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Detection of Endo-Acting Carbohydrases, Particularly

in the Presence of Exoenzymes Acting

on the Same Substrate

The detection of traces of endo-acting carbohydrases in the presence of excess of exoenzyme acting on the same substrate presents considerable difficulties. Examples are the detection of traces of a-amylase in prepa- rations of P-amylase or glucoamylase. We illustrate here how this can conveniently be done using partially oxidized substrates. The principle of the method is similar to that of a method first used by Smith and co- workers, who showed that complete periodate oxidation of laminarin (which is restricted to oxidation of the monosaccharide residues at chain ends) blocks the action of exo-/3-1,3-glucanase (1). A preliminary ac- count of this work has been published (2).

Materials and Methods. Potato amylose was purchased from Nutri- tional Biochemical Corporation. Sodium metaperiodate was from Baker. The Aspergillus niger and Rhizopus niveus glucoamylase preparations have been described elsewhere (3).

Glucoamylase activities were measured by the release of glucose from soluble starch at 37”. Glucose was determined by the glucose oxidase method of Lloyd and Whelan (4).

Experimental Procedures. Oxidation of amylose: Amylose (500 mg) was wetted with ethanol, then dissolved by addition of sodium hydroxide solution (1 N) , followed by gentle heating. The solution was adjusted to pH 6.0 by addition of acetic acid, then diluted to 40 ml. The calculated amount of sodium metaperiodate (0.033 gm) required to give an extent of oxidation of approximately 5% was dissolved in 10 ml water and slowly added to the amylose solution with stirring. The mixture was stored in the dark for 4 hr, then dialyzed against water. The oxidized amylose was isolated by freeze-drying. Determination of glucose liberated by enzymic hydrolysis: The glucose liberated from amylose and oxidized amylose by (a) A. niger glucoamylase and (b) R. niveus glucoamylase was deter- mined as follows. Polysaccharide (10 mg) , glucoamylase (1.06 units), and 100 mM acetate buffer, pH 5.0 (1.0 ml), were incubated at 37”C, together with polysaccharide-free and enzyme-free digests. Samples (10 ~1 from amylose digests, 100 ~1 from oxidized amylose digests) were removed at intervals for glucose determination.

Results. The results of hydrolysis of amylose and oxidized amylose by

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sa

cI 4a 0 B Y n =a s e 20

u

f 10

i0.a

q a

a E 6.a 1

3 4.a E P I 2.a

0

Duration of inoubstion Cmlnl

I 20 40 60 60 loo 120 140 160

Duration of incubation Cmin)

FIG. 1. Hydrolysis of amylose (a) and oxidized amylose (b) by Rhizopus niveus (0) and Aspergillus niger (0) glucoamylase preparations. Details are given under Experimental Procedures.

both glucoamylase preparations are shown in Fig. 1. While both enzyme preparations, as expected, released glucose from unoxidized amylose at approximately the same rate (Fig. la), there was a considerable difference in the release of glucose from the oxidized substrate (Fig. lb). An initial rapid release of glucose by the Rhizopus enzyme was observed, which diminished to a negligibly low rate after approximately 45% hydrolysis of the polysaccharide. This is interpreted as blockage to further hydrolysis by the oxidized glucose units. In contrast the rate of glucose release by the Aspergillus enzyme preparation, initially the same as by the Rhizopus rnzyme, did not appreciably reduce after 4% hydrolysis and was still taking place rapidly at a degree of hydrolysis at which the action of the

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latter enzyme had virtually ceased. Clearly the Aspergillus enzyme prepa- ration is capable of releasing glucose units between oxidation points, as well as those between the nonreducing end and first oxidation point in each chain.

Discussion. The oxidized amylose substrate we used is readily pre- pared. Its properties are worthy of note. In contrast to amylose itself the oxidized polysaccharide was readily soluble in cold water and showed no tendency to retrograde, even on storage in the cold. The modified solu- bility properties can be explained by the fact that introduction of oxi- dation points into the molecule results in the production of a “kinked” rather than a regular helix with consequent hindrance to intermolecular association. This situation may be compared with that of kappa- and iota-carageenans in which enzymic “dekinking” (removal of 6-sulfate groupings with formation of 3,6-anhydrogalactose or its a-sulfate) re- sults in formation of a regular helix. This modification results in increased double helix formation as shown by considerably different gelation prop- erties (5,6).

The oxidized product still stains dark blue with iodine. In view of its facile preparation it may also be useful as an alternative starch indicator to sodium starch glycolate (7).

