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Biosensing of Heavy Metal Ions Based on Specific Interactions with Apoenzymes
BIOSENSING OF HEAVY METAL IONS BASED ON SPECIFIC INTERACTIONS WITH APOENZYMES
lkuo Satoh Abstract . . . . . . . . . . . . . . . . . . . . . . . I. INTRODUCTION.. . . . . . . . . . . . . . . . . 11. PRINCIPLE . . . . . . . . . . . . . . . . . . . . . 111. CHARACTERISTICS. . . . . . . . . . . . . . . . IV. PROCEDURE.. . . . . . . . . . . . . . . . . . . V. BIOSENSING OF HEAVY METAL IONS . . . . . A. Apoenzyme Beads as Sensing Elements . . . . B. Apoenzyme Membrane as the Sensing Element VI. CONCLUSION . . . . . . . . . . . . . . . . . . .
......
. .. ... ... ... .. . ...
. .... .... .... .... .... ....
....... .......
. . . . . . . 461 . . . . . . . 462 . . . . . . . 462 . . . . . . . 463 . . . . . . . 466 . . . . . . . 467 . . . . . . . 467 . . . . . . . 470 . . . . . . . 471
ABSTRACT The present work led to a novel idea about the biosensing of heavy metal ions based on apoenzyme reactivation methods. Several kinds of immobilized metalloenzymes
Advances in Molecular and Cell Biology Volume 15B, pages 461472. Copyright 0 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0114-7
46 1
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as recognition elements for each metal ion were applied in the microdetermination of heavy metal ions in flow streams. Regeneration of the metal-free enzymes (apoenzymes) was made by loading chelating agents to the metal-bound enzymes (holoenzymes). The apoenzymes were positively reactivated by adding trace amounts of heavy metals. The recovery depended on metals trapped in the enzymes, which was shown to be a practical index for microanalysis of heavy metal ions. The proposed method was demonstrated to be convenient, safe, specific, and highly sensitive.
1. INTRODUCTION A great number of metal-dependent enzymes have already been identified (Vallee, 1980).Heavy metal ions coordinated in the active site of these enzymes play a very important role in the catalytic process. These heavy metal ions are intrinsic to each of the enzymes and most of them can be reversibly removed from the catalytic site of the enzymes. Therefore, metal-free enzymes (apoenzymes) can be used as specific and selective recognition elements for heavy metal ions in an enzymatic analysis. Use of enzymes as highly sensitive reagents is common in food and clinical analyses. Currently,immobilized enzymes in combination with transducers for monitoring their enzymatic activity are used for the construction of biosensors, and many of them are commercially available (Satoh, 1989a). Analytes have been mostly limited to substrates, products, activators, and inhibitorsin the enzyme-catalyzed reactions. In sharp contrast, we have adopted flow-injectionmicroassay for cofactors, namely heavy metal ions, based on apoenzyme reactivation (Satoh et al., 1986, 1987, 1988, 1989b, 1990ste, 1991a-e, 1992, 1993). We have tried to regenerate cofactor-free enzymes from cofactor-bound enzymes (holotype of metalloenzymes) and use them as the recognition elements for heavy metal ions in flow streams. The apoenzyme reactivation methods for flow-injection microdetermination of heavy metal ions are summarized in this paper with a special focus on our current studies.
II. PRINCIPLE Cofactors such as heaG metal ions and nucleotides are generally complexed in the active site of the metalloenzymes and flavin enzymes, respectively, and the cofactors are directly responsible for the activity of the enzymes. Therefore, these enzymes need the cofactors for expressing catalytic activity. The cofactor-bound enzyme is usually called a holoenzyme, whereas the cofactor-free enzyme is called an apoenzyme. Figure 1 schematically shows the correlation between the holoenzyme and the apoenzyme. The metalloenzymes capture the metal ions so tightly (dissociation constant: Kd< lo4 M) that they hold them throughout the purification process. In contrast with the metalloenzymes, metal-activated enzymes bind the
Biosensing of Heavy Metals
Cofactor-bound enzyme catalytically
463
Cofactor-free enzyme catalytically
[ active
[ inactive
Figure 1. lnterconversion between the holoenzyme and the apoenzyme. Cofactor: Heavy metal ions; r(lr(lS catalytic site; 000 substrate-binding site.
