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Method for assay of intestinal disaccharidases
ASALYTICAL
BIOCHEMISTRY
Method
for
7, 18-25 (1964)
Assay
of
ARNE From
the
Department
Intestinal DAHLQVIST
of Physiological Lund,
Disaccharidases
Chemistry, Sweden
Received March
Uni,versity
of Lund,
29, 1963
INTRODUCTION
The specific calorimetric assay of glucose with glucose oxidase, described by Huggett and Nixon (l), has been found useful in the assay of disaccharidase activities, since most naturally occurring disaccharides contain at least one molecule of glucose which is liberated on hydrolysis. The method is especially advantageous for the determination of the hydrolysis of reducing disaccharides, since the previously existing chemical methods, based on increase in reducing power, are not satisfactory with these substrates. With certain substrates, however, as maltose and isomaltose, the use of glucose oxidase reagents for measuring the amount of glucose liberated initially was prohibited by contaminant enzymes in the glucose oxidase preparations, which hydrolyzed these disaccharides (2). Attempts to purify the glucose oxidase from these contaminant disaccharidases were not successful (3). However, it was found, and indcpendently described from three different laboratories, that tris (hydroxymethyl) aminomethane (Tris) effectively inhibits the contaminant disaccharidases without interfering with the glucose oxidase reaction (3-5). By the addition of Tris buffer to the glucose oxidase reagent, therefore, a reagent can be prepared that can be used for the assay of all disaccharidases which liberate glucose from their substrate. In the present paper a procedure is described for the assay of the disaccharidase activities of intestinal mucosal preparations using a Trisglucose oxidase (TGO) reagent. The incubation conditions to be used for different intestinal disaccharidases are discussed, and a unit for the disaccharidase activity is defined that agrees with the recommendations for enzyme activity units recently made by the Joint Sub-Commission on Clinical Enzyme Units of the International Unions of Biochemistry and of Pure and Applied Chemistry. 18
ASSAY
OF
INTESTINAL
MATERIALS
DISACCHARIDES
AND
19
METHODS
Sugars
Maltose (4- (a-D-glucopyranosyl) -D-glucose) monohydrate, sz~crose (2(a-D-ghrcopyranosyl) -P-D-fructofuranoside) , kzctose (4 @-D-galactopyranosyl) -D-glucose) monohydrat.e, trehalose (l- (a-D-glucopyranosyl) cu-D-glucopyranoside) dihydrate, cellobiose (4- (P-D-glucopyranosyl) -Dglucose), gentiobiose (6- (/3-D-glucopyranosyl) -D-glucose) , and turanose (3- (a-D-glucopyranosyl) -D-fructose) are commercially available. Iso~~zaltose (6- (a-D-glucopyranosyl) -D-glucose) has been prepared in the laboratory (6). Palatinose (6- (a-D-glucopyranosyl) -D-fructose) monohydrate has been generously supplied by Professor R. Weidenhagen (Neuoffstein, Germany), who has described the ensymic synthesis and isolation of this disaccharide (7). Glucose oxidase, crude, was obtained from Sigma Chemical Co. (St. Louis, MO.), Worthington Biochemical Co., (Freehold, N. J.), and Nutritional Biochemicals Co. (Cleveland, Ohio). These preparations had very similar properties. More purified preparations sold by these and other companies have higher glucose oxidasc activit’y per milligram, and thus a smaller amount than t,hat recommended below can be used for the preparation of the reagent. (The amount needed of some of these preparations can be calculated from Table 1 in reference No. 3.) Horse-radish peroxidase, grade D was obtained from Worthington Biochemical Co. Other Reagents o-Dianisidine (3,3’-dimethoxybenzidine), technical was obtained from Eastman (Rochester, N. Y.), triton X-100 from Rohm & Haas Co. (Philadelphia, Pa.), and Tris (tris(hydroxymethy1) aminomet,hane j , buffe? grade from Sigma Chemical Co. Homogenates
of Intestinal
Mucosa
Pieces of human small intestine were removed during surgical operations and chilled with ice during transport to the laboratory. Pig intestine, obtained from the slaughterhouse, was removed immediately after slaughter and chilled with ice during transport.. The intestine then was cut open; t,he mucosa was scraped off with a piece of glass and homogenized in an Ultra-Turrax homogenizer with four parts (v/w) of cold buffer, saline, or distilled water. The tube was chilled with crushed ice during homogenization. Nuclei and larger cell debris were removed by
20
ARNE
centrifugation for 10 min.
in an ordinary Method
DAHLQVIST
laboratory
for Assay
centrifuge
of Disaccharidase
at 2000 to 4000 rpm Activity
A. Reagents. (1) Substrate Solution: A 0.056M solution of the appropriate disaccharide in O.lM maleate buffer (for the preparation of maleate buffer, see Table 1) of optimum pH. If the optimum pH for the
Dissolve with water
PREPARATION 1.16 gm of maleic acid to 100 ml.
