P=GlucosidaseEnzyme Determination with Ion-Selective Electrodes R. A. Llenado and G. A. Rechnitz Department of Chemistry, State Uniuersity of New York, Bufalo, N . Y. 14214
A method for the determination of the enzyme p-
glucosidase is described. The method is based upon the measurement of the rate of enzymatic hydrolysis of amygdalin as monitored with a cyanide ion-selective electrode under controlled conditions. As little as 0.01 mg of enzyme per 100 p1 can be determined with a precision of 2%, relative, and an analysis time of approximately 1 minute.
AMONGTHE NUMEROUS analytical techniques employed in conjunction with enzyme catalyzed reactions, i.e., to determine substrates, activators, inhibitors, and the enzymes themselves (I, 2), electrochemical methods seem most promising because of their simplicity of operation and susceptibility to automation. By immobilizing appropriate enzymes on suitable ion selective membrane electrodes, specific potentiometric sensors have been prepared for substrates urea (3), amino acids ( 4 ) and amygdalin (5). Furthermore, the feasibility of measuring enzyme activity with ion-selective membrane electrodes has been demonstrated. For example, Katz and Rechnitz have used the Beckman cation-sensitive glass electrode for the direct potentiometric determination of urea (6) and urease activity (7) uia the conversion of urea to NH4+. Later, Katz and Cowans (8) applied this technique to study the effect of various ions on the urease-catalyzed hydrolysis of urea. This work was extended by Guilbault and coworkers (9, 10) who used the Beckman cationic glass electrode for the assay of deaminase enzyme systems : urea-urease, glutamine-glutaminase, asparagine-asparaginase, D- and L-amino acidsamino acid oxidases. Erlanger and Sack (11) determined the operational normality of a-chymotrypsin with a fluoride electrode while Baum (12) recently reported an electrometric method for the determination of cholinesterase activity based on the use of a liquid membrane ion selective electrode which responds continuously to the concentration of acetylcholine remaining during an enzyme catalyzed hydrolysis. This work has been extended for the determination of organophosphate pesticide via the inhibition of acetylcholinesterase activity (13). (1) G. G. Guilbault, ANAL.CHEM.,42, 334 R(1970). (2) W. J. Blaedel and G. P. Hicks, Analytical Applications of Enzyme-Catalyzed Reactions in “Advances in Analytical Chemistry and Instrumentation, C. N. Reilley, ed., Vol. 3, J. Wiley and Sons, New York, N.Y., 1964. (3) G. G. Guilbault and J. G. Montalvo, Jr., J. Amer. Chem. Soc., 92, 2533 (1970). (4) G. G. Guilbault and E. Hrabankova, ANAL.CHEM.,42, 1779 ( 1970). (5) R. A. Llenado and G. A. Rechnitz, ibid., 43, 1457 (1971). (6) S. A. Katz and G. A. Rechnitz, 2. Anal. Chem., 196, 248 (1963). (7) S. A. Katz, ANAL.CHEM.,36, 2500 (1964). (8) S. A. Katz and J. A. Cowans, Biochim. Biophys. Acta., 107, 605 (1965). (9) G. G. Guilbault, R. K. Smith, and J. G. Montalvo, Jr., ANAL. CHEM.,41, 600 (1969). (IO) J. G. Montalvo, Jr., Anal. Biochem., 38, 357 (1970). (11) B. Erlanger and R. Sack, ibid., 33, 318 (1970). (12) G. Baum, ibid., 39,6.5 (1971). ’ (13) G. Baum and F. Ward, ANAL.CHEM.,43,947 (1971). 468
0
Our preliminary studies (5) have shown that a cyanide membrane electrode can be used to study the 0-glucosidase catalyzed hydrolysis of amygdaline (14) uia the reaction: CN
0 f
2CcH,,O6
+
HCN (1)
