Assay of intestinal disaccharidases

ANALYTICAL

BIOCHEMISTRY

Assay

22, 99-107

of Intestinal ARNE

Research

Department

(1968)

Disaccharidases

DAHLQVIST

of the Hospital and Department of Physiological University of Lund. Lund, Sweden Received

May

Chemistry,

1, 1967

A method for the assay of intestinal disaccharidase activity was described by Dahlqvist (1). After incubation of the enzyme preparations with the appropriate substrate the reaction was interrupted by boiling or protein precipitation, and then the glucose liberated was assayed with a Tris-buffered glucose oxidase reagent. Tris was used in order to inhibit the disaccharidases which were present as contaminants in the glucose oxidase preparations obtained commercially. When more purified, essentially disaccharidase-free, glucose oxidase preparations became available, Messer and Dahlqvist (2) developed a one-step method in which the glucose oxidase reaction took place in the same reaction mixture as the disaccharide hydrolysis. The one-step reaction was well fitted to use with a small reaction volume, thus providing a sensitive ultramicro method for the assay of disaccharidase activities in peroral biopsy preparations of the small intestine mucosa. It had, however, certain disadvantages: (a) The simultaneous-coupled reaction had a lag-phase of lo-15 min before the linear part of the reaction began, and t’herefore a 15 min blank had to be used instead of a zero-time blank. (b) The malt.ase activity, measured with the one-step method, was 405% lower than when measured with the method previously described by Dahlqvist, (1). This was found to be caused by inhibition of the glucose oxidase activity by maltose (2). The effect was not seen with other substrates. (c) The sulfuric acid used to interrupt the reaction in the one-step method was found to give difficulties because it damages the emptying system of the spectrophotometer. Therefore, a simplified two-step method is now described that is suitable for use both on the “standard” scale, with a final volume of 3.2 ml. and on t#he ‘Lultramicro’J scale, with a final volume of 320 ~1. This modified method can thus replace both of the previous ones. 99

100

ARNE

DAHLQVIST

METHODS

Preparation

of Intestinal

Homogenate

(a) When a whole piece of intestine is available, as when using experimental animals or surgically removed intestine, the mucosa is scraped off with a piece of glass. Four parts of distilled water are added, and homogenization is performed with an Ultra-Turrax homogenizer. The mucosa and water are well chilled with crushed ice for at least 5 min before and during homogenization. Although the disaccharidases are present in particle-bound form in such homogenates, centrifugation can be performed at 20004000 g for 10 min in order to remove larger cell debris, with only small loss of disaccharidase activity. Methods for the solubilization of small intestine disaccharidases by digestion with proteolytic enzymes have been described (3, 4). (5) When only very small amounts of mucosa are available, as in the analysis of perorally obtained biopsy specimens of human small intestine mucosa, homogenization is performed in a glass pestle homogenizer of Potter and Elvehjem (5) or of a similar type. We use 10 ml conical test tubes with a glass pestle of the corresponding shape. For 10-20 mg of mucosa we use 0.5 ml of water. The tube with its contents (including the pestle) are chilled with crushed ice for at least 5 min prior to homogenization and then during the whole homogenization procedure. Homogenization is performed for l-2 min with a motor giving a speed of 200-300 rpm. Higher motor speed or insufficient chilling may result in loss of enzyme activity. When the homogenates have been prepared in this way they can not be centrifuged, even at a low speed, because this will result in considerable loss of enzyme activity. I have been quoted in the literature as recommending that homogenized intestine biopsies be centrifuged prior to homogenization (6, 7). This is the result of a misunderstanding. Substrates

Maltose, sucrose, trehalose, lactose, and cellobiose are obtained commercially. Isomaltose is prepared in the laboratory by enzymic hydrolysis of dextran (8)) which is then fractionated either on carbon-Celite columns or on Sephadex G-15 (8-10). Recently also isomaltose has become commercially available (Mann Research Laboratories, Inc., New York 10006, and Koch-Light Laboratories, Ltd., Colnbrook, Bucks., England), but I have no experience with these preparations. Commercial disaccharides even of the highest purity sometimes contain measurable amounts of free glucose. This is, for instance, the case with maltose, which regularly contains 0.5-l% glucose. The glucose can be

ASSAY

OF

INTESTINAL

DISACCHARIDASES

101

removed either by incubation with glucose oxidase and catalase followed by filtration through an ion exchanger (2) or by Sephadex G-15 chromatography (10). The later procedure has the advantage that also oligosaccharides, which are present in the commercial maltose preparations, will be removed. If there is too much glucose present in a disaccharide preparation, this will be revealed by a high blank reading in the disaccharidase assay procedure. Buffers Sodium Maleate Buffer (0.1 M, pH 6.0): Dissolve 1.16 gm maleic acid in 15.3 ml 1 N NaOH and dilute with water to 300 ml. Measure the pH and adjust, if necessary, to 6.0. Substrate-Buffer Solution: An 0.056 M solution of the appropriate disaccharide in 0.1 M sodium maleate buffer, pH 6.0. Substrate-buffer solutions are stored frozen in small aliquots. Glucose A.