TABLE 1 OF O.lM MALEATE BUFFER in the amount of 1 N NaOH given
PH
18 NaOH,ml
PH
5.0 5.2 5.4 5.6 5.8
10.9 11.4 12.2 13.2 14.2
6.0 6.2 6.4 6.6 6.8 7.0
below,
and dilute
liVNaOH,ml 15.3 16.4 17.4 18.2 18.8 19.1
activity under investigation is below 5.0 or over 7.0, other kinds of buffer must be used. Toluene, about 1 ml/100 ml substrate solution, is added as a preservative. In calculating the amount of disaccharide, it should be observed that different disaccharides have different amounts of water of hydration. (2) TGO Reagent: For the preparation of the TGO (Tris-glucose oxidase) reagent, the following stock solutions are used: (a) Tris bufer, a solution of 61.0 gm (0.5 mole) of Tris in 85 ml of 5 N HCl, made up to 1000 ml with distilled water. The pH of the solution should be measured with a glass electrode and if necessary adjusted to 7. (b) Peroxidase solution, a solution of peroxidase, 1 mg/ml, in dist,illed water. This solution, when kept at -2O”C, is stable for at least several months. (c) o-Dianisidine solution, a solution of o-dianisidine, 10 mg/ml, in 95% ethanol. This solution should be kept in the dark. After a time it will become dark brown by oxidation, and then should be discarded. (d) Detergent solution, a solut,ion of 10 ml of Triton X-100 in 40 ml of 95% ethanol. Preparation of TGO Reagent: In a loo-ml volumetric flask, place 125 mg of glucose oxidase and about 50 ml of the Tris buffer. Shake for l/s min. Add 0.5 ml of peroxidase solution, 0.5 ml of o-dianisidine solution, and 1.0 ml of detergent solution. Make up with Tris buffer to 100 ml, mix, and filter. The reagent is stable for several days in the refrigerator. [Alternatively, the TGO reagent can be prepared from the Glucostat
hSShY
OF
INTESTIiVAL
21
DISACCHARIDES
glucose reagent (Worthington Biochemical Co.) in the following way: Dissolve the contents of the chromogen vial in 1 ml of the detergent solution and 2-3 ml of Tris buffer. Shake until clear. Dissolve the contents of the Glucostat vial in another 4-5 ml of the Tris buffer. Mix the two solutions and dilute with Tris buffer to 100 ml.] (8) Standard Glucose Solution: Dissolve 100 mg of glucose and 2.7g of benzoic acid in distilled water and make up to 1000 ml. The solution is stable for months at room temperature. B. Performance. Dilute an aliquot of the sample to be analyzed so that the solution contains a suitable activity of the disaccharidase to be assayed. If one molecule of glucose is formed per substrate molecule hydrolyzed, the diluted solution should contain a little less than 0.10 units/ml; if two molecules of glucose are formed it should contain somewhat less than 0.05 units/ml. (For definitions of the units, see below.) Then mix in a small test tube (7 X 120 mm) 0.1 ml of the diluted enzyme solution with 0.1 ml of the appropriate substrate solution, and immerse the tube in a constant-temperature water bath at 37°C. Add a small drop of toluene to each tube as a preservative. After 60 min of incubation, add 0.8 ml of distilled water, and then immediately interrupt the enzymic reaction by the immersion of t,he tube in boiling water for 2 min; then cool the tube with tap water. Prepare a blank with the same composition and immerse it in boiling water immediately after mixing of the enzyme and substrate. For the determination of the glucose, transfer 0.5 ml of the solution to another test tube (16 X 150 mm) and mix with 3.0 ml of the TGO reagent. Incubate the tube at 37°C for 1 hr for development of the color. At the same time incubate a standard series, containing 0.0, 0.1, 0.3, and 0.5 ml of the standard glucose solution (these tubes contain 0, 10, 30, and 50 pg, respect,ively, of glucose), distilled water to the combined volume of 0.5 ml, and 3.0 ml of the TGO reagent. After the color has been developed, measure it in spectrophotometer at 420 mp, using l-cm cuvettes, against the tube in the standard series which does not contain sugar. The color is stable for at least an hour. If the amount of glucose present in the sample of the incubated reaction mixture exceeds 50 pg, the whole assay procedure should be repeated with a more dilute sample of the enzyme solution. C. Calculation. The disaccharidase activity of the preparation analyzed is obtained by the following formula: Disaccharidase where a = amount