metal ions rather weakly (Kd < 1C3to 1O4 M), and then, the enzymes tend to lose the metal ions during purification (Wagner, 1988). Preparation of the apoenzyme lacking its catalytic activity can be made by removing the metal ion from the corresponding holoenzyme with strong chelating agents. The apoenzyme is reactivated by exposing it to the metal-containing sample so that metal ions can be taken up and trapped in the active site. Thus, the amount of metals coordinated in the catalytic center of the enzyme molecules may be closely related to the enzyme activity expressed and, in turn, be proportional to the added amount of the metals. The content of the trace metals can eventually be determined by monitoring the induced activity attributable to the reactivation of the apoenzyme. The metal ions responsible for the catalytic activity of the metalloenzymes vary with the type of enzyme. Selective determination of the metal ions depends upon choosing the appropriate metalloenzyme in which its catalytic site fits well with each metal ion.
111. CHARACTERISTICS In practice, use of metalloenzymes immobilized on supporting materials such as small glass beads and a thin polymer membrane can make assays continuous and, moreover, enhance the feasibility of handling in the process between regeneration and reactivation of the apoenzymes. Reusability and long-term stability of the immobilized enzymes may be expected. Microdetermination of heavy metal ions based on spectrophotometric monitoring was tried (Townshend and Vaugham, 1970) and a microassay for zinc(I1) ions using high performance liquid chroma-
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tographic methods (Risinger et al., 1983) as well as an electrochemical method (Mattiasson et al., 1979) were reported. We have originally developed unique biosensing of heavy metal ions based on flow-injectioncalorimetry (Satoh et al., 1986).Thermometric flow-injectionanalysis of biorelated compounds in combination with the use of a high performance semiadiabatic calorimeter were pioneered by Mosbach et al. (Danielsson and Mosbach, 1988; Danielsson, 1990). The flow-measuring system, that is, a thermal bioanalyzer, is better known as an “enzyme thermistor.” We used the calorimeter for monitoring the enzymatic activity in the assay cycle including regeneration and reactivation. The calorimetric biosensing system is schematically presented in Figure 2. The column packed with immobilized enzyme beads is interchangeable and, therefore, different kinds of metals are readily determined. In addition, other monitoring methods such as amperometry, potentiometry, and spectrophotometry are available by exchanging the thermistor with other transducers, for example, electrodes,photomultipliers, etc. The proposed biosensing methods in microanalysis of heavy metal ions do not require expensive instruments based on atomic absorption spectrophotometryand inductively coupled plasma atomic emission spectrophotometry.Furthermore, this
chela3nt
9substrate lons
I If A k m i n m bath
Figure 2. Flow-calorimetric biosensing system for heavy metal ions based on the apoenzyme reactivation method. Carrier reservoir (buffer solution), pump (flow rate 1.O ml min-’), heat exchanger (thin-walled acid-proof steel tubing: 0.8 mm, i.d.), bath (80Q x 250 mm; 303 K), enzyme column (packed with metalloenzymes immobilized on porous beads), thermistor (attachedto a gold capillary placed in a polymer holder), WB/Amp. (DC-type Wheatstone bridge with a chopper stabilized operational amplifier), Rec. (pen recorder).
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novel method does not involve pollution problems due to exhaust fumes. The characteristics of the proposed methods can be summarized as follows: 0
0 0 0
Mild assay conditions High sensitivity High selectivity Reusability of the immobilized enzymes Continuous flow use
0 0
0
0
Compact assay system Feasibility of handling Feasibility of field work Low-priced assay Separability of the metaltrapping and the activitymonitoring process
Procedure
Phenomena
Substrate
response t o Iiolocnzymcs
t Carrior
t ChelatinE t . Carrier
washing aRcnt
t
Subs t r a t c
r c g c n c r a t ion o f apociixymos washing response t o apocnzymcs
t Carricr
washing
Heavy mctal i o n s
p a r t i a l a c t i v a t i o n of apocnzymes
t
Carrier
washing
Substrate
response a t t r i b u t a b l c t o metal i o n s
t
c o o r d i n a t c d i n Clic enzymes Carrier
c
washing
Complctc r c a c t i v d t i o i i of apocnzymcs ( c o n v c r s i o o t o Iiolocnzymcs)
Carrier
washing
Figure 3. Flow chart for the assay procedure.