While the concentration of amygdalin can be calculated either by an equilibrium or rate method, the enzyme 0-glucosidase, being catalytic, can be determined only by the latter technique (15). The rate of reaction is measured and is a function of the amount of substrate and enzyme and, also, inhibitors and activators if the latter two are present. In our enzyme activity studies, we controlled conditions so that the rate of reaction becomes a function of the amount of enzyme used. Earlier methods for measuring P-glucosidase activity are based on salicin (16, 17) and 4-methylumbelliferon-/3-~glucoside (18). Salicin is cleaved to give saligenin and p-Dglucose, the rate of reaction being followed spectrophotometrically, while 4-methylumbelliferon-~-~-glucoside is split specifically by the enzyme to yield the highly fluorescent-4methylumbelliferone. Guilbault and Kramer (19) developed an amperometric technique for the assay of 0-glucosidase. We now report a new analytical procedure for the assay of &glucosidase (20,21) based on the hydrolysis of amygdalin and reaction rate measurements with a cyanide responsive electrode. This method should find applications in the diagnosis of certain enzyme abnormalities and also in plant chemistry. EXPERIMENTAL
Chemicals and Reagents. The enzyme @-glucosidase,prepared from almonds, was obtained from Sigma Chemical Co., St. Louis, Mo. 63118. Enzyme solutions were prepared on the day of use in distilled deionized water and kept cold until needed. Fresh solutions of amygdalin were prepared by sequential dilution of a stock solution with NaHZP04/ NaOH buffer to keep pH and ionic strength constant. Solutions were refrigerated in Nalgene bottles until used. ~~~
~
(14) D. R. Haisman and D. J. Knight, Biochm. J., 103, 528 (1967). (15) H. B. Mark, Jr., and G. A. Rechnitz, “Kinetics in Analytical Chemistry,” Interscience, New York, N.Y., 1968. (16) N. N. Nelson, J. Biol. Chem., 153, 375 (1944). (17) P. Baruah and T. Swain, Biochem. J . , 66, 321 (1957). (18) D. Robinson, ibid., 63, 39 (1956). (19) G. G. Guilbault and D. N. Kramer, Anal. Biochern., 18, 313 (1967). (20) J. Larner, “The Enzymes,” Vol. 4, 2nd ed., P. D. Boyer, H. Lardy, and K. Myrback, Ed., Academic Press, New York, N.Y., 1960, p 369. (21) S. Veibel. “The Enzymes,” Vol. 1, Part I, J. B. Summer and ‘ K. Myrback, Ed., Academic Press, New York, N.Y., 1950, Chapter 16.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
t
6oo
TIME,
seconds
Figure 1. Typical potential us. time plot for the enzyme reaction Conditions: 10 ml 10-aM Amygdalin, 1 mg enzyme added, 37 "C, and pH 6.4 Procedure for Rate Measurement. All measurements were carried out in a double walled cell with circulating water to keep temperature constant to within k 0 . l "C. The cell was covered with insulating foam which holds an Orion 94-06A cyanide activity electrode and an Orion 90-01 sleeve type reference electrode together with a mercury thermometer. Reaction solutions were stirred by means of a Corning LM-2 vibratory stirrer. Potentials were monitored and automatically displayed on a Beckman Model 1055 pH recorder. Ten milliliters of amygdalin solution are pipetted into the cell and allowed to equilibrate to the desired temperature, e.g., 37.0 "C. Then 100 pl of enzyme solution are rapidly delivered by means of an Eppendorf microliter pipet. The cyanide produced by the enzyme reaction is sensed by the electrodes and a plot of potential us. time is automatically recorded. A typical plot is shown in Figure 1. Such a plot expresses the rate of reaction in terms of change of potential per unit time. Conditioning the Electrode. When the electrodes have not been used for some time, proper conditioning is necessary to improve response times. Repeated soaking of the electrodes in amygdalin and then quickly introducing cyanide to a final concentration of 10-3M sufficiently conditions the electrodes to the point where response times are in the order of 3 seconds or less between f60 to -70 mV, the range at which potentials are monitored. RESULTS AND DISCUSSION
Equation 2 gives a simplified mechanism that leads to the overall enzyme catalyzed reaction in Equation 1. E
+S
kl
k-
E.S k2_ P i
+E
(2)
In this equation, the substrate S combines with the enzyme E to form an intermediate complex [ES]which subsequently breaks down into product P and liberates the enzyme. In this mechanism, first formulated by Michaelis and Menten (22), it is assumed that the overall rate of the reaction at any instant is determined by the concentration of the inter(22) L. Michaelis and M. L. Menten,'Biochern. Z . , 49, 333 (1913).