activity
(units/ml)
=
(a - b) - d n 540
of glucose (pg) found in aliquot
of the incubated
22
ARNE
DAHLQVIST
sample, b = amount of glucose (pg) found in aliquot of the corresponding blank, d = dilution factor of the enzyme solution used for mixing with the substrate, and n = number of glucose molecules that are liberated per substrate (disaccharide) molecule hydrolyzed. For maltose, isomaltose, trehalose, cellobiose, gentiobiose, and other disaccharides composed of two glucose molecules n = 2. For sucrose, lactose, turanose, and other disaccharides which contain one molecule of glucose and one of fructose or galactose n = 1. Units of activity
= pmoles disaccharide
RESULTS
AND
hydrolyzed/mm
DISCUSSIOK
(1) Incubation
Conditions
A. @. In most cases the intestinal disaccharidases have their pH optimum between 5.5 and 6.5. Enzymes with lower pH optimum have been found only in two instances: one malt,ase present in the human duodenum that had its optimum at about pH 3 (8) ; and a soluble lactase fraction in homogenates of the intestinal mucosa of small rats that had its optimum at pH 2 or lower (9). In the pig intestinal mucosa, two maltases with slightly higher pH optimum have been found (10) ; both had a rather broad optimum between pH 6.5 and 7.5.
‘0 0 4
0-O
Maltaso
0-G
lnvcrtasc I
I
I
5
6
7
I e
b 9
PH FIQ. jejunal 0.05 M 0.025 M
1. pH-activity curves for invertase and maltase activities of a homogenate 0.05 M sodium acetate, pH mucosa from an adult human. Buffers: sodium maleate, pH 5.M.5; 0.05 M sodium phosphate, pH 7-7.5; sodium Veronal, pH S-9.
of 4-5; and
ASSAY
OF
INTE23TINAL
DISACCHAHIDES
23
The maltase and invertase activities of crude pig intestinal mucosal homogenates have their optima at pH 6.5, and the lactase at 6.0 (2). The human intestinal disaccharidases have somewhat lower pH optima ; the maltase and invertase act,ivities of crude homogenates of human small intestinal mucosa have their optimum at, pH 6.0 (Fig. l), and the lactase activity at pH 5.6. For the preparation of buffers with pH around 6, maleic acid is very suitable, since its second dissociation step has a PI<, of 5.95. B. Substrate Concentration. Cert.ain of the intestinal disaccharidases show a marked “substrate inhibition” in too concentrated substrate solutions, partly as the result of transglycosylation reactions. In a study of the influence of substrate concentration on pig intestinal disaccharidases, 0.028M substrate was found to give negligible %ubstrate inhibition,” but over 50% saturat,ion of the disaccharidases with substrate (2). (2) Course of the Reaction The hydrolysis of disaccharides by intestinal enzymes has been found to follow zero-order kinetics up to 10-1570 of hydrolysis at 0.028 M substrate concentration. During this initial phase of the reaction the amount of hydrolysis products formed during a certain period of time is proportional to the amount. of enzyme present. (3) Specificity of TGO Reagent So far, all disaccharides which have been tested produce no color, or very weak color, with the TGO reagent in the disaccharidase assay procedure described here. The disaccharides which have been tested are: maltose, sucrose, isomaltose, palatinosr (isomaltulose), turanose! lactose, cellobiose, gentiobiose, and trehalosc. Both a-glucose and P-glucose react rapidly with the TGO reagent (3), but galactose and fructose do not produce a color. This has to be taken into account when calculating the degree of hydrolysis of a disaccharide, since disaccharides which are composed of two glucose molecules give twice the amount of color per molecule of disaccharide split as those which are composed of one molecule of glucose and one of fructose or galactose. Phenol interferes with the production of color by the TGO reagent, which therefore cannot be used for the assay of phenylglucosidasc activity. For this purpose the Folin-Ciocalteu phenol reagent can be used instead (2). (4) Purpose of Detergellt The colored oxidation product of o-dianisidine has a low solubility in water. To some extent, therefore, it crystallizes on the incubation of