We have shown that the introduction of a small number of oxidation points at random1 into the amylose molecule results in blockage to the action of crystalline glucoamylase after only a very small degree of hy- drolysis has taken place. Action of a highly purified preparation of glucoamylase from Aspergillus niger, prepared by a modification of the method of Fleming and Stone (11) , and previously considered to be free from a-amylase, has shown that the presence of oxidation points does not block the action of this enzyme. We can rationalize the observed dif- ferences in glucose production from the substrate by the two enzymes if the latter enzyme is contaminated by endoenzyme (cu-amylase) impurity. While the observed results could also be explained by attributing to the Aspergillus glucoamylase itself some endo action, as had been proposed in the case of an exo-/3-1,3-glucan hydrolase which can bypass branch points (12)) this would seem unlikely. We have also, subsequent to the work described here, inferred the presence of ar-amylase in the Aspergillus preparation by another method, based on the use of a chromogenic sub- strate (13). In addition, comparison of the extents of degradation of a series of starch and glycogen samples by the Aspergillus and Rhizopus

‘Drummond et al. (8) have shown by Smith degradation (9) of pullulan (10) oxidized in a similar manner that the oxidation takes place randomly. There is no reason to suspect t,hat the same is not true in the case of amylosp.

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glucoamylase preparations has been taken to indicate the presence of cu-amylase in the Aspergillus glucoamylase (3). While quantitative con- version to glucose was brought about by the Aspergillus preparation, the same extent of conversion was achieved by the Rhizopus enzyme only when wamylase was added (3).

We now routinely use the oxidized amylose substrate in this laboratory as a method to detect a-amylase in glucoamylase or a-glucosidase (14) preparations. It has been found most convenient to prepare an oxidized amylose limit dextrin by exhaustive treatment of the oxidized amylose with R. niveus glucoamylase, followed by inactivation of the enzyme and dialysis to remove glucose. Incubation of this limit dextrin with pure glucoamylase results in no further liberation of glucose; release of glucose indicates the presence of cr-amylase in the glucoamylase preparation. One of the advantages of the method is its sensitivity, the action of a trace of cr-amylase being magnified by the action of gluco- amylase. One cleavage by a-amylase is detected as a release of, on aver- age, about ten glucose molecules when the degree of oxidation is 5%.

In a similar way, the substrate can be used for testing the purity of ,8-amylase preparations. The presence of a-amylase allows maltose to be liberated, while none is liberated in its absence. Since, in this case, maltose rather than glucose is released, and the method depends on mea- surement of reducing power rather than specific determination of glucose, it is advisible to reduce the aldehyde groups in the oxidized substrate with sodium borohydride. These groups otherwise cause high blanks in re- ducing sugar measurements based on copper-reducing power (15).

Debranching enzymes such as pullulanase (16) and isoamylase (17,18) have been of immense use in studies on starch and glycogen structure, both in this laboratory and elsewhere. DefiniGve results can, however, be obtained only when these enzymes are free from even the most minute traces of a-amylase contamination. This has previously been tested for by iodine-staining measurements, looking for a decrease in the intensity of iodine stain of the debranched products, which would indicate the presence of cY-amylase. The use of oxidized amylose gives a more sensitive test for this impurity and can be carried out in a much shorter time. For this purpose it is advisable to prepare the oxidized amylose limit dextrin substrate from a sample of amylose which has been treated with pul- lulanase to remove any cy-1,6-glucosidic linkages that may be present (19,20). Since in this case the test for ol-amylase is in an enzyme prepa- ration which is not an exoenzyme, it is necessary to carry it out in the presence of added exoenzyme (R. niveus amyloglucosidase, for example) to obtain the magnification of a-amylase action which is one of the great

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advantages of the procedure. Presence of a-amylase in the debranching enzyme preparation is then observed as a release of glucose from the substrate.

While we have illustrated the use of periodate oxidation of the sub- strate to detect endo-acting impurities hydrolyzing cw-1,4-glucosidic linkages, this is clearly a general method, applicable to polysaccharides of both the (Y and /I series and the enzymes degrading them. Neither is it restricted to glucans or, for that matter, to polysaccharides. For example, using a suitable chemically modified substrate, the presence of endo- nuclease in an exonuclease preparation could be detected. Attempts to quantitate the method to measure the amount of endo activity in exo- enzyme preparations are in progress.

ACKNOWLEDGMENT

This work was eupported by a grant from the National Science Foundation (GB 8342).

