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Automatic amperometric assay of glucose oxidase
ANALYTICAL
BIOCHEMISTRY
Automatic
9,
(1964)
Amperometric
HARRY From
204-210
Assay
L. PARDUE
the Depart,ment
AND
of Che’mistry,
Received
ROBERT
Purdue
January
of Ghcose
University,
Oxidase
IX. SIMON’ Lafayette,
Indiana
27, 1964
In a recent paper Bodansky discussed the growing importance of enzyme assays in clinical laboratories (l), and emphasized the importance of automation of analytical procedures. Although his discussion dealt primarily with clinical problems it is equally applicable to other areas in which enzyme activities must be determined. In this work a rapid automatic method has been developed for the quantitative determination of enzyme activities. The method has been developed for the determination of glucose oxidase. Numerical data are obtained automatically which are easily converted to enzyme activity. Glucose oxidase catalyzes the aerobic oxidation of P-n-glucose to gluconic acid with the formation of an equivalent amount of hydrogen peroxide. Manometric methods for the enzyme based on the measurement of oxygen uptake have been described (3, 7). More recently, procedures based on the manual titration of gluconic acid produced during a preselected reaction time were developed (6, 7). All these methods involve several manual operations and require stringent control of reaction conditions. As a result, the procedures are tedious and time consuming. In the new method, hydrogen peroxide produced by the enzymic reaction rapidly oxidizes iodide to iodine (4). The formation of iodine is detected amperometrically. Automatic control equipment measures the time required for a small predetermined amount of iodine to be produced (4, 5)) the measured time interval being inversely proportional to enzyme activity. Manual operations are sharply reduced and measurement times are short, ranging from 10 to 100 set per sample. Relative standard deviations for the method are within 2%. An extensive study of possible effects of reagents required in the nonenzymic reaction on the enzyme activity demonstrated that the ‘Present address: Park, Maryland.
Department
of
Chemistry, 204
University
of
Maryland,
College
method is free from intcrferencc. Results arc report4 for the assay of several commercial preparation of glucose oxitla~c. Agreement, among the new method and comnicrcial procedures is within 25s. PRINCIPLE:S
OF
THE
METHOD
The Commission on Enzymes of the International Union of Biochemistry has outlined specific recommendation e with regard to the method of analysis, unit of activity, and conditions to be used in enzyme assays (2). These recommendations hare been carefully followed in this work. An enzyme activity unit is defined as that anlount which will catalyze the transformation of one micromole of subst~rntc per minute under specified conditions. In the glucose reaction, the rate of formation of hydrogen peroxide is equal to the rate of oxidation of glucose. The hydrogen peroxide rapidly oxidizes iodide to iodine so that the rate of formation of iodine is equal to the rat,e of oxidation of glucose. Therefore, the rate of formation of iodine in micromoles per minute is equal to the enzyme activit,y. The formation of iodine is detected by a simple amperometric system (5). A constant polarizing voltage is applied across a rotating platinum cathode and a stationary platinum anode immersed in the reaction mixture. The change in current observed is proportional to the increase in iodine concentration. Electrolysis current is calibrated in terms of the amount of iodine produced. In this work the time required in seconds for 0.0500 pmole of iodine to be produced is measured. The specific enzyme activity in units per milligram is given by:
where W is the mass of dry enzyme in milligrams used in the measurement step, At is the time in seconds required for 0.0500 pmole of iodine to be produced, and the multiplier 60 converts from ,umolesper second to pmoles per minute. For const,ant massesof enzyme the act.ivity is given as the product of a constant times the reciprocal of the automatically measured t,ime interval. EXPERIMENTAL
The experimental setup and automatic control equipment is that described in detail earlier for the amperometric determination of glucose (Fj). A Sargent reaction rat,e adapter (E. H. Sargent and Company,
206
PARDCE
AiTD
SIMON