STOCK

Oxidase Reagent

SOLUTIONS

(a) Tris buffer (0.5 M, pH 7.0) : Dissolve 61.0 gm Tris in 85 ml 5 N HCl and dilute with water to 1000 ml. Measure the pH and adjust, if necessary, to 7.0. (b) Peroxidase solution: Dissolve 10 mg peroxidase (grade D, Worthington Biochemical Co., Freehold, N. J.) in water to 10 ml. Store frozen in small aliquots. (c) Detergent solution: Dissolve 20 gm Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) in 80 gm 95% ethanol. (d) o-Dianisidine so&ion: Dissolve 100 mg o-dianisidine (technical, Eastman, Rochester, N. Y.) in ethanol to 10 ml. Store in dark. Discard when it becomes brown by oxidation. B.

TGO

REAGENT

Dissolve 2 mg glucose oxidase 130,000 (Fermco Laboratories, Chicago, Ill. 60680) in 100 ml 0.5 M Tris buffer. Add 1.0 ml o-dianisidine solution, 1.0 ml detergent solution, and 0.5 ml peroxidase solution. Mix well. This is stable for several days if stored in the refrigerator. Standard

Glucose Solutions

Prepare solutions of glucose in water containing (a) 100 pg glucose/ml, (b) 300 ,ug glucose/ml, and (c) 500 pg glucose/ml. Store frozen in small aliouots.

102

ARNE

DAHLQVIST

Assay of Disaccharidase

Activity

The enzyme preparation should be diluted to contain a suitable activity of the disaccharidase to be assayed. If there is formed 1 molecule of glucose per substrate molecule hydrolyzed, the diluted solution should contain somewhat less than 0.10 unit/ml; if 2 molecules of glucose are formed it should contain a little less than 0.05 unit/ml. When homogenates of peroral biopsy specimens are analyzed that have been prepared as described above suitable dilutions for the different activities usually are about the following: maltase 1:50; isomaltase 1:20; sucrase (invertase) 1: 10; trehalase 1: 5 ; lactase 1: 5 ; and cellobiase 1:2. The readings obtained with the incubated samples should not exceed the highest point of the standard curve (see below). If it does, the enzyme must be further diluted and a new incubation performed. The reaction is carried out in conical test tubes in which small amounts of enzyme preparation and substrate-buffer solution can be mixed and incubated adequately. When large amounts of enzyme are available, amounts of enzyme and reagents are used which will give a final reaction volume of 3.2 ml. The reaction then can be performed in ordinary conical 10 ml centrifuge tubes. When limited amounts of material are available, as in the analysis of peroral biopsy specimens, amounts are employed which give a final volume of 320 ~1. For this purpose special ultramicro test tubes are used (Fig. 1). Such tubes, made of Pyrex glass, can be obtained from Jobling & Co., Sunderland, England. 8mm.

FIG. 1. Test 320 ,ul).

tube

used

for ultramicro

assay

of disaccharidase

activity

(final

volume

ASSAY

Assay

OF

INTESTINAL

DISACCHARIDASES

with a Final

103

Volume of 52 ml

A test tube with 100 ,J diluted enzyme solution (dilution factor = d) is placed in a water bath at 37°C for a few minutes. The reaction is started by adding 100 ~1 substrate-buffer solution and mixing. After exactly 60 min, 3.0 ml TGO reagent is added. This will immediately interrupt the disaccharidase reaction. Mix well and let stand in the water bath at 37°C for further 60 min for development of the color. A blank is prepared by mixing in another test tube, in the order mentioned, 106 ~1 diluted enzyme solution, 3.0 ml TGO reagent, and 100 ~1 substrate-buffer solution. A reagent blank is prepared by mixing 200 ~1 distilled water and 3.0 ml TGO reagent. Let these tubes stand in the water bath at, 37°C for 60 min for development of the color. A glucose standard series is prepared by mixing in three test tubes 200 ~1 of standard solutions (a), (b), and (c), respectively, with 3.0 ml of TGO reagent.. These tubes will contain, in order, 20, 60, and 100 pg glucose. Incubate in the water bath at 37°C for 60 min for development of the color. After the development of color, measurement is made in a speatrophotometer at 420 rnp against the reagent blank. The calculation is performed in the following way: a = pg glucose liberated in 60 min (sample - blank). d = dilution factor for the enzyme solution of which 100 ~1 is used. n = number of glucose molecules per molecule of disaccharide (for maltose, isomaltose, trehalose, and cellobiose, n = 2; for sucrose, and lactose, n = 1). Units of disaccharidase activity: 1 unit. hydrolyzes 1 pmole disaccharide per minute. The disaccharidase activity per milliliter enzyme preparation is then calculated by the formula:

1o.a

_I. 1 (j

or

180 ’ 60 ’ n .

Disaccharidase

Activity

Assay

with

a Final Volume of S20 pl

All volumes in the procedure described above are reduced tenfold. The tubes of the standard series will contain 2, 6, and 10 pg glucose, respectively. A spectrophotometer with an ultramicro cuvet is used for the readings. The disaccharidase activity in units per milliliter enzyme preparation is calculated in the following way (for symbols, see above) or

a.d units/ml n . 108

104

ARNE

DAHLQVIST

Protein

Assay

*

When intestinal biopsy preparations are analyzed, the protein content of the homogenate is measured with a method of Lowry et al. (ll), and the disaccharidase activity is calculated as units per gram protein. The protein assay is performed with 50 ~1 homogenate and a final reaction volume of 6.5 ml. Citrate is used instead of tartrate, as recommended by Eggstein and Kreutz (12). A standard curve is prepared with freshly dissolved human serum albumen. Reading is performed in a Beckman B spectrophotometer. Attempts have been made to increase the sensitivity of the protein assay by a twentyfold reduction of all volumes, using the BeckmanSpinco ultramicrocolorimeter for the readings. For unknown reasons this did not, however, give a good proportionality for the mucosal protein, although a good standard curve was obtained with serum albumen. RESULTS

AND

Incubation

DISCUSSION

Conditions

The influence of pH and substrate concentration tivity have been discussed (1). Course

on disaccharidase

ac-

of the Reaction

The hydrolysis of disaccharides by intestinal enzymes follows zeroorder kinetics up to 10-15s of hydrolysis at 0.028M substrate concentration (Figs. 2-3). Under the conditions now described, the highest point on the standard curve corresponds to 10% hydrolysis if the substrate

0

20

40

60

MINUTES

FIG. 2. Course of reaction of intestinal homogenate (at two different concentrations) with sucrose. The homogenate has been incubated with sucrose for different times before addition of the TGO reagent, and then the color has been allowed to develop for 60 min.

ASSAY

OF

INTESTINAL

DILUTION

DISACCHARIDASES

OF

105

uOMOGENATE

FIQ. 3. Proportionality between amount of enzyme present and amount of glucose liberated in 60 min. Sucrose was used as substrate and a 1:5 homogenate of human mucosa containing 40 mg/ml of protein as enzyme. The final volume was 320 ~1. Reading was made in a Beckman-Spine0 spectrocolorimeter with a cuvet with a light pathway of 6 mm. Up to an increase of optical density of 1.100 (sample minus blank), which corresponds to 12 gg glucose liberated (12% hydrolysis of the substrate), the increase is proportional to the amount of homogenate present.

contains 1 molecule of glucose, and to 5% hydrolysis if it contains 2 glucose molecules. This is thus well within the limits of proportionality between amount of enzyme and amount of glucose liberated in the reaction. In the one-step method there was a lag phase of 10-15 min after the mixing of the reagents before the reaction started (2). A similar lag phase has been observed in assaying disaccharidase activity with another simultaneous-coupled enzymic assay system (hexokinase and glucose-6-phosphate dehydrogenase) (13). When disaccharide hydrolysis and glucose oxidase reaction are performed as two separate steps, as in the presently described method, no such lag phase is seen (Fig. 2).

Disaccharidase

Activity

ITnits

The disaccharidase unit is defined as the activity hydrolyzing 1 pmole of disaccharide per minute (14) under the conditions used, i.e., temperature 37”C, substrate concentration 0.028 M, pH 6.0. Disaccharidase activity units measured with the method previously described by Dahlqvist (1) can be compared with the activity measured with the method described here. This is also true for the one-step method of Messer and Dahlqvist (2), with the important exception that the maltasc activity