REFERENCES

1. NELSON, T. E., SCALETTI, J. V., SMITH, F., AND KIRKWOOD, S., Can. J. Chem. 41, 1671 (1963).

2. SMITH, E. E., DRUMMOND, G. S., MARSHALL, J. J., AND WHELAN, W. J., Fed. Proc., Fed. A.mer. Sot. Ezp. Biol. 29, 930 (1970).

3. MARSHALL, J. J., AND WHELAN, W. J., FEBS Letters 9,85 (1970). 4. LLOYD, J. B., AND WHELAN, W. J., Anal. Biochem. 30,467 (1969). 5. LAWSON, C. J., AND REES, D. A., Nature (London) 227,392 (1970). 6. REES, D. A., Advan. Carbohydrate Chem. 24, 267 (1969). 7. PENT, S., BOURNE, E. J., AND THROWER, R. D., Nature (London) 159, 810 (1947).

8. DRUMMOND, G. S., SMITH, E. E., .~ND WHELAN, W. J.. FEBS Letters. in press (1971).

9. GOLDSTEIN, I. J., HAY, G. W.. LEWIS, B. A., AND SMITH, F.. in “Methods in Car- bohydrate Chemistry” (R. L. Whist,ler, ed.), Vol. V, 361. Academic Press. New York (1966).

10. BENDER, H., LEHMANN, J., AND WALLENFELS. K., Biochem. Biophys. Acta 36, 309 (1959).

11. FLEMING, I. D., AND STONE, B. A., Biochem. J. 97, 13P (1965). 12. NELSON, T. E., JOHNSON, J., JANTZEN, E., AND KIRKWOOD, S., J. Biol. Chem. 244,

5972 (1969).

13. MARSHALL, J. J., Anal. Biochem. 37, 466 (1970). 14. MARSHALL, J. J., AND TAYLOR, P. M., Biochem. Biophys. Res. Commun. 42, 173

(1971).

15. NELSON, N., J. Biol. Chem. 153, 375 (1944). SOMOGYI. M., J. Biol. Chem. 195, 19 (1952).

16. ABDULLAH, M.. CATLEY, B. J., LEE, E. Y. C.. ROBYT, J., WALLENFELS, K., AND WHELAN. W. J., Cereal Chem. 43, 111 (1966).

17. GUNJA-SMITH, Z., MARSHALL, J. J., SMITH. E. E., AND WHELAN, W. J., FEBS Letters 12, 96 (1970).

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18. GUNJA-SMITH, Z., MARSHALL, J. J., MERCIER, C.. SMITH, E. E.. AND WHELAN, W. J., FEBS Letters 12, 101 (1970).

19. KJ@LBERG, O., AND MANNERS, D. J., Biochem. J. 86,258 (1963). 20. BAXKS, W., AND GREENWOOD, C. T., Arch. Biochem. Biophys. 117, 674 (1966).

Depnrtment of Biochemistry University of Miami School of Medicine Minmi, Florida 33136

Received March .24,1971

J.J. MARSHALL W. J. WHELAN

Sensitive Polarographic Estimation of Thiol Groups with N-Ethylmaleimide

The reagent N-ethylmaleimide (NEM) has been used by a number of investigators in the study of thiols and thiol groups in proteins. The method commonly employed for following the interaction of NEM with these groups depends on the decrease in the NEM absorbance in the region of 300 nm accompanying its reaction with SH (13). However, the relatively low value of the NEM extinction coefficient renders this method somewhat insensitive, particularly when only small amounts of a protein are available.

We have found that NEM gives a well-defined polarographic reduction wave and that measurement of this can form the basis of a more sensitive procedure for estimating SH groups. Reference has previously been made (4) to unpublished work of Loening on the polarographic behavior of NEM, but no further details have been reported.

Materials and Methods. NEM, cysteine, and 5,5’-dithiobis- (Z-nitro- benzoic acid) were obtained from Sigma Chemical Company. Solutions of NEM were made up in water just prior to use. Insulin was a gift from Dr. J. Schlichtkrull (Novo, Copenhagen).

The dropping mercury electrode was used, together with the saturated calomel electrode, to which all potentials are referred. Polarographic measurements were made with a Radiometer PO4 recording polarograph using a reaction vessel and procedure as previously described (5).

Results and Discussion. Figure 1 shows the polarographic wave given by NEM, due to the reduction of the double bond, and Fig. 2 shows the linear relationship between wave height and NEM concentration. On pro-