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IV. PROCEDURE A flowchart for the assay procedure is shown in Figure 3. Three rotary valves for injection are connected in series between a damper and a drain valve to avoid their mutual contamination. At first the catalytic activity of the immobilized holoenzymes at the reactor (column or membrane) is measured by injecting its substrate. The change in output is a measure of the activity attributable to the holoenzymes, as schematically illustrated in Figure 4. After exposing the enzymesto the chelating agent, a drastic decrease in the activity is observed. This means that the cofactors, namely heavy metal ions complexed in the catalytic site of the holoenzymes, are mostly removed. The amount of chelating agent required for regenerating the
Scan L
60 min
Y
Figure 4. Schematic presentation of response curves for a reaction cycle.
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467
apoenzymes varies with the kind of metalloenzyme and with the conditions of the chelating agents (variables such as concentration, pH, volume, etc.). Subsequent injection of a trace amount of the cofactors reactivates the apoenzymes. Thus, the recovery of the activity is determined by adding the substrate. While sufficient cofactors are introduced to the mixture of the cofactor-free and cofactor-bound enzymes, all of the enzymes may be completelyconverted to the holoenzymes. The system is then ready for another assay. A reaction cycle generally takes 40 to 60 minutes, but application of a membrane-type reactor reduces the cycle time (within 30 min).
V. BIOSENSING OF HEAVY METAL IONS A. Apoenzyme Beads as Sensing Elements Alkaline Phosphatase as the Recognition Element
Alkaline phosphatase as the recognition element in combination with a couple of monitoring devices was feasible in the most sensitive assay of zinc(I1) and cobalt(I1) ions. The enzyme purified from Escherichia cofi (EC 3.1.3.1., Asahi Chemical Industry Co., Ltd., Ohito-cho, Tagata-gun, Shizuoka-ken, Japan) was immobilized on epoxide acrylic beads (Eupergit C: 100-200 pm particle diameter; 40 nm pore diameter; 180m2g-' surface area; Rohm Pharma, Darmstadt, Germany) and then packed into a small polymer column (0.3 ml). The enzyme is often employed as a labeling reagent for enzyme immunoassay. Hydrolysis of p-nitrophenyl phosphate top-nitrophenol and orthophosphate was monitored for measuring the enzyme activity: p-nitrophenyl phosphate + H,O
+ p-nitrophenol + orthophosphate
(1)
Tris-HC1 100mM buffer @H 8.0, containing 1.OM NaC1) was used and the catalytic activity was calorimetrically determined by injecting 0.1 ml of 100 mM substrate (p-nitrophenylphosphate) (Satoh et al., 1991d). Exposing the column to 5 ml of20 mM 2,6-pyridine dicarboxylate solution (PH 6.0) as the cofactor-complexingagent almost virtually converted the holoenzymes to the apoenzymes. The effect of pH on the chelating agent in the regeneration reaction was tested. No noticeable variation in the level of the regeneration was observed over the pH range of 4.0 to 8.0. The recovery of the activity attributable to the partial reactivation of the apoenzymes was a function of the added amount of zinc(I1) ions. The calibration graph for zinc(I1) ions demonstrated a sigmoidal curve. Zinc(I1) ions ranging from 0.01 to 1.O mM were calorimetrically determined for a 0.5 ml injection. The effect of pH on the reactivation of the apoenzyme was investigated in the weakly acidic pH region (from 4.0 to 6.0 in steps of 0.5). The recovery was unaffected by variations
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in pH; this means the proposed biosensing using alkalinephosphatasedoes not need any critical pH adjustment, which is very practical. The column retained much more long-term stability than that of a column packed with immobilized bovine carbonic anhydrase. The reactor was repeated over 120 times during the two months of operation. The spectrophotometric approach to measuring enzyme activity based on detecting changes in absorbance at 405 nm attributable to p-nitrophenol formation provided similar results, except for the highly sensitive assay for zinc(I1) ions in submicromolar levels (Satoh et al., 1990b, c). Potentiometric monitoring of the activity with a flow-through ISFET for detecting pH shifts attributable to orthophosphate released in the hydrolytic reaction was also carried out to determine zinc(I1) ion concentrations in the range of 0.01 to 1.O mM (Satoh et al., 1990d). Cobalt-substituted alkaline phosphatase is also capable of hydrolyzing the orthophosphate ester. Therefore, the same immobilized preparations were applied in the microdetermination of cobalt(I1) ions. Biosensing based on spectrophotometry gave excellent results (Satoh, 1992b),and cobalt(I1) ions ranging in concentration from 1-200 pM were determined. Use of a calorimetric sensing system was possible in the concentration range of 0.04 to 1.O mM. Ascorbate Oxidase as the Recognition Element
