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A specific method for quantitative determination of glucose
ANALYTICAL
A
BIOCHF,MISTRY
Specific
14, 258-260 (1966)
Method
for of
Quantitative
Determination
Glucose
R. R. BARTON From the Enzymology Research Laboratory, Chemicals Division, Elkhart,
Miles Indiana
Laboratories,
Inc.,
Received August 25, 1965
During studies on the action of amylolytic enzymes on starch, it was necessary to have a procedure for the accurate determination of glucose in the presence of other sugars. The procedures employing glucose oxidase described by Mansford and Opie (1) and by Fleming and Pegler (2) appeared to offer the most promise. Both procedures had a serious drawback, however, in that they used impure glucose oxidase preparations containing significant quantities of maltase. To suppress maltase activity, the oxidation of glucose was carried out in the presence of tris buffer at pH 7.0. Underkofler (3) reported that the optimum pH for the action of glucose oxidase is between pH 4.0 and 6.0. At higher pH values the rate of the enzymic reaction is much slower. In order to obtain oxidation of glucose at pH 7.0, it was necessary, therefore, to run the reaction for extended time periods. In unaerated systems, the possibility of inadequate quantities of oxygen for lengthy reactions existed. This could lead to erratic results and lack of sensitivity. If a purified glucose oxidase preparation free of maltase is used, the reaction can be carried out at the optimum pH and for a shorter period of time. This should make it possible to increase the sensitivity and improve the reproducibility of the procedure. There is now available a highly purified glucose oxidase preparation free of all traces of maltase, invertase, and amylase. On electrophoresis of this preparation, the only material detected, other than glucose oxidase, was a trace of catalase. Glucose oxidase of this purity can be prepared by the procedure of Pazur and Kleppe (4). We have, therefore, used this purified enzyme in a modification of the procedure of Fleming and Pegler (2) which permits rapid and accurate determination of glucose in the presence of maltose and other sugars. 258
QUANTIT$TIVE
DETERMINATION
OF
GLUCOSE
259
METHOD
Reagents Enzymic reagent. Dissolve 10 mg of o-dianisidine hydrochloride, 10 mg of horse-radish peroxidase (Worthington Biochemical Co.), and 0.1 ml of purified glucose oxidase (Miles Chemical Co.) containing 1000 glucose oxidase units/ml in O.lM acetate buffer, pH 5.5, and make to a volume of 100 ml. Standard glucose solution. Prepare a standard glucose solution using analytical-grade anhydrous glucose containing 100 pg glucose/ml. This solution should stand at room temperature for at least 1 hr prior to use to allow completion of the mutarotation of glucose. Sulfuric acid, 10 .V. Procedure Pipet 4.0 ml of the enzyme reagent into a 25 X 150 mm test tube, place in a 30°C water bath, and allow reagent to come to temperature. Pipet 2.0 ml of the test solution containing up to 200 pg of glucose into the reagent and mix. After exactly 5 min reaction time, stop the reaction by adding 8.0 ml of 10N sulfuric acid. Measure the optical density of the solution against a reagent blank consisting of 4.0 ml of enzyme reagent and 2.0 ml of water at 525 rnp. For precise results, it is desirable to run a glucose standard control with each set of unknowns, employing 2.0 ml of the standard glucose solution (200 pg glucose). pg glucose in 2.0 ml sample =
OD unknown OD standard ’ 2oo
Calculation RESULTS
To determine the sensitivity and reproducibility of this procedure, a series of glucose concentrations ranging from 100 to 400 pg were analyzed. The results are shown in Fig. 1. It can be seenthat the results are linear over the concentrations of glucose employed. We have also found that the results are uniform from day to day. In order to determine the effect of maltose on the determination of glucose, several concentrations of glucose were dissolved in a 2% maltose solution. These solutions were then diluted with water so that they contained approximately 100 pg glucose,/ml and analyzed. The results are shown in Table 1. These results are in close agreement, check well with the levels of glucose added to the maltose solution, and show that maltose
260
R. R. BARTON
FIG. 1. Relationship
between
log lo/Z
at 525 mp and glucose
TABLE
concentrations.
1
DETERMINATION OF GLUCOSE IN THE PRESENCE OF MALTESE Glucose 1
0 2 4 6 8 10 12
found,
n&ml
2
0 2.07 4.00 6.00 8.15 10.16 12.20
3
0 2.00 4.00 5.88 8.05 10.06 11.90
0 2.08 4.08 6.20 7.95 9.95 11.95
does not affect the accuracy of this method for det,ermining glucose. By proper dilution, so that the test solutions contain lessthan 100 pg glucose/ ml, a wide range of glucose concentrations can be measured accurately and rapidly with this procedure. REFERENCES 1. MANSFORD, K. R. L., AND OPIE, R. K., Analyst 88,646-649 (1963). 2. FLEMINQ, I. D., AND PEGLER, I-I. F., Analyst 88, 967-968 (1963). 3. UNDERKOFLER, L. A., Proc. Intern. Symp. Enzyme Chem. Tokyo 48G490 ( 1958). 4. PAZUR, J. H., AND KLEPPE, K., Biochemistry 3, 578-583 (1964).