106

ARNE

DAHLQVIST

will be 40% lower when measured with the one-step method than with the two other methods. This is the case both for the maltase of crude mucosal homogenates and for each of the maltase peaks that can be isolated on chromatography of solubilized enzymes (15), which accords with the concept that t,he lower maltase activity measured with the onestep method is caused by interference between maltose and glucose oxidase, i.e., with the assay system, and not with the disaccharidase activity itself. Action

of Tris

Tris (13, 16) is a powerful inhibitor of the intestinal disaccharidase activity. The addition of the TGO reagent to the reaction mixture will instantaneously interrupt the disaccharide hydrolysis, and boiling or pro-

/

PHOSPHATE

m

maltose sucrose

MINUTES

FIG. 4. Intestinal homogenate and disaccharide substrates (maltose and sucrose) incubated in the presence of glucose oxidase-o-dianisidine reagent, prepared with 0.5M phosphate buffer and Tris buffer, respectively. The disaccharide hydrolysis is completely abolished in the presence of Tris.

tein precipitation after incubation of the disaccharidase with its substrate is unnecessary (Fig. 4). The color development of free glucose with glucose oxidase-o-dianisidine is not influenced by Tris (17). Other Possible Disaccharidase Inhibitors

Tris is only one of a large number of polyalcohols that inhibit disaccharidase activity (18). Glycerol was tested as an alternative substance to be used. Up to 10% glycerol added to the glucose oxidase reagent (prepared in phosphate buffer) did not interfere with the glucose oxidase reaction. It inhibited disaccharidase activity, but less efficiently than Tris. Glycerol can therefore not replace Tris in the preparation of the reagent.

ASSAY

OF INTESTINAL

DISACCHARIDASES

107

The addition of 10% glycerol to the Tris-buffered glucose oxidase reagent will increase the inhibitory effect on disaccharidases, and may be of value if it is desired to assay preparations containing very high disaccharidase activity, using short incubation times for the hydrolysis. When the reaction is performed as described here, addition of glycerol is meaningless. Action

of Detergent

The detergent (Triton X-100) will bot,h keep in solution the oxidized o-dianisidine, which has a very limited solubility in water without detergent, and exert a clearing action on the intestinal homogenate, thus reducing the blank value. SUMMARY

A simplified method for the assay of small intestine disaccharidase activity is described which is suitable for use both on the ‘lstandard” scale (final volume 3.2 ml) and on the “ultramicro” scale (final volume 320 ~1). ACKNOWLEDGMENTS This investigation has been supported by grants from the Swedish Medical Research Council (Grant 13X-157), Loo and Hans Osterman’s Foundation, A. Robbert’s Foundation, and Dir. A. PBhlsson’s Foundation. Miss M. B. Dahl, Mrs. A. Olson, and Miss B. Klintebgck contributed skillful technical assistance. REFERENCES A., Anal.

1. DAHLQvIST,

M.,

Biochem.

7, 18 (1964).

A., Anal. Biochem. 14, 376 (1966). A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. VIII, “Complex Carbohydrates” (E. F. Neufeld and V. Ginsburg, eds.). Academic Press, New York-London, 1966. 4. AURICCHIO, S., DAHLQVIST, A., AND SEMENZA, G., Biochim. Biophys. Acta 73, 582 2. MESSER, 3. DAHLQVIST,

AND

DAHLQVIST,

(1963).

5. 6. 7. 8.

V. R., AND ELVEHJEM, C. A., J. Biol. Chem. 114, 495 (1936). W. W., Gastroenterology 48, 299 (1965). H. B., WEBB, J., AND DAWSON, A. M., Brit. Med. J. 2, 1037 (1966). JEANES, A., WILHAM, C. A., JONES, R. W., TSUCHIYA, H. M., AND RIST, C. E., J. Am. Chem. Sot. 75, 5911 (1953). 9. DAHLQVIST, A., Acta Chem. &and. 14, 72 (1960). 10. DAHLQVIST, A., ANP MCMICHAEL, H. B., in preparation. 11. LOWRY, 0. H., ROSEBROUGH, N. J., F.~RR, A. L., .~ND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 12. EGGSTEIN, M., AND KREUTZ, F. H., Klin. Wochschr. 33, 879 (1955). 13. LARNER, J., AND MCNICKLE, C. M., J. Biol. Chem. 215, 723 (1955). 14. FREEMAN, M. E., Clin. Chim. Acta 6, 300 (1961). 15. DAHLQVIST, A., AND TELENIUS, U., in preparation. 16. DAHLQVIST, A., Acta Chem. &and. 12, 2012 (19.58). 17. DAHLQVIST, A., Biochem. J. 80, 547 (1961). 18. KELEMEN, N. V., AND WHELAN, W. J., Biochem. J. 100, 5P (1966). POTTER,

PETERNEL, MCMICHAEL,