Ascorbate oxidase is one of the most typical copper-dependentenzymes involved in the oxidation of L-ascorbate to dehydroascorbate. The reaction is described by 2L-Ascorbate + 0, + 2dehydroascorbate + 2H,O
(2)
Ascorbate oxidase from cucumber (EC 1.10.3.3, Asahi Chemical Industry Co., Ltd.) immobilized onto porous glass beads with controlled pore size (CPG; 5 1.5 nm pore diameter, 120-200 mesh, 44 m'g-', Electronucleonics Inc., Fairfield, NJ, USA) was packed into a column and then mounted in the proposed flow-calorimetric system (Satoh et al., 1987). Since oxidative reactions involving molecular oxygen are usually accompanied by considerable heat generation, highly sensitive and precise biosensing of copper(I1) ions attributable to the significant exothermic reaction was anticipated. Thus, micromolar levels of copper(I1) ions were calorimetrically determined. Regeneration of the apoenzymes was achieved with exposure of 20 mM NJV-diethyldithiocarbamate solution (PH 8.0) to the column. The apoenzymes derived from the ascorbate oxidase was selectively responsive to copper(I1) ions and not to divalent cations in 1 mM level such as Ca(II), Co(II), Mg(II), Ni(II), and zinc(I1). Trace amounts of copper(I1) ions in human blood sera were analyzed by the calorimetric method and compared with those obtained by atomic absorption spectrophotometry. There was satisfactory agreement between these methods. Amperometric monitoring of the enzyme activity with a polarographic oxygen electrode showed a more rapid and sensitive determination of copper(I1) ions. The
Biosensing of Heavy Metals
469
assay covered concentrationsranging from 0.5 to 2.0 pM and required about half an hour. L-Ascorbate can be determined by detectingabsorbance at 265 nm, which is used for monitoring the oxidase activity.Thus, copper(I1)ions measured photometrically ranged from 0.1 to 10 pM. A series of these monitoring methods validated the use of immobilized ascorbate oxidase beads as an excellent recognition element for copper(I1) ions. Carbonic Anhydrase as the Recognition Element
Carbonic anhydrase purified from erythrocytes (EC 4.2. 1.1) is known for its remarkably high turnover numbers. The enzyme is a dominant factor in equilibrium between carbon dioxide and bicarbonate in blood as shown in Equation 3, which also expresses esterase activity as seen in Equation 4. CO, + H,O p-nitrophenyl acetate + H,O
-+
H,CO,
(31
3
p-nitrophenol + acetate
(4)
Application of bovine carbonic anhydrase immobilized on the same sort of porous glass beads (CPG) for the specific determination of zinc(I1) and cobalt(I1) ions in combination with flow-calorimetric monitoring turned out to be feasible for the first time (Satoh et al., 1986, 1989b). Injection of 0.5 ml ofp-nitrophenyl acetate into the carrier streams (Tris-HC1 buffer, 0.1 M, pH 8.0) in the calorimetric system resulted in an exothermic response. Since values of changes in enthalpy for ester hydrolysis are normally zero, we considered that the exothermic change resulted from the protonizing heat of acetic acid enzymatically formed in the tris buffer. Exposing the immobilized enzyme beads in a column (packed volume, 0.3 ml) to 5 ml of 10 mM 2,6-pyridine dicarboxylate solution (pH 5.0) as the chelating agent caused regeneration of the apoenzymes. Zinc(I1) ions in the range of 25 to 250 pM could be calorimetrically measured using 0.5 ml injections. Generally, zinc(I1) ions complexed in the active site of zinc enzymes (e.g., hydrolases) are reversibly exchanged with cobalt(I1) ions, and the cobalt-coordinated enzymes still retain their catalytic activity. We also succeeded in assaying submillimolar levels of cobalt(I1) ions (0.05 to 0.2 mM) using cobalt-substituted enzymes (Satoh, 1989b). In this case, less volume of the chelating agent (2.5 ml) was sufficient for regeneration of the enzymes. Galactose Oxidase as the Recognition Element
Galactose oxidase (EC 1.1.3.9) catalyzes oxidation of D-galactose as follows: D-Galactose + 0, 3 D-galacto-hexodialdose + H,O,
(5)
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This enzyme isolated from Dactilium dendroidesreadily converted to the apoenzyme under water soluble conditions while it takes a much longer time for reactivation of the apoenzyme. Utilization of the immobilized preparation in combination with the flow-injection technique for monitoring of enzyme activity resolved the problem. Copper(I1) ions were calorimetrically determined in millimolar levels (Satoh, 1990e). Amperometric monitoring with an oxygen electrode and a hydrogen peroxide electrode made the assay more sensitive and rapid (Satoh et al., 1990a). There is room for improvement for a more sensitive response.