24
ARNE
DAHLQVIST
Hugget and Nixon’s (1) reagent with glucose, yielding opalescent solutions which give somewhat inconsistent readings in the spectrophotometer. Added detergent keeps the colored substance in a clear solution. Several detergents tested yielded a brown color with the TGO reagent due to contaminant peroxides, but Triton X-100, recommended by Sols and de la Fuente (4), is free of peroxides. Addition of this detergent does not interfere with the cnzymic reactions. (5) Influence
of Protein
on Glucose Determination
Proteins do not interfere with the reaction of glucose with the TGO reagent, and therefore protein precipitation is usually not necessary. In some cases, however, where the disaccharidase activity is low, and subsequently concentrated mucosal preparations are used, the protein present may cause a considerable turbidity, especial!y after the disaccharidase activity has been interrupted by boiling before determination of the amount of glucose liberated, This will interfere with the spectrophotometric reading. In these cases protein precipitation can be performed with Somogyi’s (11) zinc-barium-reagents before the mixing of the solution with the TGO reagent, or, alternatively, the TGO reaction mixture can be centrifuged clear before the reading is made. (6) Definition
of Unit of Disaccharidase
Activity
In accord with agreements recently made for the definition of clinical enzyme activity units (12), the activity hydrolyzing 1 pmole of disaccharide per minute is taken as the unit. In several previous publications the activity liberating 1 mg of monosaccharides during 60 min has been used as the unit (2, 3, 13, 14). One unit as now defined (,moles/‘min) equals 21.6 units of those previously used (mg/60 min). (7) Disaccharidase
Activity
of Human
Small
Intestinal
Mucosa
Assay of disaccharidase activities in homogenates of human small intestinal mucosa, obtained from pieces of apparently normal jejunum and ileum, removed from adults during surgical operations, have shown the following range of activity, expressed as units (pmoles substrate split per minute) per gram (wet weight) of mucosa: maltase, 13-54; invertase, 6-17; isomaltase, 4-13; palatinase (isomaltulase) 1.54; turanase, l.O3.4; lact.ase, 0.2-19; cellobiase, 0.03-3.6; gentiobiase, 0.01-0.11; and trehalase, l-10. Some of the substrates are hydrolyzed by a mixture of several enzymes (14).
ASSAY
OF
INTESTINAL
DISACCHARIDES
25
SUMMARY
1. A procedure is described for assay of disaccharidase activities in extracts of intestinal mucosa, using a Tris-glucose oxidase reagent for assay of the glucose liberated from the substrate The incubation conditions are discussed. 2. A unit for disaccharidase activity is defined that is in accord with recommendations made by the Joint Sub-Commission on Clinical Enzyme Units of the International Unions of Biochemistry and of Pure and Applied Chemistry. ACKNOWLEDGMENTS Grants gratefully Karstensson
that have been obtained from the Swedish acknowledged. Skillful technical assistance and Miss Karin Iilang.
Medical Research Council are has been given by Mrs. Alice
REFERENCES 1. HUGGETT, A. ST. G., AND NIXON, D. A., Lawet ii, 368 (1957). 2. DAHLQVIST, A., Acta Chem. Sand. 14, 1797 (1960). 3. DAHLQVIST, A., Biochem. J. SO, 547 (1961). 4. SOLS, A., AND DE ~.4 FUENTE, G., in “Methods in Biochemical Research” tel, ed.), p. 302. Year Book Publishers, Chicago, 1961. 5. WHITE, J. W., JR., AND SUBERS, M. H., Anal. Biochem. 2, 380 (1961). 6. DAHLQ~IST, A., Acta Chem. Stand. 14, 72 (1960). 7. WEIDENHAGGN, R., AND LORENZ, S., Angelo. Chew 69, 641 (1957). 8. AURICCHIO, S., personal communication. 9. DOELL, R. G., AND KRETCHMER, N., Biochim. Biophys. Acta 62, 353 10. DAHLQVIST, A., Acta Chem. &and. 14, 1 (1960). 11. SOMOGYI, M., J. Biol. Chem. 160, 69 (1945). 12. FREEMAN, M. E., CIzn. Chim. Acta 6, 300 (1961). 13. DAHLQVIST, A., AND BORGSTR~~M, B., Biochem. J. 81,411 (1961). 14. DAHLQVIST, A., J. Clin. Invest. 41, 463 (1962).
