available and continuous variation of the offsetting constant current source were possible, greater sensitivities could be reported. Note that since the working electrode is grounded, pickup of 60 Hz line frequency is eliminated. Ground-loop problems are circumvented since the digipotentiogrator is very small and can be mounted next t o the cell. Also, the leads to the cell are kept short. Figure 9, trace A shows a n anodic stripping polarogram of lead and copper taken a t the 2.5 and 12 nanomolar levels, respectively. The very large peak a t the right is due to the stripping of the added mercury plated during the amalgam accumulation step. The copper and lead peaks are barely discernible in the background. After a ten-fold display expansion of trace A shown in trace B , the lead and copper peaks are much more in evidence. High resolution digital recording is an almost indispensible aid to anodic stripping analysis. It permits the recording of an entire sweep with the option of expanding fullscale, after the fact, any portion thereof for visual inspection. This convenience feature is entirely lacking when analog signal recording is employed. Further, after transferring t o magnetic tape, the digital data can be processed with a computer to remove the sloping background contribution and to integrate the peak areas. The digipotentiogrator is presently being used in a n environmental study t o determine lead in natural waters. The present device with its *lO-V pulsed current sources does not have sufficient potential drive to operate the rotated mercury coulometry cell ( 4 ) over the full requisite 1 2 V us. SCE range; however, it is perfectly stable over the *500mV range it can now control. Future design of higher voltage sources and gates will result in a more suitable instrument for coulometric applications.
-
FUTURE WORK
Considering the many threats t o the ecology from human, industrial, and transportation wastes, it will be importunate upon the scientific and engineering community to develop adequate means for making the necessary measurements of pollutants. These measurements must be made with adequate accuracy and at a minimum cost since large numbers of analyses will be required t o force compliance. The direct conversion of charge to numerical form technique described herein seems t o be quite promising in fulfilling these needs and should be considered. A single digipotentiogrator could be made to serve many different measurement or control functions. It could, for example, measure sequentially, current related spectrometric data, perform polarography, d o constant current titrations, sense temperature, humidity, digitize specific-ion electrode and pH information, plus a host of other functions. The digital output data could be sent continuously, conveniently, and inexpensively by telephone or micro-wave relay t o a central processing center for data reduction and analysis. The fact that the instrument is inexpensive, easy to construct, and requires little power makes it very attractive for the above mentioned uses. ACKNOWLEDGMENT
The authors wish to thank E. H. Huffman and E. K. Hyde for their support in this undertaking.
RECEIVED for review December 27, 1971. Accepted March 17, 1972. Work performed under the auspices of the U. S. Atomic Energy Commission.
Rhodanese Enzyme Determination Using Ion-Selective Membrane Electrodes R. A. Llenado and G . A. Rechnitz Department of Chemistry, State Unicersity of
New Y o r k , Buffalo, N . Y . 14214
A rapid method for the determination of the animal enzyme rhodanese is described. The method uses cyanide-sensitive membrane electrodes to monitor rates of enzyme catalyzed reactions under controlled conditions. Data on electrode parameters and experimental conditions are critically evaluated for analytical purposes. THEANALYTICAL CHEMISTRY of enzyme catalyzed reactions (I, 2)-i.e., the determination o f substrates, activators, inhibitors, and the enzymes themselves-has recently’received considerable attention, especially in conjunction with the use of ion selective membrane electrodes (3, 4). The large number of recenr publications (5-15) indicates that membrane (1) G. G. Guilbault, ANAL.CHEM., 42, 334R(1970). (2) G. G. Guilbault, “Enzymatic Methods of Analysis,” Pergamon Press, New York, N.Y., 1970. (3) R. A. Durst, “Ion Selective Electrodes,” NBS Spec. Pub/. 314, U.S.Government Printing Office, Washington, D.C., 1969. (4) G. A. Rechnitz, ANAL.CHEW,41 (12), 109A (1969). (5) S. A. Katz and G. A. Rechnitz, Z. Anal. Chem., 196, 248 (1963). (6) S . A. Katz, ANAL.CHEW, 36,2500 (1964). 1366
ANALYTICAL CHEMISTRY, VOL. 44, NO. 8, JULY 1972
