue amylose although it cowl liberate a limited amount of glucose. The exe action #of the enzyme ceases when a glucose residue substituted with

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the chromophore is reached. If, however, amyloglucosidase contaminated with cu-amylase is used, the substituted glucose units are by-passed by the amylase with consequent release of colored material and a consider- ably larger amount of glucose.

Three samples of amyloglucosidase were tested: (a) three-times crystallized Rhizopus niveus amyloglucosidase (Miles Laboratories, Inc.), (b) Aspergillus niger amyloglucosidase prepared as by Fleming and Stone (3)) and Qureshi (4)) the second enzyme peak from a DEAE-cellulose fractionation being used, (c) a crude culture filtrate of Aspergillus nwamori (Glaxo Laboratories Ltd., Greenford, Middlesex, England). The Rhizopus enzyme had been shown previously (5) to be free from o(- amylase; the Aspergillus n.iger preparation is contaminated with this endo acting enzyme (5).

Cibachron Blue amylose was incubated with equal amounts of the three amyloglucosidases (6.2 International Units, measured by the release of glucose from soluble starch (4) ) . Samples were removed at intervals, and enzyme action stopped by addition to 0.5 M Tris, pH 10.25. After centrifugation, the supernatant solutions were analyzed for gluco’se and the optical densities read at 625 nm.

From a standard graph, prepared using an aqueous solution of Ciba- chron Blue dye (Ciba Chemical and Dye Co.), the amount of dye released into solution was determined (2). The results are shown in Figures 1 and 2. It can be seen that the Rhizopus enzyme releases glucose

DURATION OF INCUBATION [mn~

FIG. 1. Release of dye from Cibachron Blue amylose by M&opus niveu.s (e), Aspergillus niger (01, and Aspergillus awamori (0) amyloglucosidases. The digests contained substrate (200 mg), 0.1 M sodium acetate/acetic acid buffer, pH 4.5 (10.0 ml), and 62 International Units of amyloglucosidaze in a total volume of 12.0 ml. 1.0 ml samples were removed and added to 1.5 ml 0.5 M Tris, pH 1025. A control digest without enzyme was included to correct for nonenzymic release of dye. The digests were incubated in a. shaking water bath (SO strokes/min) at 40°C.

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30 -

20 -

,,,‘.A-

",./ o-o 10 - o/y-o-

Od &gS- .-.-.-.

I I I 100 200 300

DURATION OF INCUBATION (m”)

FIQ. 2. Release of glucose from Cibachron Blue amylose by M&opus niveus (@), Aspergillus niger (O), and Aspergilh awamori (0) amyloglucosidases. Conditions and compositions of the digests are given in the legend to Figure 1. After termination of enzyme action and centrifugation, 20 ~1 samples were analyzed for glucose by the glucose oxidase method of Fleming and Pegler (6) as modified by Catley (7).

from the substrate without any concomitant release of color into the supernatant solution. The rate of glucose release diminishes rapidly, be- cause of the exo action being arrested by substituted glucose residues. Both the Aspergillus preparations release a considerably larger amount of glucose, together with colored soluble products. This is attributed to the endo action of the contaminating cY-amylase.

The effect of amyloglucosidase on the cu-amylase assay procedure using chromogenic substrate (2) was then tested by allowing porcine pancre- atic cu-amylase to act on the substrate in the presence and absence of a

DURATION OF INCUBATION tmtnl

FIG. 3. Release of dye from Cibaehron Blue amylose by porcine pancreatic a- amylase (8) in the presence and absence of amyloglucosidase. The digests contained substrate (200 mg), 0.04 M phosphate buffer, pH 7.0, containing 0.02M sodium chloride (10.0 ml), and 0.05 International Units of a-amylase in a total volume of 12.0 ml, with (0) and without (0) OS International Unit of Rhizopus niveus amylo- glucosidase. 1.0 ml samples were removed, added to 6.0 ml 0.1 M phosphate buffer, pH 4.3, centrifuged and the optical densities read at 625 nm (2). A control digest was included to correct for nonensymic release of dye. Digest conditions as de- scribed in the legend to Figure 1.

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large excess of Rhizopus niveus amyloglucosidase. The amount of colored material released was the same in both cases (Fig. 3). The amount of reducing sugar determined by the Nelson-Somogyi procedure (10) was, as expected, considerably greater in the digest containing the amyloglu- cosidase (Fig. 4).

It is readily apparent that this substrate and the cu-amylase assay procedure described by Klein (2) provide a convenient method for the specific assay of cw-amylase in the presence of amyloglucosidase, and presumably other exo a,cting enzymes such as P-amylase. This is other- wise very difficult to do. Moreover it is clear that this substrate can be used for the detection of trace amounts of cY-amylase in other carbo- hydrase preparatilons. We recently developed a similar method based on blocking exo action by periodate oxidation of a small proportion of the anhydroglucose units in the polysaccharide chain (9).

DURATION OF INCUBATION (min)

FIG. 4. Release of reducing sugars from Cibachron Blue amylose by porcine pan- creatic a-amylase in the presence (0) and absence (0) of Rhizopus niveus amylo- glucosidase. Digest compositions are given in the legend to Figure 3; conditions were as in the legend to Figure 1. After termination of enzyme action and centrifugation, 0.5 ml samples were analyzed for reducing sugar by the Nelson-Somogyi pro- cedure ( 10).

ACKNOWLEDGMENTS

I wish to thank Dr. Bernard Klein for providing the Cibachron Blue amylose and the Ciba Chemical and Dye Company for Cibachron Blue F3GA dye. I am par- ticularly grateful to Dr. W. J. Whelan for encouragement and stimulating discussion. This work was supported by a grant from the National Science Foundation (NSF GB 8342).

REFEERENCES

1. RINDERKNECHT, H., WILDING, P., AND HAVERBACK, B. J., Experientia 23, 805 (1967).

2. KLEIN, B., FOREMAN, J. A., AND SEARCY, R. L., Anal. Biochem. 31, 412 (1969).

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3. FLJZMINQ, I. D., AND STONE, B. A., B&hem. J. 97, 13P (1965). 4. QURFSHI, M. Y., Ph.D. Thesis, University of London, 1967. 5. MARSHALL, J. J., AND WHELAN, W. J., FEBS Letters, 9, 85 (1970). 6. FLEMING, I. D., AND PEGLER, H. F., Analyst (London) 88, 967 (1963). 7. CATLEY, B. J., Ph.D. Thesis, University of London, 1967. 8. FISHER, E. H., AND STEIN, E. A., Arch. Sci. (Geneva) 7, 131 (1954). 9. SMITH, E. E., DFUJMMOND, G. S., MARSHALL, J. J., AND WHELAN, W. J., Federa-

tion Proc. 29, 930 (1970). MARSHALL, J. J., AND WHELAN, W. J., in preparation. 10. ROBYT, J. F., AND WHNLAN, W. J., in “Starch and Its Derivatives” (J. A. Radley,

ed.), p. 430. Chapman & Hall, London, 1968.

Department of Biochemistry University of Miami School of Medicine Miami, Florida 3316.8

Received June 6, 1970

J. J. MARSHALL