(J. Quas-
(1962).
BIOCHEMISTRY
Method
for
7, 18-25 (1964)
Assay
of
ARNE From
the
Department
Intestinal DAHLQVIST
of Physiological Lund,
Disaccharidases
Chemistry, Sweden
Received March
Uni,versity
of Lund,
29, 1963
INTRODUCTION
The specific calorimetric assay of glucose with glucose oxidase, described by Huggett and Nixon (l), has been found useful in the assay of disaccharidase activities, since most naturally occurring disaccharides contain at least one molecule of glucose which is liberated on hydrolysis. The method is especially advantageous for the determination of the hydrolysis of reducing disaccharides, since the previously existing chemical methods, based on increase in reducing power, are not satisfactory with these substrates. With certain substrates, however, as maltose and isomaltose, the use of glucose oxidase reagents for measuring the amount of glucose liberated initially was prohibited by contaminant enzymes in the glucose oxidase preparations, which hydrolyzed these disaccharides (2). Attempts to purify the glucose oxidase from these contaminant disaccharidases were not successful (3). However, it was found, and indcpendently described from three different laboratories, that tris (hydroxymethyl) aminomethane (Tris) effectively inhibits the contaminant disaccharidases without interfering with the glucose oxidase reaction (3-5). By the addition of Tris buffer to the glucose oxidase reagent, therefore, a reagent can be prepared that can be used for the assay of all disaccharidases which liberate glucose from their substrate. In the present paper a procedure is described for the assay of the disaccharidase activities of intestinal mucosal preparations using a Trisglucose oxidase (TGO) reagent. The incubation conditions to be used for different intestinal disaccharidases are discussed, and a unit for the disaccharidase activity is defined that agrees with the recommendations for enzyme activity units recently made by the Joint Sub-Commission on Clinical Enzyme Units of the International Unions of Biochemistry and of Pure and Applied Chemistry. 18
ASSAY
OF
INTESTINAL
MATERIALS
DISACCHARIDES
AND
19
METHODS
Sugars
Maltose (4- (a-D-glucopyranosyl) -D-glucose) monohydrate, sz~crose (2(a-D-ghrcopyranosyl) -P-D-fructofuranoside) , kzctose (4 @-D-galactopyranosyl) -D-glucose) monohydrat.e, trehalose (l- (a-D-glucopyranosyl) cu-D-glucopyranoside) dihydrate, cellobiose (4- (P-D-glucopyranosyl) -Dglucose), gentiobiose (6- (/3-D-glucopyranosyl) -D-glucose) , and turanose (3- (a-D-glucopyranosyl) -D-fructose) are commercially available. Iso~~zaltose (6- (a-D-glucopyranosyl) -D-glucose) has been prepared in the laboratory (6). Palatinose (6- (a-D-glucopyranosyl) -D-fructose) monohydrate has been generously supplied by Professor R. Weidenhagen (Neuoffstein, Germany), who has described the ensymic synthesis and isolation of this disaccharide (7). Glucose oxidase, crude, was obtained from Sigma Chemical Co. (St. Louis, MO.), Worthington Biochemical Co., (Freehold, N. J.), and Nutritional Biochemicals Co. (Cleveland, Ohio). These preparations had very similar properties. More purified preparations sold by these and other companies have higher glucose oxidasc activit’y per milligram, and thus a smaller amount than t,hat recommended below can be used for the preparation of the reagent. (The amount needed of some of these preparations can be calculated from Table 1 in reference No. 3.) Horse-radish peroxidase, grade D was obtained from Worthington Biochemical Co. Other Reagents o-Dianisidine (3,3’-dimethoxybenzidine), technical was obtained from Eastman (Rochester, N. Y.), triton X-100 from Rohm & Haas Co. (Philadelphia, Pa.), and Tris (tris(hydroxymethy1) aminomet,hane j , buffe? grade from Sigma Chemical Co. Homogenates
of Intestinal
Mucosa
Pieces of human small intestine were removed during surgical operations and chilled with ice during transport to the laboratory. Pig intestine, obtained from the slaughterhouse, was removed immediately after slaughter and chilled with ice during transport.. The intestine then was cut open; t,he mucosa was scraped off with a piece of glass and homogenized in an Ultra-Turrax homogenizer with four parts (v/w) of cold buffer, saline, or distilled water. The tube was chilled with crushed ice during homogenization. Nuclei and larger cell debris were removed by
20
ARNE
centrifugation for 10 min.
in an ordinary Method
DAHLQVIST
laboratory
for Assay
centrifuge
of Disaccharidase
at 2000 to 4000 rpm Activity
A. Reagents. (1) Substrate Solution: A 0.056M solution of the appropriate disaccharide in O.lM maleate buffer (for the preparation of maleate buffer, see Table 1) of optimum pH. If the optimum pH for the
Dissolve with water
PREPARATION 1.16 gm of maleic acid to 100 ml.
