Nakai for encouragement, and to Sumiko Kurobane for her assistance throughout the investigation. LITERATURE CITED
Collection Czech Chem. Commun. 24, 1723 (19591. -\
I
(67 Connick, R. D., I;. S. At. Energy Comm., Rept. MDDC-1245 (1947). ( 7 ) Day, H. O., Gill, J. S., Jones, E. V., Marshall. W. L., Zbid.. Rent. AECD3506 (1963). (8) Dizdar, Z. I., Obrenovic, I. D., Anal. Chim. Acta 21,560 (1959). (9) Jensen, K. J., Mundv, R. J., “Analytical Chemistry of t h e Manhattan Proiect.” n. 214, hIcGraw-Hill, New Yoik, 1956. (101 Jones. M.E.. Rider. B. F.. Hendrick‘ son, H. ’C., U . ‘ S . A i . Eneigy Comm., Rept. KAPL-1497 (1956). (11) Katz, J. K., Seaborg, G I., “The I
(1) Ahaland, S., Acta Chem. Scand. 14, 2035 (1960). (2) Armson, J., Mason, H., Cockaday, R. E.. Worthineton. R. E.. U . S . At. Enerou Comm.. Reot‘ SCS-d-4 (19491. (3)Bh&agar, D. P. J . Sci. Znd. Res. (India) 16B, 23 (1957). (4) Booman, G. L., ELliott, M. C., Kimball, R. B., Cartan, F. O., Rein, J. E., ANAL.CHEM.30,284 (1958). ( 5 ) CepelBk, J., Maly, J., T’esely, V.,
.
Chemistry of the Actinide Elements,” p. 186, Wiley, Sew York, 1957.
(12) Menis, O., Manning, D. L., Goldstein, G., U . S. At. Energy Comm., Rept. ORNL-2178 (1956). (13) Moore, R. L., Schmidt, H. R., Ibid., Rept. HW-14603 (1949). (14) Parlour, A. K., Ibid., Rept. CC-1432 (1955). (15).Rygan, J. A., Sanderson, J. R., Winsor, D., Zbid., Rept. SCS-R-378 (1955). (16) Sanderson, J. R., Ibid., Rept. SCSM-104 (1948). (17) Ibid., Rept. SCS-M-129 (1950). (181 Ibid.. R e d . SCS-R-146 (19501. (19) Schneidkr, R. A., Rasmussen; M.J., Ibid., Rept. HW-53368 (1959).
RECEIVED for review September 23, 1963. Accepted December 30, 1963.
A n Automatic Potentiometric Method for the Determination of the Absolute Activity of Glucose Oxidase HARRY
L.
PARDUE cind ROBERT K.
SIMON’
Department of Chemixtry, Purdue University, Lafayette, Ind.
HOWARD V. MALMSTADT Department o f Chemidry and Chemical Engineering, University of Illinois, Urbana, 111.
,A rapid and simple automatic method is described for the assay of glucose oxidase. The method is based on the specific catalysis by glucose oxidase of the oxidation of glucose. Hydrogen peroxide produced by the enzymatic reaction rapidly oxidizes iodide to iodine in the presence of Mo(VI). The rate of oxidation of glucose, and therefore the rate of production of iodine, is equal to the glucose oxidase activity. The formation of iodine is detected potentiometrically. Automatic control equipment provides direct readout of the time required for a predetermined amount of iodine to be produced. The reciprocal of the measured time interval is proportional to the glucose oxidase activity. A simple calibration procedure is described for evaluating the proportionality constant. Automatic results for commercial samples show relative standard deviations of about 270. Measurement times tire between 10 and 100 seconds.
I
there has been increasing interest in the development of automatic methods for measuring the rates of enzyme catalyzed reactions (1, 3,6, 7 ) . Applications of these methods t o the determination of enzyme activiN RECENT YEARS
Present address. Chemistrv Deuartment, University (9 I/Iarylan&, ~d11ege Park, Md.
ties t o date have been restricted to the measurement of relative activities. I n many cases reliable enzyme standards are not, available and relative values of enzyme activity are not satisfactory. I n this work i t is shown that the method for measuring reaction rates described by Malmstadt and Pardue (6) is ideally suited to the automatic measurement of absolute enzyme activities. A simple procedure is described for the measurement of the absolute activity of glucose oxidase. The method is independent of any enzyme standards. It offers the advantages of simplicity, speed, and improved reliability over conventional procedures (6,8,9). The method is based on the specific catalysis by glucose oxidase of the oxidation of p-D-glucose by oxygen. Hydrogen peroxide produced by the enzymatic reaction rapidly oxidizes iodide to iodine in the presence of Mo (VI). The rate of formation of iodine is detected potentiometrically and the time required for a small predetermined amount of iodine to be produced is measured automatically (6). The measured time is inversely proportional to glucose oxidase activity. T o ensure that the method provides accurate values of absolute enzyme activity the possible influence on the enzyme reaction of each reagent used in the reaction system was investigated. Under the conditions described there is no interference from a n y of the reagents.