Chicago, Ill.) can be substituted for the relay system with equivalent results. The study of interferences was performed using a commercial pH-stat (Radiometer, Copenhagen, Denmark) to follow the rate of formation of gluconic acid. All reagents are handled with tuberculine type hypodermic syringes. The reaction mixture and wash water are removed from the sample compartment by an aspirator tube. Temperature is controlled to within -+O.Ol”C using conventional control equipment and a circulating pump. Reagents All reagents are prepared in distilled water, which is passed over a mixed cation-anion exchange resin bed before use. Iodine and iodide solutions are stored in brown glass-stoppered bottles at 4°C. Glucose Oxidase. Aqueous enzyme samples are prepared by dissolving appropriate amounts of the dry preparation in water and diluting to give concentrations of 0.03 to 0.3 units/ml. Enzyme solutions which are kept for several days are stored at 4°C immediately after preparation. Five commercial samples were investigated. Specific activities in units per milligram determined at 35.O”C, pH 5.10, and final glucose concentration of 0.17M were reported as 1.30, 16.8, 39.0, 88.7, and 37.8. Buffer. Sodium acetate (0.25 M) is prepared by dissolving 20.5 gm of Baker’s anhydrous powder in water and diluting to 1 liter. Acetic acid (0.25 M) is prepared by mixing 14.2 ml of glacial acetic acid (99.8%) with water and diluting to 1 liter. An acetate buffer of pH 5.05 is prepared by mixing these solutions in a ratio of 2: 1. Final adjustment of pH is made using a pH meter. Iodide. A fresh (0.25 M) stock solution is prepared every 2-3 days by dissolving 4.15 gm of Baker’s analyzed potassium iodide in water and diluting to 100 ml. Iodine Solution. A standard iodine solution is prepared by dissolving 0.0602 gm of USP resublimed crystals in a KI slurry (41.5 gm/50 ml) and diluting to 1 liter. Standardization against primary standard As&, gave a value of 2.31 X lo-” M. Composite Reagent. A composite reagent is prepared by dissolving 45.0 gm of D (+) -dextrose. (Pfanstiehl Laboratories Inc., Waukegan, Ill.) and 0.185 gm of ammonium molybdate [ (NH,),Mo,O,~*~H,O] in 250 ml of acetate buffer (initial pH 5.05) and diluting to 500 ml with water. One milliliter of this reagent diluted to a final volume of 3.0 ml in the measurement step gives the following final conditions and concentrations: glucose (0.17 M), Mo(VI) (5.45 X lo-” AI), and pH (5.10).
AUTOMATIC
ASSAY
Preparation
CJF
GLUCOSE
C)XIDASE
207
of Equipment
The measurement and control equipment is connected as discussed in detail earlier (5). Electrolysis current is measured as the voltage drop across a lOOO-ohm resistor so that each millivolt represents 1 pa of current. The prebias and bias voltages are adjusted to 5.00 mv each (corresponding to 5.00 pa each). The concentration comparator is turned on and the function switch set on PXP and t,he reagent selector switch in position 2. Amperome tric Calibration Amperometric response is calibrated in terms of micromoles of iodine by adding aliquots of a standard iodine solution to the sample compartment containing all reagents in the same concentrations used in the analysis step except the enzyme. Under the conditions of this work the amperometric sensitivity was 0.0100 pmole of iodine (in 3.00 ml) per pa or 0.05 pmole of iodine for the 5-pa interval measured. This standardization was shown to be reproducible to about 1.6% over a ten-day period. Plots of iodine concentration versus current, arc linear with zero intercept. Measurement
Step
One milliliter of composite reagent and 1 ml of iodide solution arc added to the sample compartment and the rotating electrode is turned on. Then 1 ml of enzyme solution is added to the rapidly stirred solution and the semiautomatic switch on the comparator is closed momentarily. After the measurement is completed automatically, the reaction time is read from the t,imer and the enzyme activity is computed using Eq. (1). RESULTS
Recorded curves of indicator current versus time for several enzyme concentrations within the range reported are linear. Table 1 lists data for several dilutions of a commercial enzyme preparation. These data, typical of many similar sets obta’ined over many months, demonstrate both the reproducibility of the method and the validity of the proportionality expressecl in Eq. (1). Table 2 lists data for five commercial enzyme preparations. Comparative data are shown for the amperometric method and the reported assay values provided by the suppliers. The amperometric data were obtained two days after receipt of the enzyme preparations. The activities reported by suppliers were determined by titration of the gluconic acid produced during a preset reaction time at 35°C. These activities were normalized
208
PARDUE AND SIMON TABLE
1
AUTOMATIC RESULTS WH .\QUEO~S DILUTIONS OF A SIN~;LE ENZYME PREPARATION
71.5 71.5 36.3 24.3 18.5 14.9 10.0 7.3 a Results
Enzyme units/mg
Reciproral time, set-’ x 102
Time, E3ee
1.40 1.40 2.75 4.12 5.4 6 .7 10.0
13.7 computed
using
activity, X 102
Taken
Founda
0.83 0.83 1.66 2.49 3.32 4.15 6.2 8.3
0.86 0.86 1.69 2.52 Standard 4.11 6.15 8.35
the 0.0332
unit/mg.
sample
TABLE
Rel. Std. Dev. (%)
1.1 1.1 0 .7 2.1 2.3 1.4 1.8 1.1
EWX. %
______
3.6 3.6 1.8 1.2 1.0 0.8 0.6
as a standard.