Figure 3. Lineweaver-Burk plot of the data in Figure 2
mediate complex [E.S]. Thus [ES] is assumed to be at a steady state or to be in rapid equilibrium with S and E. It is also assumed that the rate of reaction is measured near the initiation of the reaction. With these assumptions, a simple rate equation was derived. (3)
It can be seen from Equation 3 that the initial rate is a function of the maximum attainable rate, R, at any particular enzyme concentration, the Michaelis constant, Km,and the substrate concentration, S. Effect of Substrate Concentration, Figure 2 shows the effect of amygdalin concentration on the initial rate of the &glucosidase catalyzed reaction. At a fixed enzyme concentration, the initial rate increases with amygdalin concentration until a nonlimiting excess of substrate is reached, after which additional substrate causes no further increase in the rate. This maximum attainable rate is R,,,. Examination of Equation 3 gives a physical interpretation of K,, the Michaelis constant. K, is that substrate concentration which gives an initial rate equal to R,,,. For the enzyme reaction, therefore, the apparent value of K, is 2.5 X 10-4M. A Lineweaver-Burk plot (23) of l/Ri us. l / [ a is shown in Figure 3. The straight line plot gives a slope equal to Km/ R,,, and an intercept l/Rn,ax. From these values, K , was found to be 2.5 X 10-4M. These Km values are useful in (23) H. Lineweaver and D. J. Burk, J. Amer. Chem. Soc., 56, 658 (1934). ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
469
Figure 4. pH dependence of the enzyme reaction Conditions: 10 ml of l o + MAmygdalin used at 37 "C and pH 6.4 Upper curve: 2 mg enzyme added Lower curve: 1mg enzyme added
Figure 6. Calibration of Orion cyanide electrode at varying pH 12.5 pH 10.2 pH 9.2 D. pH 7.4 E. pH 6.4 A.
B. C.
5 r2 b
Figure 5. Temperature dependence of the rate of enzyme reaction
predicting the range at which enzyme reactions are analytically useful ( I , 2). Effect of pH. The rate at which an enzyme reaction occurs is markedly dependent on the pH of the system. It is known that the reaction rate is maximal at a certain pH range. This optimum pH, however, is not of fundamental interest, since it is the result of the operation of several different factorsi.e., substrate and enzyme concentration, presence of activators and inhibitors, nature of the buffer, and method of measuring the rate of reaction. In the enzyme reaction studied, an optimum pH of 6.4 was found as shown in Figure 4. The pH optimum is higher than reported by others (16-19) and may reflect rhe difference in rate measuring techniques. Effect of Temperature. The effect of temperature on the activity of enzymes is very complex and is interrelated with pH, nature of the buffer, and substrate concentration. With increasing temperature, the activity increases and at the same time inactivation becomes appreciable. The temperature at which denaturation becomes decisive varjes from enzyme to enzyme. While for most enzymes, heat inactivation is negligible below 30 "Csome enzymes can be heated to >60 "C without appreciable loss of activity. Inactivation by heat also depends on the time an enzyme is exposed to heat. In 470
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
d
d
/
i
2
3
1
S
b
7
GLUCOSE UNITS
Figure 7. Correlation of cyanide and glucose units all our experiments, reactions were run and completed in 5 1 minute, not enough time for inactivation to be significant. Hence, the initial rate of reaction increases with the temperature. Results are shown in Figure 5 . Although the sensitivity of the enzyme activity measurement will increase with temperature, working above 40 OC results in appreciable solvent evaporation. Effect and Measurment of 0-Glucosidase Activity. From the Michaelis-Menten equation, the initial rate of an enzyme reaction is proportional to the initial enzyme concentration when a nonrate-limiting concentration of substrate is used. Hence, the rate can be. taken as a measure of 0-glucosidase activity when the concentration of amygdalin used is 2 10-2M. Figure 1 shows a typical E us. time curve. By varying the chart speed, various initial amounts of enzyme can be