Also, recommendations of the International Union of Biochemistry’s Commission on Enzymes ( 4 ) for the method of analysis, unit of activity, and conditions to be used have been strictly followed. The commission recommends that wherever possible enzyme activities be based upon initial rates of reaction. An enzyme activity unit U is defined as that amount which will catalyze the transformation of one micromole of substrate per minute under defined conditions. Conditions to be defined are: temperature which is to be 25.0” C. where practicable, pH which is to be optimum, and substrate concentration which is to be sufficient to saturate the enzyme. The concentration of an enzyme is expressed in units per milliliter and the specific activity in units per milligram of protein. The described procedure yields results with relative standard deviations within 2y0 over periods of ten days. Since there is no interference in the chemical step the accuracy is limited only by the calibration step and is about 2% relative. Measurement times range from 10 to 100 seconds for enzyme concentrations between 0.03 and 0.003 units per milliliter in the assay solution. PRINCIPLES OF THE METHOD
According to the reaction sequence described earlier ( 6 ) the rate of formation of iodine is equal to the rate of oxidation of glucose. The activity is VOL. 36,
NO. 4, APRIL 1964
735
by definition equal to the rate of transformation of glucose and therefore equal to the rate of formation of iodine. The concentration of enzyme in the reaction mixture is determined simply as the rate of increase of iodine concentration. The rate of change in iodine concentration is determined by measuring the time required for the cell voltage to change over a predetermined amount (6). The voltage interval is calibrated in terms of iodine concentration. It is observed experimentally t h a t at a given enzyme concentration the reaction proceeds at a constant rate for periods of time much longer than the measurement intervals used here. Therefore the iodine concentration interval divided by the measured time interval gives the enzyme concentration directly.
where [ E ]is the enzyme concentration in units per ml., A I I z ]is the iodine concentration interval in pmoles per ml. (constant), and At is the measured time interval in minutes. EXPERIMENTAL
Instrumentation. T h e experimental setup a n d automatic control equipment is t h a t described for the potentiometric determination of glucose ( 6 ) . The quantitative data reported here were obtained using the auxiliary relays system described originally. However, a Sargent Reaction Rate Adapter for the Model Q Concentration Comparator (E. H. Sargent and Co., Chicago, Ill.) gave comparable results. Temperature is controlled to within =1=0.01"C. using conventional control equipment and a circulating pump. Samples and reagents are handled with hypodermic syringes. The reaction mixture is removed from the sample compartment with an aspirator tube. 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 0.01 to 0.1 units per ml. Enzyme solutions which are kept for several days are stored a t 4" C. immediately after preparation. Five commercial samples were investigated. Specific activities in units per milligram determined at 35.0" C., p H 5.10, and final glucose concentration of 0 . 1 i M were reported as 1.30, 16.8, 39.0, 88.7, a n d 37.8. BUFFER. Sodium acetate (0.25J4) is prepared by dissolving 20.5 grams of Baker's anhydrous powder in water and diluting to 1 liter. Acetic acid (0.25M) is prepared by mixing 14.2 ml. of glacial acetic acid (99.8%) with water and diluting to one liter. An acetate buffer
736
ANALYTICAL CHEMISTRY
of p H 5.05 is prepared by mixi ng these solutions in a ratio of two to one. Final adjustment of p H is made using a p H meter. IODIDE..4 fresh (0.25M) stock solution is prepared every 2-3 days by dissolving 4.15 grams 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 gram of U.S.P. resublimed crystals in a K I slurry (41.5 grams per 50 ml.) and diluting to 1 liter. Standardization against primary standard AszOa gave a value of 2.31 X 10-4Alf. COMPOSITEREAGENT. A composite reagent is prepared by dissolving 45.0 grams of D (+) - Dextrose (Pfanstiehl Labs., Inc., Waukegan, Ill.) and 0.185 grams of ammonium molybdate [ (XH& M 0 ~ 0 ~ ~ . 4 Hin~ 0250 ] 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 (0.17M), Mo(V1) (5.45 X 10-5M) and p H (5.10). REFERENCESOLUTION. A reference iodine solution (98.ipM) is prepared by dissolving 0.0125 gram of U.S.P. resublimed crystals and 6.9 grams of potassium iodide in 500 ml. of the composite reagent. Potentiometric Calibration. The potentiometric response of the concentration cell is calibrated by adding aliquots of the standard iodine solution t o t h e sample compartment containing t h e final assay composition of all reactants except the enzyme. Deionized water is added to replace the enzyme solution added in the assay step. The amount of iodide solution added in each calibration step is adjusted so that the final volume is always 3.00 ml. and the final iodide concentration is always the same as in the assay step. The calibration is carried out in the region of a concentration null-point between the sample and reference solutions ( [121ref. = 98.7pM). The voltage interval used in this work was from 0.00 to +5.00 millivolts with respect to the reference electrode. The average calibrated value for the concentration interval corresponding to this voltage interval was found to be 45.3phl compared to a theoretical value of 46.3pM computed from the Nernst equation. Procedure.