B. Apoenzyme Membrane as the Sensing Element Table 1 summarizes successful biosensing of heavy metal ions based on the apoenzyme reactivation method using bead-type immobilized enzymes. Application of the apoenzyme column to flow-injection biosensing demonstrated their long-term stability. Practical use has already been found in clinical and food analyses. In order to obtain a more rapid assay, a contact type of apoenzyme sensor has been developed (Satoh et al., 1992, 1993). Ascorbate oxidase immobilized onto a porous polymer membrane (partially aminated polyacrylonitrile,50 pm thickness, Asahi Chemical Industry Co. Ltd.) was directly attached to a flow-through polarographic oxygen electrode and used as the recognition element for copper(I1) Table 1. Flow-Injection Biosensing of Heavy Metal Ions Based on the Apoenzyme Reactivation Methods
Metal
Zn(I1) Zn(I1) Zn(I1) Zn(I1) Cu(I1) Cu(I1) Cu(I1) CU(I1) Cu(I1) Cu(I1) Co(I1) Co(I1) Co(I1)
Recognition Element
ALP ALP ALP BCA ASOD ASOD ASOD GalOD GalOD GalOD ALP ALP BCA
Monitoring Method
Ranae lmMl
Calonmetry Potentiometry Spectrophotometry Calorimetry Amperometry Calorimetry spectrophotometry Amperometry Amperometry Calorimetry Calorimetry Spectrophotometry Calorimetry
/T /I /P /T 10 /T /P
0.010-1.0 0.010-1.0
0.00014.010 0.0254.25
0.000~.002 0.0014.05
0.00014.010
10
0.1-10.0
/H /T
0.01-10.0 5.0-20.0 O.O&I.O 0.0014.2 0.0054.2
/T /P /T
Nofes: Enzyme:ALP, alkalinephosphatase; ASOD, Ascorbate oxidase; IBCA, Bovine carbonic anhydrase; GalOD,
Galactose oxidase. Method T (thermistor), I (pH-ISFET),P (photomultiplier), 0 (polarographic oxygen electrode), H (hydrogen peroxide electrode).
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471
ions (Satoh et al., 1992a). Use of the system mounted with the apoenzymemembrane sensor resulted in a more rapid assay (26 min). Furthermore, microdetermination of dual heavy metal ions was tried with hybrid enzymes immobilized onto the same kind of membrane (Satoh, 1993). Alkaline phosphatase and ascorbate oxidase were coimmobilized onto the polymer membrane and tightly attached to a polarographic oxygen electrode. Ascorbate oxidase functioned not only as the recognition element for copper(I1) ions but also as an indicator enzyme for amperometric monitoring of alkaline phosphatase activity on the same membrane. For sensing zinc(I1) ions, the following coupled reactions were amperometrically monitored: L-Ascorbyl-2 phosphate + H,O
-+ L-ascorbate + orthophosphate
(6)
2 L-Ascorbate + 0, + 2 dehydroascorbate + 2 H,O
(7) The apoenzyme membrane was regenerated by pumping cofactor-complexing agents for removing each cofactor, namely copper(I1) and zinc(I1) ion from the catalytic site of alkaline phosphatase or ascorbate oxidase. Thus, zinc(I1) ions in 2 to 200 pM concentrations and copper(I1) ions in 2 to 100 pM concentrations were determined through activation of each of the immobilized apoenzymes on the same supporting membrane.