electrodes are gaining significant utility in this area of analysis. We now report on the development of a n electrode method for the assay of rhodanese enzyme activity. The enzyme rhodanese, so named by Lang (16) and characterized extensively (17, la),catalyzes the formation of thiocyanate from the substrates cyanide and thiosulfate according to the equation (7) S . A. Katz and J. A. Cowans, Biochim. Biopliys. Acta., 107, 605 (1965). (8) G. G. Guilbault, R. K. Smith, and J. G. Montalvo, Jr., ANAL. CHEM., 41,600 (1969). (9) G. G. Guilbault and J. G. Montalvo, Jr., J. Amer. Cliem. SOC., 92,2533 (1970). (10) G . G. Guilbault and E. Hrabankova, ANAL.CHEM.,42, 1779 (1970). (11) B. F. Erlanger and R. A. Sack, Anal. Biochem., 33, 318 (1970). (12) G. Baum and F. Ward, ibid., 42,487 (1971). ( 1 3 ) G. Baum and F. Ward, ANAL.CHEM., 43,947 (1971). (14) R. A. Llenado and G. A. Rechnitz. ibid., p 1457. (15) Zbid., 44,468 (1972). (16) K. Lang, Biocliem. Z., 259,243 (1933). (17) P. Horowitz and F. Detoma, J. Biol. Chem., 245, 984 (1970). (18) P. Horowitz and J. Westley, ibid., p 986.
Thus, it is clearly possible to measure rhodanese enzyme activity by following the rate of disappearance of cyanide or the appearance of thiocyanate, both ions that can be sensed by commercially available membrane electrodes, although it will be shown below that it is more advantageous to monitor the consumption of cyanide than the production of thiocyanate for the purposes of the present method. EXPERIMENTAL Reagents. The enzyme rhodanese (thiosulfate : cyanide sulfur transferase, EC 2.8.1.1) from beef liver was obtained from Sigma Chemical Co., St. Louis, M o . 63118. Enzyme solutions were prepared with the usual precautions on the day of use in distilled deionized water and kept cold until needed. Aqueous solutions of cyanide and thiosulfate were prepared in buffer from Fisher ACS certified sodium salts. Solutions of cyanide and thiosulfate were combined on the day of use, aliquots taken and diluted with buffer to keep pH and ionic strength constant. The latter solution as calculated is taken as the final substrate composition. Procedure for Rate Measurement. All measurements were carried out in a double walled thermostated cell with temperature control of 1 0 . 1 "C. The cell was covered with insulating foam which served as a holder for the Orion 94-06A cyanide activity electrode and an Orion 90-01 sleeve type reference electrode together with a mercury thermometer as shown in Figure 1. Reaction solutions were stirred by means of a Corning L M - 2 vibratory stirrer. Potentials were monitored and automatically displayed on a Beckman model 1055 pH recorder. To measure rhodanese activity, 10 ml of the substrate solution is pipetted into the cell and allowed to equilibrate to the desired temperature, e.g., 35.0 "C. Then 100 pl of the rhodanese enzyme preparation is rapidly delivered by means of an Eppendorf microliter pipet into the system at equilibrium. The cyanide consumed during the reaction is monitored by the electrodes and a plot of potential us. time is automatically recorded. Typical E us. t plots are shown in Figure 2. Such plots express the rate of reaction in terms of change of potential per unit time.
.
TT
I
Figure 1. Enzyme determination set-up A . Orion 94-06A electrode B. Reference electrode C. Corning LM-2 vibratory stirrer D. To Beckman pH recorder E. Thermometer F. Insulating cap G . Substrate H . Inlet for enzyme addition 1.
J.
K.
Double walled beaker Circulating water inlet Outlet
RESULTS AND DISCUSSION
To evaluate the feasibility of using the cyanide membrane electrode for monitoring the rate of rhodanese catalyzed reaction 1, isopotential selectivity ratios of cyanide over the other ions were measured by the method of Srinivasan and Rechnitz (19). Values calculated from Figure 3 show that selectivity ratios for cyanide over thiosulfate, thiocyanate, and sulfite are approximately 10, 1000, and 10,000, respectively. It is thus possible for the cyanide electrode to monitor the disappearance of cyanide in reaction 1 without serious interference from the other ions present during the course of the reaction. For best results, the thiosulfate concentration must be kept as close as possible to the concentration of cyanide. This is so because thiosulfate and cyanide react on a 1 : l basis as shown in Equation 1, and Equations 3 and 4 will be valid only as both [CN-] and [S2032-] change continuously and homogeneously in the same proportions to each other. This effectively cancels out thiosulfate interference and its effect is only to shift the potential. As with any enzyme assay, critical control of the kinetic parameters is required, and we proceeded to investigate the effect of pH, substrate concentration, and temperature. (19) K. Srinivasan and G. A. Rechnitz, ANAL.CHEW,41, 1203 (1969).