TABLE 1 OF O.lM MALEATE BUFFER in the amount of 1 N NaOH given
PH
18 NaOH,ml
PH
5.0 5.2 5.4 5.6 5.8
10.9 11.4 12.2 13.2 14.2
6.0 6.2 6.4 6.6 6.8 7.0
below,
and dilute
liVNaOH,ml 15.3 16.4 17.4 18.2 18.8 19.1
activity under investigation is below 5.0 or over 7.0, other kinds of buffer must be used. Toluene, about 1 ml/100 ml substrate solution, is added as a preservative. In calculating the amount of disaccharide, it should be observed that different disaccharides have different amounts of water of hydration. (2) TGO Reagent: For the preparation of the TGO (Tris-glucose oxidase) reagent, the following stock solutions are used: (a) Tris bufer, a solution of 61.0 gm (0.5 mole) of Tris in 85 ml of 5 N HCl, made up to 1000 ml with distilled water. The pH of the solution should be measured with a glass electrode and if necessary adjusted to 7. (b) Peroxidase solution, a solution of peroxidase, 1 mg/ml, in dist,illed water. This solution, when kept at -2O”C, is stable for at least several months. (c) o-Dianisidine solution, a solution of o-dianisidine, 10 mg/ml, in 95% ethanol. This solution should be kept in the dark. After a time it will become dark brown by oxidation, and then should be discarded. (d) Detergent solution, a solut,ion of 10 ml of Triton X-100 in 40 ml of 95% ethanol. Preparation of TGO Reagent: In a loo-ml volumetric flask, place 125 mg of glucose oxidase and about 50 ml of the Tris buffer. Shake for l/s min. Add 0.5 ml of peroxidase solution, 0.5 ml of o-dianisidine solution, and 1.0 ml of detergent solution. Make up with Tris buffer to 100 ml, mix, and filter. The reagent is stable for several days in the refrigerator. [Alternatively, the TGO reagent can be prepared from the Glucostat
hSShY
OF
INTESTIiVAL
21
DISACCHARIDES
glucose reagent (Worthington Biochemical Co.) in the following way: Dissolve the contents of the chromogen vial in 1 ml of the detergent solution and 2-3 ml of Tris buffer. Shake until clear. Dissolve the contents of the Glucostat vial in another 4-5 ml of the Tris buffer. Mix the two solutions and dilute with Tris buffer to 100 ml.] (8) Standard Glucose Solution: Dissolve 100 mg of glucose and 2.7g of benzoic acid in distilled water and make up to 1000 ml. The solution is stable for months at room temperature. B. Performance. Dilute an aliquot of the sample to be analyzed so that the solution contains a suitable activity of the disaccharidase to be assayed. If one molecule of glucose is formed per substrate molecule hydrolyzed, the diluted solution should contain a little less than 0.10 units/ml; if two molecules of glucose are formed it should contain somewhat less than 0.05 units/ml. (For definitions of the units, see below.) Then mix in a small test tube (7 X 120 mm) 0.1 ml of the diluted enzyme solution with 0.1 ml of the appropriate substrate solution, and immerse the tube in a constant-temperature water bath at 37°C. Add a small drop of toluene to each tube as a preservative. After 60 min of incubation, add 0.8 ml of distilled water, and then immediately interrupt the enzymic reaction by the immersion of t,he tube in boiling water for 2 min; then cool the tube with tap water. Prepare a blank with the same composition and immerse it in boiling water immediately after mixing of the enzyme and substrate. For the determination of the glucose, transfer 0.5 ml of the solution to another test tube (16 X 150 mm) and mix with 3.0 ml of the TGO reagent. Incubate the tube at 37°C for 1 hr for development of the color. At the same time incubate a standard series, containing 0.0, 0.1, 0.3, and 0.5 ml of the standard glucose solution (these tubes contain 0, 10, 30, and 50 pg, respect,ively, of glucose), distilled water to the combined volume of 0.5 ml, and 3.0 ml of the TGO reagent. After the color has been developed, measure it in spectrophotometer at 420 mp, using l-cm cuvettes, against the tube in the standard series which does not contain sugar. The color is stable for at least an hour. If the amount of glucose present in the sample of the incubated reaction mixture exceeds 50 pg, the whole assay procedure should be repeated with a more dilute sample of the enzyme solution. C. Calculation. The disaccharidase activity of the preparation analyzed is obtained by the following formula: Disaccharidase where a = amount