2
AUTOMATIC RESULTS FOR COMMERCLLL GL~JCOSE OXIDASE PREP.\RATIONS Activity, SaNmdlle
I II III IV V 0 The reported b The difference
-
(Units/mg..
25°C)
Amp.
Rep.~
0.86 10.2 23.8 23.5 53.8
0.80 10.3 23 .9 23.2 54.4
values are normalized from hetween the amperometric
35°C to 25°C and normalized
Rel. Dev.
0.9 2.2 0.4 3.0 0.8
Std. (%)
Diff.b
+7.5 -1.0 -0.4 +1.3 -1.1
by multiplying by 0.614. reported results expressed
a,8percent.
to 25’ for comparative purposes by multiplying by an empirically determined temperature coefficient of 0.614 (AZaOe= A2,0, X 0.614). These data demon&rate relative standard deviations for the amperometric method of about 2%. Agreement between the amperometric and the commercial methods is within 2% for all except preparation No. I. This was a crude preparation which was incompletely soluble in water. Samples were prepared by grinding a weighed amount of enzyme in a few milliliters of water and then diluting to volume. Differences may have resulted from failure in some cases to get all of the enzyme into solution. It should be noted that the relative standard deviation of the amperometric results for this sample is within 1%. Sample No. II was assayed by the amperometric method several times during a three-month period. The dry enzyme preparation was stored in a desiccator at 4°C during this period. The average specific activity obtained for the first month was 10.1 + 0.3 u/mg. After about one
AUTOMATIC
ASSAY
OF
GLUCOSE
OXIDASE
209
month, the activity began to drop, reaching a value of 85% of the original activity after t.hree months. Similar observations were made on other preparations. Similar experiments demonstrated that aqueous preparations retained 100% activity for about four days when stored at 4°C. The aqueous solutions usually lost about 10% activity after one week. DISCUSSION
During the course of this work assays were run by the conventional gluconic acid titration procedure, a pH-stat procedure, a potentiometric method for detecting the iodine produced by the reaction sequence described here, and the amperometric method. Both the amperometric and potentiometric methods were much simpler and more rapid than either the titration or pH-stat procedures. Both methods were more sensitive than the pa-stat method. Once set up, the amperometric and potentiometric procedures give comparable results, The initial setup and maintenance of equipment is simpler for the amperometric than for the potentiometric method. Also, the amperometric method does not require a reference solution. These points make the amperometric method the preferred one. The pH-stat method proved to be a valuable tool for studying the effects on the enzyme reaction of variables such as pH, temperature, substrate and oxygen concentrations, and inorganic reagents. The optimum pH for the method was observed to be 5.1 at 25”C, in agreement with other investigators (3). Temperature studies indicated a maximum at about 40°C. The ratio of activity at 25” to that at 35” was found to be quite reproducible at 0.614. Glucose present at 0.05 J4 or greater is sufficient to saturate the enzyme at the concentrations investigated here (0.01-0.1 units/ml). A glucose concentration of 0.17&l is used in the assay procedure to be consistent with conventional procedures. The air-saturated solutions contain sufficient oxygen to give maximum enzyme activity throughout the mcasurement interval. Bubbling air or oxygen through the solutions caused no change in activity. Iodide concentrations up to 0.2 M, micromolar amounts of iodine produced in the reaction, and Mo(V1) at concentrations below 5.5 X 1OF M had no effect on the enzyme activity. M (VI) above 1 X 1F M caused an increase in reaction velocity. Also molybdenum above the millimolar range caused a shift of the optimum pH to higher values. The Mo(VI) concentration of 5.5 X 10-j M used in this work causes no interference. Catalase does not interfere with the method. These observations, combined with the good agreement among the
210
PARDUE
AND
SIMON