Figure 2. Effect of heat on rhodanese catalyzed reaction Effect of pH. The rate at which an enzyme catalyzed reaction occurs is dependent on the pH of the system. It can be shown that the enzymatic rate is maximal in a certain pH range. The experimentally determined pH dependence of the rhodanese catalyzed reaction is shown in Figure 4. The optimum pH range for such a reaction is, however, a function of the operation of several different factors, i.e., substrate and enzyme concentration, presence of activators or inhibitors, nature of the buffer used, and the method of measuring the rate of reaction. Thus, the pH optima observed using phosphate buffer, pH 7.9, differed from those observed using borate and Tris buffers, e.g., pH 8.6-8.7. ANALYTICAL CHEMISTRY, VOL. 44, NO. 8, JULY 1972
1367
Figure 3. Selectivity plots of the Orion cyanide electrode (A) (C)
-
Cyanide, ( B ) Thiosulfate, Thiocyanate, (D) Sulfite
I
I
!
1
7
8
9
10
I
PH
Figure 4. pH dependence of rhodanese catalyzed reactions at 35 "C. [CN-] = [S203?-] = 1 X M ( A ) N ~ H Z P O ~ . H ~ O / Nbuffer ~ O Husing 3 mg enzyme ( B ) Na2B10710H20/HCI buffer using 3 mg enzyme (0 Tris/HCI buffer using 3 mg enzyme (a) NaH2P04.H20/NaOH buffer using 1 mg enzyme (6) Na2B4O7lOH20/HCI buffer using 1 mg enzyme ( c ) Tris/HCl buffer using 1 mg enzyme
Experimental p H optima for borate and Tris agreed well with literature values. Effect of Substrate Concentration. The concentration of substrate is an important factor affecting the rate of a given enzymatic reaction. This dependence is shown by the Michaelis-Menten equation below where u i is the initial rate, V,,,, is the maximum attainable rate at a given set of conditions, Km is the Michaelis constant and [s] is the substrate concentration.
K,,, values relate to substrate concentrations and are useful in predicting the range a t which enzyme catalyzed reactions are analytically practical ( I , 2), e.g., ct is directly proportional to [SI when [SI Km, the rate of reaction can be taken as a measure of the enzyme activity. In our study of reaction 1, with other variables (e.g., pH, temperature, and amount of enzyme) constant, we initially made measurements with solutions having [CN-] constant at to 10-4M and [S203*-]varied from 1.3 X lo-* to 4.0 X 10-4M. Measurements above 10-2Mare difficult because of a quick erosion of the electrode surface which gives drifting potentials, in addition to the fact that it is difficult to measure small changes in [CN-] at high initial background [CN-1. This was particularly troublesome when the initial [S2032--]used was low, i.e., [S2032-]< 0.2 [CN-1. On the other hand the initial [S2032-]used must not exceed the initial [CN-] used by more than a factor of 5 since serious interference is observed with K C S - I S ~selectivity O~ ratio of approximately 10 for the cyanide electrode. The use of more dilute solutions of cyanide and thiosulfate minimized interference due to the latter effect, but also reduced both sensitivity and electrode response characteristics. A compromise substrate composition was therefore needed, one that would give maximum sensitivity as well as smooth and rapid response in the whole range of enzyme activity. We studied the rate of reaction of solutions which had inito tial concentration of [CN] = [S203]between 1.2 X 4.0 x lO-4M. The results are illustrated in Figure 5 and show that there is a decrease in the rate of reaction at high substrate concentration. This rate depression is not predictable from the Michaelis equation although it has been observed in a variety of enzyme reactions. It may be due to a number of causes : namely, competitive inhibition, formation of ineffective complexes, or it may reflect a limitation in the rate measurement technique. The results of Mintel (20) (20) R. Mintel and J. Westley, J. Biol. Chem.,241, 3386 (1966).