Table I. ,%Glucosidase Activity Determination (All measurements were carried out with 10 ml of 10-*M amygdalin as substrate at 37.0 "C and pH 6.4 using 1OO-pl enzyme samples) Glucose Enzyme units" Cyanide unitscld per 100 pl, mg per 100 p1 AE/minb per 100 p1 7.4 5.6 3.7 1.8 0.74 0.37 0.18 0.074 0.037
2.0 1.5 1 .o 0.50 0.20 0. IO 0.050 0.020 0.010
670 510 350 170 90.0 60.5 35.5 30.0 7.2
2.41 1.83 1.26 0.610 0.323 0.217 0.127 0.108 0.026
1 reaction volume d[CN] d E - dt X 3.59 X dt
added to a constant volume of 10-2M amygdalin solution at pH 6.4 and 37 "C and appropriate rate curves obtained with good precision. The electrodes used to monitor the rate of the reaction develop a potential proportional to the cyanide activity. =
E"
RT
- -- InacNnF
=
nF
(5)
The Nernst slope RT/nF has a theoretical value of 61 mV/ decade change in [CN] at 37 "C and an experimental value of 64 at 37 "C and pH 6.4 (see Figure 6). The greater experimental slope is due to the complexation of CN- by H+ to form HCN (K, = 4 X This is reflected in the cyanide calibration curves in Figure 6. Differentiation of Equation 5 with respect to time yields dt
2.303
= pmoles CN- min-l
(9)
In enzyme units, Enzyme activity
=
dE/dt X 3.5 grams X
(10)
expressed as pmoles CN- liberated per min per 100 4 sample or per mg of enzyme preparation. Typical values obtained are shown in Table I. The Beckman Model 1055 recorder which was used to monitor the rate of the enzyme reaction, has as its fastest and slowest chart speed, 10 and 0.1 inch/min, respectively. At its fastest chart speed, the recorder limits the enzyme activity that can be assayed to 5 3 cyanide units. While 0.001 enzyme unit can still be detected, 0.01 cyanide unit seems the analytical limit when the recorder chart speed is at its slowest speed. By varying the chart speed to suit the experimental rate curve, precision of 2 or better is easily obtainable. CONCLUSION
RT E " - - In [CN]
d E- - -64 . - .1-
106 pmoles/mole (8)
(4)
or concentration (at constant pH and ionic strength)
E
x
and thus,
One glucose unit corresponds to I pmole of glucose liberated per minute from salicin at pH 5.25 and 37 "C by 1 mg of enzyme. b Average values of at least 5 determinations. c One cyanide unit corresponds to 1 pmole of cyanide liberated per minute from amygdalin at pH 6.4 and 37.0 "C by 1 mg of enzyme or 100 pl of enzyme preparation. d Correlation between glucose and cyanide units is shown in Figure 7.
E
The negative sign means the potential is becoming more negative with time. All rate values, dE/dt, were obtained from tangents drawn at -0 mV on the rate curve. This point is close to the initiation of the reaction and corresponds to a cyanide level of -10-5 mole/liter (see Figures 1 and 6). Introducing the reaction solution volume of 10 ml and converting moles to pmoles, we get
[CNI
d[CN] dt
A new electrochemical method is described for the determination of P-glucosidase activity. The introduction of the cyanide unit is not intended to complicate the already numerous and arbitrary enzyme units but is an attempt to establish an activity unit consistent with the method. With this new method, results are available in one minute compared to the 30 minutes required by the fluorimetric technique of Robinson (18) and the methods based on salicin (16, 17). Though fluorometric techniques may be more sensitive, this faster technique hopefully will stimulate further studies of &glucosidase content and action in both animals and plants. It would also be interesting to develop an electrochemical transducer capable of directly measuring p-glucosidase activity in the blood stream or other biological fluids. Such efforts are currently under way in this laboratory.
Rearranging, we have d[CN] -dE/dt __ - _ ___ dt 27.8
. [CNI
(7)
RECEIVED for review July 6, 1971. Accepted October 13, 1971. We gratefully acknowledge support of NIH grant GM-17576.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
471