VI. CONCLUSION Flow-injection microassay of heavy metal ions such as cobalt(II), copper(II), and zinc(I1) ions based on apoenzyme reactivation methods was found feasible with immobilized metalloenzymes. Regardless of the shape of the immobilized metalloenzymes, that is, beads or membrane, this method of unique monitoring is now widely applied in several analytical areas. Further developmental studies towards establishing the generality and versatility of these analytical techniques involving immobilized apoenzymes in flow systems are currently in progress.
REFERENCES Danielsson, B. (1990). Calorimetric biosensors. J. Biotechnol. 15, 187-200. Danielsson, B. & Mosbach, K. (1988). Enzyme thermistors. In: Methods in Enzymology (Mosbach, K., Ed.), Vol. 137, pp. 181-197. Academic Press, New York. Mattiasson, B., Nilsson, H., & Olsson, B. (1979). An apoenzyme electrode. J. Appl. Biochem. I , 377-3 84. Risinger, L., Ogren. L., & Johansson, G. (1983). Determination ofzinc(I1) ions with a reactor containing immobilized carboxypeptidase A in a flow system. Anal. Chim. Acta 154,251-257. Satoh, I. (1989a). Biosensing using calorimetric devices. In: Chemical Sensor Technology (Seiyama, T., Ed.), Vol. 2, pp. 26%282. Kodansha Ltd., Tokyo, Japan. Satoh, I. (1989b). Continuous biosensing of heavy metal ions with use of immobilized enzyme-reactors as recognition elements. In: Proceedings of the MRS International Meeting on Advanced Materi-
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als, Vol. 14 (Biosensors, Karube, I., Ed.), pp. 4550. Material Research Society, Pittsburgh, Pennsylvania, USA. Satoh, I. (19904. Apoenzyme reactivation microassay for zinc(1I) ions with flow-through transducers. In: Proceedings of the 3rd International Meeting on Chemical Sensors, pp. P/lO&P/l07. The Organizing Committee of the 3rd International Meeting on Chemical Sensors, Cleveland, OH, USA. Satoh, I. (1990e). Calorimetric biosensing of heavy metal ions with the reactors containing the immobilized apoenzymes. AM. N.Y. Acad. Sci. 613,401404. Satoh, I. (1991d). An apoenzyme thermistor microanalysis for zinc(I1) ions with use of an immobilized alkaline phosphatase reactor in a flow system. Biosensors and Bioelectronics6(4), 375-379. Satoh, I. (1991a). Flow-injection calorimetry of heavy metal ions using apoenzyme-reactors. Netsusokutei (Calorimetry and Thermal Analysis) (in Japanese) 18(2), 89-96. Satoh, I. (1991e). Flow-injection microdetermination of heavy metal ions using a column packed with immobilized apoenzyme beads. J. Flow Injection Anal. 8(2), 111-126. Satoh, I. (1993). Amperometric biosensing of heavy metal ions using a hybrid type of apoenzyme membrane in flow streams. Sensors and Actuators, in press. Satoh, I. (1992b). Use of immobilized alkaline phosphatase as an analytical tool for flow-injection biosensing of zinc(I1) and cobalt(I1) ions. AM. N.Y. Acad. Sci. 672,240-244. Satoh, I., Abe, R., & Nambu, T. (1988). Bioelectrochemical sensing of copper(I1) ions using an immobilized apoenzyme column. Denki Kagaku 56(12), 10451049. Satoh, I. & Aoki, Y. (1990d). Biosensing of zinc(I1) ions using an apoenzyme reactor and an ISFET detector inflow streams. DenkiKagaku58(12), 1114-1118. Satoh, I., Ikeda, K., & Watanabe, N. (1986). Microanalysis of zinc@) ion by using an apoenzyme thermistor. In: Proceedings of the 6th Sensor Symposium (Takahashi, K., Ed.), pp. 203206. Institute of Electrical Engineers of Japan, Tokyo, Japan. Satoh, I., Itoh, H., & Anzai, H. (1992a). Flow-injection amperometric biosensing of copper (11) ions using a contact-type of an apoenzyme sensor. In: Proceedings of the 2nd World Congress on Biosensors, pp. 1 8 H 9 0 . Elsevier