PREPARATION
with two one-milliliter portions of water between runs. ANALYSIS STEP. Exactly 1.00 ml. each of composite reagent and potassium iodide solutions are added to the sample cell and the stirrer is started. Then 1.00 ml. of the enzyme sample is added to the sample cell and the semiautomatic switch is closed momentarily. After the measurement is completed automatically the reaction time is read from the timer and the enzyme activity is computed. CALCULATIONO F RESULTS. The specific activity of the dry enzyme preparation is calculated using Equation 2. A = -li = (
w
0.0453 X
where 0.0453 is the iodine concentration interval expressed as pmoles per ml., 3.00 is the final volume of the reaction mixture in ml., W is the weight of dry enzyme taken expressed in mg., At is the measured time in seconds and the miltiplier 60 converts the time base from seconds to minutes. RESULTS
Plots of reciprocal time us. enzyme concentration give linear curves with zero intercept. This was verified b y both the potentiometric method described here and by a pH-stat method based on the detection of gluconic acid formed by the enzymatic reaction. The potentiometric method was used to determine the activity of five commercial glucose oxidase preparations. All runs were made two days after receipt of the enzyme. Results for these preparations are given in Table I along with comparative data reported by the suppliers. -4ctivities reported by the suppliers were determined by titration of gluconic acid produced at 35" C. and were corrected to 25" C. by multiplying by a n empirically determined temperature coefficient of 0.614. The relative standard deviation for the potentiometric method is demonstrated to be in the range of 2%. Agreement between the commercial methods and the more rapid and simple potentiometric method is quite good.
OF
EQUIPMENT. Electrical connections are made as described in t h e original paper (6). The function switch on the Model Q Concentration Comparator is set at the P N P *0.01 position. The zero adjust is set at 2.5 (5.00 mv. bias). The reagent selector switch is set in position 2. The reference solution is changed once daily. After adding reference solution to the reference compartment it is adjusted to 25.0"C. by circulating water from the constant temperature bath. All samples and reagents are adjusted to the working temperature by immersion in the water bath. The sample compartment is rinsed
DISCUSSION
I n order that the assay method provide accurate values for glucose oxidase activity it is necessary that none of the reagents used interfere n i t h the enzyme reaction. Also, i t is necessary to establish optimum reaction conditions. A pH-stat method which detected the formation of gluconic acid was used t o provide independent information on each of these points. Results of this work are discussed here. Glucose Concentration. Plots of reaction rate us. glucose concentration have the general shapes expected from
Michaelis-Menton kinetics. A final glucose concentration above 0.05M is necessary to provide maximum glucose oxidase activity in the enzyme concentration range exainined here. The final assay concentration of 0.17M used in this work is in a region of zero slope and is consistent with the value used in commercial procedures (8). Optimum pH. Th(h pH of maximum activity under t h e conditions of this work is observcd t o he a t 5.1. This is in agreement with t h ? work of Franke and Lorenze (8) and others. This is the p H selected for the assay procedure. Oxygen Concentration. T h e normal oxygen content of t h e air saturated solutions is sufficient t o sustain t h e reactions over thl3 intervals measured in this work. This point is verified by observations that there is no increase in reaction rate with continuous passage of oxygen t h u g h the reaction mixture. This is in marked contrast t o conventional procedures u hich require continuous aeration during the reaction. Mo(V1) Concentration. hlo(V1) concentrations betneen 5.5 X 10-6 and 5.5 X lO-4M have no effect on the enzymatic reaction. However, for molybdenum concentrations above 1 X 10-3M the reaction is accelerated up to three times its n o r m d rate depending upon the actual concentration. Also these higher molybd1:num concentrations shifted the p H niaximum in phosphate buffer from 5.1 u p to 6.0. This
Table 1. Automatic Assay Results for Commercial Enzyme Preparation
Glucose oxidase activity Reported“ Found 0.80 10.3 23.9 23.2 54.4
0.83 10.4 23.4 23.4 54.1
Rel. std. dev.,
Difference,
o/G
%
1.0 3.0 1.2 2.0 0.5
f3.8 $1.0 -2.1 $0.9 -0.6 a Suppliers values reported at, 35” C. normalized to 25’ C. by multiplying by a temperature coefficient of 0.614.