activity
(units/ml)
=
(a - b) - d n 540
of glucose (pg) found in aliquot
of the incubated
22
ARNE
DAHLQVIST
sample, b = amount of glucose (pg) found in aliquot of the corresponding blank, d = dilution factor of the enzyme solution used for mixing with the substrate, and n = number of glucose molecules that are liberated per substrate (disaccharide) molecule hydrolyzed. For maltose, isomaltose, trehalose, cellobiose, gentiobiose, and other disaccharides composed of two glucose molecules n = 2. For sucrose, lactose, turanose, and other disaccharides which contain one molecule of glucose and one of fructose or galactose n = 1. Units of activity
= pmoles disaccharide
RESULTS
AND
hydrolyzed/mm
DISCUSSIOK
(1) Incubation
Conditions
A. @. In most cases the intestinal disaccharidases have their pH optimum between 5.5 and 6.5. Enzymes with lower pH optimum have been found only in two instances: one malt,ase present in the human duodenum that had its optimum at about pH 3 (8) ; and a soluble lactase fraction in homogenates of the intestinal mucosa of small rats that had its optimum at pH 2 or lower (9). In the pig intestinal mucosa, two maltases with slightly higher pH optimum have been found (10) ; both had a rather broad optimum between pH 6.5 and 7.5.
‘0 0 4
0-O
Maltaso
0-G
lnvcrtasc I
I
I
5
6
7
I e
b 9
PH FIQ. jejunal 0.05 M 0.025 M
1. pH-activity curves for invertase and maltase activities of a homogenate 0.05 M sodium acetate, pH mucosa from an adult human. Buffers: sodium maleate, pH 5.M.5; 0.05 M sodium phosphate, pH 7-7.5; sodium Veronal, pH S-9.
of 4-5; and
ASSAY
OF
INTE23TINAL
DISACCHAHIDES
23
The maltase and invertase activities of crude pig intestinal mucosal homogenates have their optima at pH 6.5, and the lactase at 6.0 (2). The human intestinal disaccharidases have somewhat lower pH optima ; the maltase and invertase act,ivities of crude homogenates of human small intestinal mucosa have their optimum at, pH 6.0 (Fig. l), and the lactase activity at pH 5.6. For the preparation of buffers with pH around 6, maleic acid is very suitable, since its second dissociation step has a PI<, of 5.95. B. Substrate Concentration. Cert.ain of the intestinal disaccharidases show a marked “substrate inhibition” in too concentrated substrate solutions, partly as the result of transglycosylation reactions. In a study of the influence of substrate concentration on pig intestinal disaccharidases, 0.028M substrate was found to give negligible %ubstrate inhibition,” but over 50% saturat,ion of the disaccharidases with substrate (2). (2) Course of the Reaction The hydrolysis of disaccharides by intestinal enzymes has been found to follow zero-order kinetics up to 10-1570 of hydrolysis at 0.028 M substrate concentration. During this initial phase of the reaction the amount of hydrolysis products formed during a certain period of time is proportional to the amount. of enzyme present. (3) Specificity of TGO Reagent So far, all disaccharides which have been tested produce no color, or very weak color, with the TGO reagent in the disaccharidase assay procedure described here. The disaccharides which have been tested are: maltose, sucrose, isomaltose, palatinosr (isomaltulose), turanose! lactose, cellobiose, gentiobiose, and trehalosc. Both a-glucose and P-glucose react rapidly with the TGO reagent (3), but galactose and fructose do not produce a color. This has to be taken into account when calculating the degree of hydrolysis of a disaccharide, since disaccharides which are composed of two glucose molecules give twice the amount of color per molecule of disaccharide split as those which are composed of one molecule of glucose and one of fructose or galactose. Phenol interferes with the production of color by the TGO reagent, which therefore cannot be used for the assay of phenylglucosidasc activity. For this purpose the Folin-Ciocalteu phenol reagent can be used instead (2). (4) Purpose of Detergellt The colored oxidation product of o-dianisidine has a low solubility in water. To some extent, therefore, it crystallizes on the incubation of