and
Kyoto,
1967,
A
BIOCHF,MISTRY
Specific
14, 258-260 (1966)
Method
for of
Quantitative
Determination
Glucose
R. R. BARTON From the Enzymology Research Laboratory, Chemicals Division, Elkhart,
Miles Indiana
Laboratories,
Inc.,
Received August 25, 1965
During studies on the action of amylolytic enzymes on starch, it was necessary to have a procedure for the accurate determination of glucose in the presence of other sugars. The procedures employing glucose oxidase described by Mansford and Opie (1) and by Fleming and Pegler (2) appeared to offer the most promise. Both procedures had a serious drawback, however, in that they used impure glucose oxidase preparations containing significant quantities of maltase. To suppress maltase activity, the oxidation of glucose was carried out in the presence of tris buffer at pH 7.0. Underkofler (3) reported that the optimum pH for the action of glucose oxidase is between pH 4.0 and 6.0. At higher pH values the rate of the enzymic reaction is much slower. In order to obtain oxidation of glucose at pH 7.0, it was necessary, therefore, to run the reaction for extended time periods. In unaerated systems, the possibility of inadequate quantities of oxygen for lengthy reactions existed. This could lead to erratic results and lack of sensitivity. If a purified glucose oxidase preparation free of maltase is used, the reaction can be carried out at the optimum pH and for a shorter period of time. This should make it possible to increase the sensitivity and improve the reproducibility of the procedure. There is now available a highly purified glucose oxidase preparation free of all traces of maltase, invertase, and amylase. On electrophoresis of this preparation, the only material detected, other than glucose oxidase, was a trace of catalase. Glucose oxidase of this purity can be prepared by the procedure of Pazur and Kleppe (4). We have, therefore, used this purified enzyme in a modification of the procedure of Fleming and Pegler (2) which permits rapid and accurate determination of glucose in the presence of maltose and other sugars. 258
QUANTIT$TIVE
DETERMINATION
OF
GLUCOSE
259
METHOD
Reagents Enzymic reagent. Dissolve 10 mg of o-dianisidine hydrochloride, 10 mg of horse-radish peroxidase (Worthington Biochemical Co.), and 0.1 ml of purified glucose oxidase (Miles Chemical Co.) containing 1000 glucose oxidase units/ml in O.lM acetate buffer, pH 5.5, and make to a volume of 100 ml. Standard glucose solution. Prepare a standard glucose solution using analytical-grade anhydrous glucose containing 100 pg glucose/ml. This solution should stand at room temperature for at least 1 hr prior to use to allow completion of the mutarotation of glucose. Sulfuric acid, 10 .V. Procedure Pipet 4.0 ml of the enzyme reagent into a 25 X 150 mm test tube, place in a 30°C water bath, and allow reagent to come to temperature. Pipet 2.0 ml of the test solution containing up to 200 pg of glucose into the reagent and mix. After exactly 5 min reaction time, stop the reaction by adding 8.0 ml of 10N sulfuric acid. Measure the optical density of the solution against a reagent blank consisting of 4.0 ml of enzyme reagent and 2.0 ml of water at 525 rnp. For precise results, it is desirable to run a glucose standard control with each set of unknowns, employing 2.0 ml of the standard glucose solution (200 pg glucose). pg glucose in 2.0 ml sample =
OD unknown OD standard ’ 2oo
Calculation RESULTS
To determine the sensitivity and reproducibility of this procedure, a series of glucose concentrations ranging from 100 to 400 pg were analyzed. The results are shown in Fig. 1. It can be seenthat the results are linear over the concentrations of glucose employed. We have also found that the results are uniform from day to day. In order to determine the effect of maltose on the determination of glucose, several concentrations of glucose were dissolved in a 2% maltose solution. These solutions were then diluted with water so that they contained approximately 100 pg glucose,/ml and analyzed. The results are shown in Table 1. These results are in close agreement, check well with the levels of glucose added to the maltose solution, and show that maltose
260
R. R. BARTON
FIG. 1. Relationship
between
log lo/Z
at 525 mp and glucose
TABLE
concentrations.
1
DETERMINATION OF GLUCOSE IN THE PRESENCE OF MALTESE Glucose 1
0 2 4 6 8 10 12
found,
n&ml
2
0 2.07 4.00 6.00 8.15 10.16 12.20
3
0 2.00 4.00 5.88 8.05 10.06 11.90
0 2.08 4.08 6.20 7.95 9.95 11.95
does not affect the accuracy of this method for det,ermining glucose. By proper dilution, so that the test solutions contain lessthan 100 pg glucose/ ml, a wide range of glucose concentrations can be measured accurately and rapidly with this procedure. REFERENCES 1. MANSFORD, K. R. L., AND OPIE, R. K., Analyst 88,646-649 (1963). 2. FLEMINQ, I. D., AND PEGLER, I-I. F., Analyst 88, 967-968 (1963). 3. UNDERKOFLER, L. A., Proc. Intern. Symp. Enzyme Chem. Tokyo 48G490 ( 1958). 4. PAZUR, J. H., AND KLEPPE, K., Biochemistry 3, 578-583 (1964).
and
Kyoto,
1967,