Advanced Technology, Oxford. UK. Satoh, I., Kasahara, T., & Goi, N. (199Oa). Amperometric biosensing of copper(I1) ions with use of an immobilized apoenzyme reactor. Sensors and Actuators BI, 499-503. Satoh, I., Kimura, S., & Nambu, T. (1987). Biosensing of copper(I1) ions with an apoenzymethermistor containing immobilized metalloenzymes in flow system. In: Digest of Technical Papers, The 4th International Conference on Solid-state Sensors and Actuators (Transducers '87, Matsuo, T., Ed.), pp. 789-790. Institute of Electrical Engineers of Japan, Tokyo, Japan. Satoh, I. & Masumura, T. (1990b). Flow-injection biosensing of zinc(I1) ions with use ofan immobilized alkaline phosphatase reactor. In: Technical Digest of the 9th Sensor Symposium (Sasaki, A., Ed.), pp. 197-200. Institute of Electrical Engineers of Japan, Tokyo, Japan. Satoh, I. & Nambu, T. (1991b). Flow-injection photometric biosensing of copper(I1) ions with use of an immobilized ascorbate oxidase column. In: Technical Digest of the 10th Sensor Symposium (Nakamura, T., Ed.), pp. 77-80. Institute of Electrical Engineers of Japan, Tokyo, Japan. Satoh, I. & Yamada, Y. (1991~).Flow-injection biosensing of cobalt(I1) ions with use of an immobilized alkaline phosphatase reactor in a flow system. In: Digest of Technical Papers of the 6th International Conferenceon Solid-state Sensorsand Actuators (Transducers '91, Chang, S.-C., Ed.), pp. 699-702. The Institute of Electrical and Electronics Engineers, Inc. Piscataway, NJ, USA. Townshend, A. & Vaugham, A. (1970). Application of enzyme-catalysed reactions in trace analysis - V Determination of zinc and calcium by their activation of the apoenzyme of calf-intestinal alkaline phosphatase. Talanta 17,289-298. Vallee, B.L. (1980). Zinc and other active metals as probes of local conformation and htnction of enzymes. Carlsberg Res. Commun., 15,423-441. Wagner, F.W. (1988). Preparation of metal-free enzymes. In: Methods in Enzymology (Riordan, J.F. & Vallee, B.L., Eds.), Vol. 158 A, pp. 21-32. Academic Press, San Diego.
lkuo Satoh Abstract . . . . . . . . . . . . . . . . . . . . . . . I. INTRODUCTION.. . . . . . . . . . . . . . . . . 11. PRINCIPLE . . . . . . . . . . . . . . . . . . . . . 111. CHARACTERISTICS. . . . . . . . . . . . . . . . IV. PROCEDURE.. . . . . . . . . . . . . . . . . . . V. BIOSENSING OF HEAVY METAL IONS . . . . . A. Apoenzyme Beads as Sensing Elements . . . . B. Apoenzyme Membrane as the Sensing Element VI. CONCLUSION . . . . . . . . . . . . . . . . . . .
......
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....... .......
. . . . . . . 461 . . . . . . . 462 . . . . . . . 462 . . . . . . . 463 . . . . . . . 466 . . . . . . . 467 . . . . . . . 467 . . . . . . . 470 . . . . . . . 471
ABSTRACT The present work led to a novel idea about the biosensing of heavy metal ions based on apoenzyme reactivation methods. Several kinds of immobilized metalloenzymes
Advances in Molecular and Cell Biology Volume 15B, pages 461472. Copyright 0 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0114-7
46 1
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as recognition elements for each metal ion were applied in the microdetermination of heavy metal ions in flow streams. Regeneration of the metal-free enzymes (apoenzymes) was made by loading chelating agents to the metal-bound enzymes (holoenzymes). The apoenzymes were positively reactivated by adding trace amounts of heavy metals. The recovery depended on metals trapped in the enzymes, which was shown to be a practical index for microanalysis of heavy metal ions. The proposed method was demonstrated to be convenient, safe, specific, and highly sensitive.