1368
0
ANALYTICAL CHEMISTRY, VOL. 44, NO. 8, JULY 1972
Table I. Initial [CN-1, M
x x x 2.0 x 8.0 x 1.0 x 5.0 x
4.0 1.0 1.0
a
Summary of Effect of Substrate Concentration on the Rhodanese Catalyzed Reaction Initial [ S Z O S ~M -], Km5#"3'-, M Remarks
10-4 10-3
10-3 10-3 10-3 10-2
Concentration range used for
x x 2.0 x 7.0 x 1.0 8.0
10-3 to 2 . 0 x 10-4 to 2 . 5 x 10-3 to 8 . 0 x 10-3 to 1 . 3 x
10-3
Lowest useful concentration Optimum concentration K , = [s] at V = V,, Lineweaver-Burk plot (22) Lineweaver-Burk plot ( 2 2 ) Too high concentration Reference 21 Reference 20
1 . 2 x 10-3 5.9 x 10-3
10-a 10-3a 10-25
x 10-3 6.30 x 10-3
8.7
6.67 X 1 0 - 3 Kms203'-
measurement.
35 c
'5
z>
t
30-
E
-f
25-
L
8
* 0
20.
t
e
15
-
101 20
30
25
35 40 Temperature, 'C
45
50
Figure 5. Substrate concentration dependence of rhodanese catalyzed reaction at 35"C, pH 8.6 borate buffer, [CN] = [S*032-I = [SI
Figure 6. Temperature dependence of rhodanese catalyzed reaction. pH8.6 borate buffer, [CN] = 1 X lov3M , [S2032--] = 2 x 10-3 M
and Blumenthal (21) who carried out rate measurements on the same reaction, using a different technique, suggest to us that the rate depression in our work is primarily due to the limitation of our technique. Our succeeding work has done with initial [CN-] at 1.0 X 10-3M as the experimentally derived optimum concentration from Figure 4. To find the best [S?03*-]concentration when [CN-] = 1.0 X 10-3M, rate measurements were made with solutions 8.0 X to 2.5 X 10-3M in thiosulfate, keeping other variables constant. Optimum [S2032--]were found to be 1.0 X lOP3Mto 2.0 X 10-3M in terms of maximum rate as well as overall electrode response characteristics. Table I summarizes the data on our substrate studies. Effect of Temperature. The effect of temperature on the activity of enzymes manifests itself as two forces acting simultaneously but oppositely. While the overall effect is interrelated with pH, buffer system, substrate concentration, etc., enzyme activity generally increases with increasing temperature. However, the rate of denaturation of the enzyme also increases at elevated temperatures. Our study of the effect of temperature on the rate of reaction 1 is graphically illustrated in Figure 6. An optimum temperature is observed at 40 "C beyond which rapid enzyme denaturation occurs with a sharp decrease in the rate of
reaction. This temperature is not necessarily the best temperature to obtain the optimum activity of the enzyme. This is so because inactivation by heat depends also on the time an enzyme is exposed to heat as demonstrated in Figure 2 which shows progress curves of the enzyme reaction 1 at various temperatures. The initial velocity increases with the temperature, but overall linearity of the reaction is maintained only at 25 "C over the period of observation of 4 minutes. At 30, 35, and 40 "C, the bending curves indicate that the amount of active enzyme available decreases continuously. At 43 "C, the curve bends sharply after a short period indicating a rapid enzyme denaturation process. Measurement of Rhodanese Activity. To measure rhodanese activity, 100 pl of enzyme solution is rapidly delivered into 10 ml of substrate previously equilibrated at the desired temperature-e.g., 35.0 "C in the cell shown in Figure 1. The electrodes used to monitor the reaction develop a potential proportional to the cyanide activity or concentration at constant pH and ionic strength.
(21) K. M. Blumenthal and R. L. Heinrikson, J . Biof. Ckem., 246, 2430 (1971). (22) H. Lineweaver and D. J. Burk, J. Amer. Chem. Soc., 56, 658 (1934).