24
ARNE
DAHLQVIST
Hugget and Nixon’s (1) reagent with glucose, yielding opalescent solutions which give somewhat inconsistent readings in the spectrophotometer. Added detergent keeps the colored substance in a clear solution. Several detergents tested yielded a brown color with the TGO reagent due to contaminant peroxides, but Triton X-100, recommended by Sols and de la Fuente (4), is free of peroxides. Addition of this detergent does not interfere with the cnzymic reactions. (5) Influence
of Protein
on Glucose Determination
Proteins do not interfere with the reaction of glucose with the TGO reagent, and therefore protein precipitation is usually not necessary. In some cases, however, where the disaccharidase activity is low, and subsequently concentrated mucosal preparations are used, the protein present may cause a considerable turbidity, especial!y after the disaccharidase activity has been interrupted by boiling before determination of the amount of glucose liberated, This will interfere with the spectrophotometric reading. In these cases protein precipitation can be performed with Somogyi’s (11) zinc-barium-reagents before the mixing of the solution with the TGO reagent, or, alternatively, the TGO reaction mixture can be centrifuged clear before the reading is made. (6) Definition
of Unit of Disaccharidase
Activity
In accord with agreements recently made for the definition of clinical enzyme activity units (12), the activity hydrolyzing 1 pmole of disaccharide per minute is taken as the unit. In several previous publications the activity liberating 1 mg of monosaccharides during 60 min has been used as the unit (2, 3, 13, 14). One unit as now defined (,moles/‘min) equals 21.6 units of those previously used (mg/60 min). (7) Disaccharidase
Activity
of Human
Small
Intestinal
Mucosa
Assay of disaccharidase activities in homogenates of human small intestinal mucosa, obtained from pieces of apparently normal jejunum and ileum, removed from adults during surgical operations, have shown the following range of activity, expressed as units (pmoles substrate split per minute) per gram (wet weight) of mucosa: maltase, 13-54; invertase, 6-17; isomaltase, 4-13; palatinase (isomaltulase) 1.54; turanase, l.O3.4; lact.ase, 0.2-19; cellobiase, 0.03-3.6; gentiobiase, 0.01-0.11; and trehalase, l-10. Some of the substrates are hydrolyzed by a mixture of several enzymes (14).
ASSAY
OF
INTESTINAL
DISACCHARIDES
25
SUMMARY
1. A procedure is described for assay of disaccharidase activities in extracts of intestinal mucosa, using a Tris-glucose oxidase reagent for assay of the glucose liberated from the substrate The incubation conditions are discussed. 2. A unit for disaccharidase activity is defined that is in accord with recommendations made by the Joint Sub-Commission on Clinical Enzyme Units of the International Unions of Biochemistry and of Pure and Applied Chemistry. ACKNOWLEDGMENTS Grants gratefully Karstensson
that have been obtained from the Swedish acknowledged. Skillful technical assistance and Miss Karin Iilang.
Medical Research Council are has been given by Mrs. Alice
REFERENCES 1. HUGGETT, A. ST. G., AND NIXON, D. A., Lawet ii, 368 (1957). 2. DAHLQVIST, A., Acta Chem. Sand. 14, 1797 (1960). 3. DAHLQVIST, A., Biochem. J. SO, 547 (1961). 4. SOLS, A., AND DE ~.4 FUENTE, G., in “Methods in Biochemical Research” tel, ed.), p. 302. Year Book Publishers, Chicago, 1961. 5. WHITE, J. W., JR., AND SUBERS, M. H., Anal. Biochem. 2, 380 (1961). 6. DAHLQ~IST, A., Acta Chem. Stand. 14, 72 (1960). 7. WEIDENHAGGN, R., AND LORENZ, S., Angelo. Chew 69, 641 (1957). 8. AURICCHIO, S., personal communication. 9. DOELL, R. G., AND KRETCHMER, N., Biochim. Biophys. Acta 62, 353 10. DAHLQVIST, A., Acta Chem. &and. 14, 1 (1960). 11. SOMOGYI, M., J. Biol. Chem. 160, 69 (1945). 12. FREEMAN, M. E., CIzn. Chim. Acta 6, 300 (1961). 13. DAHLQVIST, A., AND BORGSTR~~M, B., Biochem. J. 81,411 (1961). 14. DAHLQVIST, A., J. Clin. Invest. 41, 463 (1962).
(J. Quas-
(1962).