amperometric, potcntiometric, and titrimetric procedures, indicate that the accuracy of the ampcrometric method is as good as the reproducibility of 2% relative. The instrument settings used in this work are somewhat arbitrary. The 5.00-pa premeasurement interval provides a minimum of 10 set for the highest enzyme concentrations examined (0.1 u/ml), during which time stirring and temperature equilibrium are established. The 5.00-pa measurement interval provides measurement times between 10 and 100 sec. The amperometric sensitivity for iodine is a function of several variables including stirring rate, applied polarizing voltage, and indirectly the dropping resistor. The conditions described here provide optimum signalto-noise ratios. The amperometric sensitivity under these conditions is reproducible to within 2% over periods up to one month and only infrequent calibrations are required. The method as described here should be applicable to the many enzyme reactions involving electroactive reactants or products. ABSTRACT
A rapid, simple, automatic method is described for the measurement of glucose oxidase. Hydrogen peroxide produced by the enzymic oxidation of glucose rapidly oxidizes iodide to iodine. The rate of formation of iodine, which is equal to the enzyme activity, is measured by an automatic amperometric system. Measurement times for the determination of enzyme concentrations between 0.01 and 0.1 units per milliliter are between 10 and 100 sec. Relative standard deviations are within 2%. Interference studies and comparisons with independent methods indicate that the accuracy of the method is within 2%. ACKNOWLEDGMEXTS This investigation was supported Grant GM-16681 from the Xational samples from Eli Lilly and Company,
in part by Public Health Service Research Institutes of Health. The receipt of enzyme Indinnapolis, Ind.. was greatly appreciated.
REFEREPiCES 1. BODAN~KY, O., Am. J. Clin. Path. 38, 343 (1962). 2. “International Union of Biochemistry, Commission on Enzymes,” Vol. 20, pp. 10, 11. Pergamon Press, New York, 1961. 3. KEILIN, D., AND HARTREE, E. F., Biochem. J. 42, 221 (1948). 4. MALMSTADT, H. V., AND PARDUE, H. L., Anal. Chem. 33, 1040 (1961). 5. PARDUE, H. L., Anal. Chem. 35, 1240 (1963). 6. “Sigma Assay Form 322, Glucose Oxidase,” Sigma Chemical Co., St. Louis 18, MO. 7. UNDERKOFLER, L. H., Proc. Intern. Symp. E,rlzyme Chem., Tokyo, Kyoto 2, 486 ( 1957).
BIOCHEMISTRY
Automatic
9,
(1964)
Amperometric
HARRY From
204-210
Assay
L. PARDUE
the Depart,ment
AND
of Che’mistry,
Received
ROBERT
Purdue
January
of Ghcose
University,
Oxidase
IX. SIMON’ Lafayette,
Indiana
27, 1964
In a recent paper Bodansky discussed the growing importance of enzyme assays in clinical laboratories (l), and emphasized the importance of automation of analytical procedures. Although his discussion dealt primarily with clinical problems it is equally applicable to other areas in which enzyme activities must be determined. In this work a rapid automatic method has been developed for the quantitative determination of enzyme activities. The method has been developed for the determination of glucose oxidase. Numerical data are obtained automatically which are easily converted to enzyme activity. Glucose oxidase catalyzes the aerobic oxidation of P-n-glucose to gluconic acid with the formation of an equivalent amount of hydrogen peroxide. Manometric methods for the enzyme based on the measurement of oxygen uptake have been described (3, 7). More recently, procedures based on the manual titration of gluconic acid produced during a preselected reaction time were developed (6, 7). All these methods involve several manual operations and require stringent control of reaction conditions. As a result, the procedures are tedious and time consuming. In the new method, hydrogen peroxide produced by the enzymic reaction rapidly oxidizes iodide to iodine (4). The formation of iodine is detected amperometrically. Automatic control equipment measures the time required for a small predetermined amount of iodine to be produced (4, 5)) the measured time interval being inversely proportional to enzyme activity. Manual operations are sharply reduced and measurement times are short, ranging from 10 to 100 set per sample. Relative standard deviations for the method are within 2%. An extensive study of possible effects of reagents required in the nonenzymic reaction on the enzyme activity demonstrated that the ‘Present address: Park, Maryland.