E
=
RT Eo - - In [CN-]
nF
(3)
The Nernst slope has a theoretical value of 61 and an experimental value of 62 mV/decade at this temperature. Differentiating Equation 3 with time and rearranging in a manner we have previously described (151, we arrive at -
d[CN-1 - dE - - X 0.372 dt dt
ANALYTICAL CHEMISTRY, VOL. 44, NO. 8, JULY 1972
(4) 1369
Table 11. Typical Rhodanese Activity Determinationsa
Mg enzyme preparation per 100-pl sample used Enzyme assay with electrodes (Values are calculated from Equation 4)
3.00
2.18
Trial I I1 I11 IV
49.1 34.4 42.8 34.4 42.8 33.5 44.6 35.3 V 42.8 35.3 Averageb 44.4 i 2.73c 34.6 f 0.753 Re1 Std Deviation, 6.15 2.18 Values are at 35 "C, pH 8.6 borate buffer (see Experimental section). b Range is std deviation. Assay values >60 enzyme units are unreliable. Lowest limit of detection is 0.10 enzyme unit.
-Log
Figure 7.
0.575
0.104
20.1 20.8 18.6 19.3 20.1 19.8 i 0.847 4.28
8.93 8.18 8.18 8.93 8.56 8.56 f 0.375 4.38
1.04 1.08 1.12 1.19 1.08 1.10 f 0.0346d 3.15
M
Orion 94-58 thiocyanate electrode calibration plots ( A ) Cyanide
( B ) Thiocyanate
and enzyme activity equals (-d[CN-]/dt) expressed as pnoles cyanide converted to thiocyanate per minute per 100 pl sample or per mg of enzyme preparation. The rate of reaction, dEldt, is taken as the change in potential per unit time at the initiation of the reaction when [CN-] is 1.0 x 10-3M. Typical values are shown in Table 11. The proposed method is useful over a roughly 600-fold range of enzyme activity. Our method appears to be more rapid and convenient, although less sensitive, than the earlier colorimetric or spectrophotometric methods (23, 24). Since these methods also require reaction termination and color development steps, it seems that the electrode method would be more readily adaptable for routine and automated analyses. The sensitivity limit of 0.10 enzyme unit is based on an assay time of 5 1 min. Longer incubation times (>30 min) lower the limit to 0.01 enzyme unit. The use of lower [CN-] and [S203*-]improved the detection limit but at the expense of longer incubation times. Guilbault et al. (25-27) have described similar work. Experiments with the Orion 94-58 Thiocyanate Electrode.
We also investigated the possibility of using the Orion 94-58 (23) B. H. Sorbo, Acta Cliem. Scand., 7,1129 (1953). (24) J. Westley and T. Nakamoto, J. Biol. Chem., 237, 547 (1962). (25) W. F. Gutknecht and G. G. Guilbault, Enciron. Lett., 2(2), 51 (1971). (26) G. G. Guilbault, W. F. Gutknecht, S. S. Kuan, and R. Cochran, Anal. Biochem., in press. (27) W. R. Hussein, L. H. vonStrop, and G. G. Guilbault, Anal. Chim. Acta, in press. 1370
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 8, JULY 1972
Figure 8. Comparison of cyanide (curve A ) and thiocyanate (curve B ) electrodes as sensors for rhodanese catalyzed reaction under identical conditions
thiocyanate electrode. Initial work involved the study of the response of the electrode to both SCN- and CN-. The calibration curves shown in Figure 7 illustrate the fact that the electrode is more selective for CN- than for SCN-. When the Orion 94-58 thiocyanate electrode was used in a manner similar to what we have just described for the determination of rhodanese, both sensitivity and response characteristics were less attractive than those of the cyanide electrode. This is graphically shown in Figure 8. Response time measurements also show that the thiocyanate electrode is more sluggish than the cyanide electrode. This and the fact that the disappearance of cyanide and the appearance of thiocyanate can both be sensed by the electrode lead to compensating potential changes which will be difficult to differentiate. Hence, it is more advantageous to monitor the disappearance of CN- with the cyanide electrode than the appearance of SCN- with the thiocyanate electrode for the purposes of the present analysis. ACKNOWLEDGMENT
We thank G. G . Guilbault for preprints of unpublished results. RECEIVED for review January 12, 1972. Accepted March 17, 1972. We gratefully acknowledge the support of a grant from the National Institutes of Health.