ing rise to erroneous results. However, i t has been demonstrated t h a t catalase does not compete effectively for hydrogen peroxide in this reaction sequence (7). ACKNOWLEDGMENT
The receipt of enzyme samples from Eli I A y and Co. of Indianapolis, Ind., was greatly appreciated. LITERATURE CITED
( 1 ) Blaedel, W. J., Hicks, G. P., Anal. Biochem. 4, 476 (1962).
latter effert was not observed in acetate buffers. The Mo(V1) concentration of 5.45 X 10-5M used in this work is well below the range where interference occurs. Iodide Concentration. Iodide and chloride added as t h e potassium salts u p t o 0.2M had no effect on t h e enzyme reaction. T h e final iodide concentration of 0.08M used in this work is sufficient t o cause rapid reduction of t h e hydrogen peroxidc as i t is produced. Iodine. T h e micromolar amount of iodine produced during t h e reaction interval does not affect t h e reaction velocity or p H nor does i t cause detectable oxidation of glucose a t p H 5.1. Catalase. Catalase in t h e enzyme preparations could compete with iodide for the hydrogen peroxide giv-
(2) Franke, W., Lorenze, F., Liebig’s Ann. der Chemie 532, 1 (1937). (3) Guilbault, G. G., Tyson, B. C., Jr., Kramer, I>. N., Cannon, P. L., Jr., ANAL. CHEM.35, 582 (1963). (4) “International Union of Biochem-
istrv. Commission on Enzvmes.” T’ol. 20, pp. 10, 11, Pergamon“PresA, New “
I
York, 1961. ( 5 ) Keilin, D., Hartree, E. F., Biochem. J., 42, 221 (1948). (6) Malmstadt, H. V., Pardue, H. L., ANAL.CHEM.33, 1040 (1961). ( 7 ) Pardue. H. L.. Zbid.. 35. 1240 (1963). (8) “Sifima Assay ’Form 322,‘Glucose Oxi-
dase, Sigma Chemical Co., St. Louis 18, Mo. (9) Underkofler, L. A., Proc. Intern. Symp. Enzyme Chem., Tokyo and Kyoto, 2,486 (1957).
REWIVEDfor review November 4, 1963. Accepted January 10, 1964. This investigation was supported in part by Public Health Service Research Grant GM-10681 from the National Institutes of Health.
Potentiometric Titration of Metal Ions Using a Silver Electrode JAMES S. FRITZ and BARBARA 8. GARRALDA Institute for Atomic Re.search and Department of Chemistry, Iowa State University, Ames, Iowa
b Metal cations can be titrated potentiometrically with (cthylenedinitri1o)tetraacetic acid (EDTA) using a silver indicator electrode if CI trace of silver ( I ) is added before the titration. Magnesium(ll), calcium(ll), strontium(ll), and barium(l1) are titrated directly at pH 9.0 to 10.5. Many metal cations can be determined by addition of excess EDTA, followed by backtitration with calcium nitrate. The direct titration has been applied to the determination of total hardness in water.
R
Strafelde, (3) published a method for the potentiometric titration of silver(1) with (ethylenedinitri1o)tetraacetic acid (EDTA) using ECEKTLY
a silver metal indicator electrode. H e obtained very good titration curves a t p H 9 to 10, and indicated that the electrode potential reaches a constant value very quickly. The titration is limited to rather dilute solutions because of the limited solubility of silver (1)-EDTA. Reilley and coworkers (1, 2 ) have made extensive use of a mercury metal indicator electrode for titration of metal ions with EDTA. I n their method, a small amount of mercury(I1) is added to set up a reversible potential with the mercury electrode. We have found that the silver electrode can be used for the potentiometric titration of various metal ions with EDTA in a somewhat analogous manner. However, the potential of the silver electrode
is largely independent of the substance titrated, while in most cases the potential of the mercury electrode depends on the stability constant of the E D T A complex of the metal titrated. Thus, the silver electrode is an electrode of the second type in our method; in most EDT.1 titrations the mercury electrode functions as a n electrode of the third type. The silver electrode attains equilibrium more rapidly in basic solutions than does the mercury electrode. I n some cases, especially in the titration of barium(I1) with EDTA, the potentiometric end point is sharper using the silver indicator electrode. I%ecause silver(1)-EDTA is a rather weak titrations must complex ( K = be carried out in alkaline solution, and VOL. 36, NO. 4, APRIL 1964
737