Department
of
Chemistry, 204
University
of
Maryland,
College
method is free from intcrferencc. Results arc report4 for the assay of several commercial preparation of glucose oxitla~c. Agreement, among the new method and comnicrcial procedures is within 25s. PRINCIPLE:S
OF
THE
METHOD
The Commission on Enzymes of the International Union of Biochemistry has outlined specific recommendation e with regard to the method of analysis, unit of activity, and conditions to be used in enzyme assays (2). These recommendations hare been carefully followed in this work. An enzyme activity unit is defined as that anlount which will catalyze the transformation of one micromole of subst~rntc per minute under specified conditions. In the glucose reaction, the rate of formation of hydrogen peroxide is equal to the rate of oxidation of glucose. The hydrogen peroxide rapidly oxidizes iodide to iodine so that the rate of formation of iodine is equal to the rat,e of oxidation of glucose. Therefore, the rate of formation of iodine in micromoles per minute is equal to the enzyme activit,y. The formation of iodine is detected by a simple amperometric system (5). A constant polarizing voltage is applied across a rotating platinum cathode and a stationary platinum anode immersed in the reaction mixture. The change in current observed is proportional to the increase in iodine concentration. Electrolysis current is calibrated in terms of the amount of iodine produced. In this work the time required in seconds for 0.0500 pmole of iodine to be produced is measured. The specific enzyme activity in units per milligram is given by:
where W is the mass of dry enzyme in milligrams used in the measurement step, At is the time in seconds required for 0.0500 pmole of iodine to be produced, and the multiplier 60 converts from ,umolesper second to pmoles per minute. For const,ant massesof enzyme the act.ivity is given as the product of a constant times the reciprocal of the automatically measured t,ime interval. EXPERIMENTAL
The experimental setup and automatic control equipment is that described in detail earlier for the amperometric determination of glucose (Fj). A Sargent reaction rat,e adapter (E. H. Sargent and Company,
206
PARDCE
AiTD
SIMON
Chicago, Ill.) can be substituted for the relay system with equivalent results. The study of interferences was performed using a commercial pH-stat (Radiometer, Copenhagen, Denmark) to follow the rate of formation of gluconic acid. All reagents are handled with tuberculine type hypodermic syringes. The reaction mixture and wash water are removed from the sample compartment by an aspirator tube. Temperature is controlled to within -+O.Ol”C using conventional control equipment and a circulating pump. Reagents All reagents are prepared in distilled water, which is passed over a mixed cation-anion exchange resin bed before use. Iodine and iodide solutions are stored in brown glass-stoppered bottles at 4°C. Glucose Oxidase. Aqueous enzyme samples are prepared by dissolving appropriate amounts of the dry preparation in water and diluting to give concentrations of 0.03 to 0.3 units/ml. Enzyme solutions which are kept for several days are stored at 4°C immediately after preparation. Five commercial samples were investigated. Specific activities in units per milligram determined at 35.O”C, pH 5.10, and final glucose concentration of 0.17M were reported as 1.30, 16.8, 39.0, 88.7, and 37.8. Buffer. Sodium acetate (0.25 M) is prepared by dissolving 20.5 gm of Baker’s anhydrous powder in water and diluting to 1 liter. Acetic acid (0.25 M) is prepared by mixing 14.2 ml of glacial acetic acid (99.8%) with water and diluting to 1 liter. An acetate buffer of pH 5.05 is prepared by mixing these solutions in a ratio of 2: 1. Final adjustment of pH is made using a pH meter. Iodide. A fresh (0.25 M) stock solution is prepared every 2-3 days by dissolving 4.15 gm of Baker’s analyzed potassium iodide in water and diluting to 100 ml. Iodine Solution. A standard iodine solution is prepared by dissolving 0.0602 gm of USP resublimed crystals in a KI slurry (41.5 gm/50 ml) and diluting to 1 liter. Standardization against primary standard As&, gave a value of 2.31 X lo-” M. Composite Reagent. A composite reagent is prepared by dissolving 45.0 gm of D (+) -dextrose. (Pfanstiehl Laboratories Inc., Waukegan, Ill.) and 0.185 gm of ammonium molybdate [ (NH,),Mo,O,~*~H,O] in 250 ml of acetate buffer (initial pH 5.05) and diluting to 500 ml with water. One milliliter of this reagent diluted to a final volume of 3.0 ml in the measurement step gives the following final conditions and concentrations: glucose (0.17 M), Mo(VI) (5.45 X lo-” AI), and pH (5.10).
AUTOMATIC
ASSAY
Preparation
CJF
GLUCOSE
C)XIDASE
207
of Equipment
The measurement and control equipment is connected as discussed in detail earlier (5). Electrolysis current is measured as the voltage drop across a lOOO-ohm resistor so that each millivolt represents 1 pa of current. The prebias and bias voltages are adjusted to 5.00 mv each (corresponding to 5.00 pa each). The concentration comparator is turned on and the function switch set on PXP and t,he reagent selector switch in position 2. Amperome tric Calibration Amperometric response is calibrated in terms of micromoles of iodine by adding aliquots of a standard iodine solution to the sample compartment containing all reagents in the same concentrations used in the analysis step except the enzyme. Under the conditions of this work the amperometric sensitivity was 0.0100 pmole of iodine (in 3.00 ml) per pa or 0.05 pmole of iodine for the 5-pa interval measured. This standardization was shown to be reproducible to about 1.6% over a ten-day period. Plots of iodine concentration versus current, arc linear with zero intercept. Measurement
Step
One milliliter of composite reagent and 1 ml of iodide solution arc added to the sample compartment and the rotating electrode is turned on. Then 1 ml of enzyme solution is added to the rapidly stirred solution and the semiautomatic switch on the comparator is closed momentarily. After the measurement is completed automatically, the reaction time is read from the t,imer and the enzyme activity is computed using Eq. (1). RESULTS
Recorded curves of indicator current versus time for several enzyme concentrations within the range reported are linear. Table 1 lists data for several dilutions of a commercial enzyme preparation. These data, typical of many similar sets obta’ined over many months, demonstrate both the reproducibility of the method and the validity of the proportionality expressecl in Eq. (1). Table 2 lists data for five commercial enzyme preparations. Comparative data are shown for the amperometric method and the reported assay values provided by the suppliers. The amperometric data were obtained two days after receipt of the enzyme preparations. The activities reported by suppliers were determined by titration of the gluconic acid produced during a preset reaction time at 35°C. These activities were normalized
208
PARDUE AND SIMON TABLE
1
AUTOMATIC RESULTS WH .\QUEO~S DILUTIONS OF A SIN~;LE ENZYME PREPARATION
71.5 71.5 36.3 24.3 18.5 14.9 10.0 7.3 a Results
Enzyme units/mg
Reciproral time, set-’ x 102
Time, E3ee
1.40 1.40 2.75 4.12 5.4 6 .7 10.0
13.7 computed
using
activity, X 102
Taken
Founda
0.83 0.83 1.66 2.49 3.32 4.15 6.2 8.3
0.86 0.86 1.69 2.52 Standard 4.11 6.15 8.35
the 0.0332
unit/mg.
sample
TABLE
Rel. Std. Dev. (%)
1.1 1.1 0 .7 2.1 2.3 1.4 1.8 1.1
EWX. %
______
3.6 3.6 1.8 1.2 1.0 0.8 0.6
as a standard.
2
AUTOMATIC RESULTS FOR COMMERCLLL GL~JCOSE OXIDASE PREP.\RATIONS Activity, SaNmdlle
I II III IV V 0 The reported b The difference
-
(Units/mg..
25°C)
Amp.
Rep.~
0.86 10.2 23.8 23.5 53.8
0.80 10.3 23 .9 23.2 54.4
values are normalized from hetween the amperometric
35°C to 25°C and normalized
Rel. Dev.
0.9 2.2 0.4 3.0 0.8
Std. (%)
Diff.b
+7.5 -1.0 -0.4 +1.3 -1.1
by multiplying by 0.614. reported results expressed
a,8percent.
to 25’ for comparative purposes by multiplying by an empirically determined temperature coefficient of 0.614 (AZaOe= A2,0, X 0.614). These data demon&rate relative standard deviations for the amperometric method of about 2%. Agreement between the amperometric and the commercial methods is within 2% for all except preparation No. I. This was a crude preparation which was incompletely soluble in water. Samples were prepared by grinding a weighed amount of enzyme in a few milliliters of water and then diluting to volume. Differences may have resulted from failure in some cases to get all of the enzyme into solution. It should be noted that the relative standard deviation of the amperometric results for this sample is within 1%. Sample No. II was assayed by the amperometric method several times during a three-month period. The dry enzyme preparation was stored in a desiccator at 4°C during this period. The average specific activity obtained for the first month was 10.1 + 0.3 u/mg. After about one
AUTOMATIC
ASSAY
OF
GLUCOSE
OXIDASE
209
month, the activity began to drop, reaching a value of 85% of the original activity after t.hree months. Similar observations were made on other preparations. Similar experiments demonstrated that aqueous preparations retained 100% activity for about four days when stored at 4°C. The aqueous solutions usually lost about 10% activity after one week. DISCUSSION
During the course of this work assays were run by the conventional gluconic acid titration procedure, a pH-stat procedure, a potentiometric method for detecting the iodine produced by the reaction sequence described here, and the amperometric method. Both the amperometric and potentiometric methods were much simpler and more rapid than either the titration or pH-stat procedures. Both methods were more sensitive than the pa-stat method. Once set up, the amperometric and potentiometric procedures give comparable results, The initial setup and maintenance of equipment is simpler for the amperometric than for the potentiometric method. Also, the amperometric method does not require a reference solution. These points make the amperometric method the preferred one. The pH-stat method proved to be a valuable tool for studying the effects on the enzyme reaction of variables such as pH, temperature, substrate and oxygen concentrations, and inorganic reagents. The optimum pH for the method was observed to be 5.1 at 25”C, in agreement with other investigators (3). Temperature studies indicated a maximum at about 40°C. The ratio of activity at 25” to that at 35” was found to be quite reproducible at 0.614. Glucose present at 0.05 J4 or greater is sufficient to saturate the enzyme at the concentrations investigated here (0.01-0.1 units/ml). A glucose concentration of 0.17&l is used in the assay procedure to be consistent with conventional procedures. The air-saturated solutions contain sufficient oxygen to give maximum enzyme activity throughout the mcasurement interval. Bubbling air or oxygen through the solutions caused no change in activity. Iodide concentrations up to 0.2 M, micromolar amounts of iodine produced in the reaction, and Mo(V1) at concentrations below 5.5 X 1OF M had no effect on the enzyme activity. M (VI) above 1 X 1F M caused an increase in reaction velocity. Also molybdenum above the millimolar range caused a shift of the optimum pH to higher values. The Mo(VI) concentration of 5.5 X 10-j M used in this work causes no interference. Catalase does not interfere with the method. These observations, combined with the good agreement among the
210
PARDUE
AND
SIMON
amperometric, potcntiometric, and titrimetric procedures, indicate that the accuracy of the ampcrometric method is as good as the reproducibility of 2% relative. The instrument settings used in this work are somewhat arbitrary. The 5.00-pa premeasurement interval provides a minimum of 10 set for the highest enzyme concentrations examined (0.1 u/ml), during which time stirring and temperature equilibrium are established. The 5.00-pa measurement interval provides measurement times between 10 and 100 sec. The amperometric sensitivity for iodine is a function of several variables including stirring rate, applied polarizing voltage, and indirectly the dropping resistor. The conditions described here provide optimum signalto-noise ratios. The amperometric sensitivity under these conditions is reproducible to within 2% over periods up to one month and only infrequent calibrations are required. The method as described here should be applicable to the many enzyme reactions involving electroactive reactants or products. ABSTRACT
A rapid, simple, automatic method is described for the measurement of glucose oxidase. Hydrogen peroxide produced by the enzymic oxidation of glucose rapidly oxidizes iodide to iodine. The rate of formation of iodine, which is equal to the enzyme activity, is measured by an automatic amperometric system. Measurement times for the determination of enzyme concentrations between 0.01 and 0.1 units per milliliter are between 10 and 100 sec. Relative standard deviations are within 2%. Interference studies and comparisons with independent methods indicate that the accuracy of the method is within 2%. ACKNOWLEDGMEXTS This investigation was supported Grant GM-16681 from the Xational samples from Eli Lilly and Company,
in part by Public Health Service Research Institutes of Health. The receipt of enzyme Indinnapolis, Ind.. was greatly appreciated.
REFEREPiCES 1. BODAN~KY, O., Am. J. Clin. Path. 38, 343 (1962). 2. “International Union of Biochemistry, Commission on Enzymes,” Vol. 20, pp. 10, 11. Pergamon Press, New York, 1961. 3. KEILIN, D., AND HARTREE, E. F., Biochem. J. 42, 221 (1948). 4. MALMSTADT, H. V., AND PARDUE, H. L., Anal. Chem. 33, 1040 (1961). 5. PARDUE, H. L., Anal. Chem. 35, 1240 (1963). 6. “Sigma Assay Form 322, Glucose Oxidase,” Sigma Chemical Co., St. Louis 18, MO. 7. UNDERKOFLER, L. H., Proc. Intern. Symp. E,rlzyme Chem., Tokyo, Kyoto 2, 486 ( 1957).