1. INTRODUCTION A great number of metal-dependent enzymes have already been identified (Vallee, 1980).Heavy metal ions coordinated in the active site of these enzymes play a very important role in the catalytic process. These heavy metal ions are intrinsic to each of the enzymes and most of them can be reversibly removed from the catalytic site of the enzymes. Therefore, metal-free enzymes (apoenzymes) can be used as specific and selective recognition elements for heavy metal ions in an enzymatic analysis. Use of enzymes as highly sensitive reagents is common in food and clinical analyses. Currently,immobilized enzymes in combination with transducers for monitoring their enzymatic activity are used for the construction of biosensors, and many of them are commercially available (Satoh, 1989a). Analytes have been mostly limited to substrates, products, activators, and inhibitorsin the enzyme-catalyzed reactions. In sharp contrast, we have adopted flow-injectionmicroassay for cofactors, namely heavy metal ions, based on apoenzyme reactivation (Satoh et al., 1986, 1987, 1988, 1989b, 1990ste, 1991a-e, 1992, 1993). We have tried to regenerate cofactor-free enzymes from cofactor-bound enzymes (holotype of metalloenzymes) and use them as the recognition elements for heavy metal ions in flow streams. The apoenzyme reactivation methods for flow-injection microdetermination of heavy metal ions are summarized in this paper with a special focus on our current studies.
II. PRINCIPLE Cofactors such as heaG metal ions and nucleotides are generally complexed in the active site of the metalloenzymes and flavin enzymes, respectively, and the cofactors are directly responsible for the activity of the enzymes. Therefore, these enzymes need the cofactors for expressing catalytic activity. The cofactor-bound enzyme is usually called a holoenzyme, whereas the cofactor-free enzyme is called an apoenzyme. Figure 1 schematically shows the correlation between the holoenzyme and the apoenzyme. The metalloenzymes capture the metal ions so tightly (dissociation constant: Kd< lo4 M) that they hold them throughout the purification process. In contrast with the metalloenzymes, metal-activated enzymes bind the
Biosensing of Heavy Metals
Cofactor-bound enzyme catalytically
463
Cofactor-free enzyme catalytically
[ active
[ inactive
Figure 1. lnterconversion between the holoenzyme and the apoenzyme. Cofactor: Heavy metal ions; r(lr(lS catalytic site; 000 substrate-binding site.
metal ions rather weakly (Kd < 1C3to 1O4 M), and then, the enzymes tend to lose the metal ions during purification (Wagner, 1988). Preparation of the apoenzyme lacking its catalytic activity can be made by removing the metal ion from the corresponding holoenzyme with strong chelating agents. The apoenzyme is reactivated by exposing it to the metal-containing sample so that metal ions can be taken up and trapped in the active site. Thus, the amount of metals coordinated in the catalytic center of the enzyme molecules may be closely related to the enzyme activity expressed and, in turn, be proportional to the added amount of the metals. The content of the trace metals can eventually be determined by monitoring the induced activity attributable to the reactivation of the apoenzyme. The metal ions responsible for the catalytic activity of the metalloenzymes vary with the type of enzyme. Selective determination of the metal ions depends upon choosing the appropriate metalloenzyme in which its catalytic site fits well with each metal ion.
111. CHARACTERISTICS In practice, use of metalloenzymes immobilized on supporting materials such as small glass beads and a thin polymer membrane can make assays continuous and, moreover, enhance the feasibility of handling in the process between regeneration and reactivation of the apoenzymes. Reusability and long-term stability of the immobilized enzymes may be expected. Microdetermination of heavy metal ions based on spectrophotometric monitoring was tried (Townshend and Vaugham, 1970) and a microassay for zinc(I1) ions using high performance liquid chroma-
IKUO SATOH
464
tographic methods (Risinger et al., 1983) as well as an electrochemical method (Mattiasson et al., 1979) were reported. We have originally developed unique biosensing of heavy metal ions based on flow-injectioncalorimetry (Satoh et al., 1986).Thermometric flow-injectionanalysis of biorelated compounds in combination with the use of a high performance semiadiabatic calorimeter were pioneered by Mosbach et al. (Danielsson and Mosbach, 1988; Danielsson, 1990). The flow-measuring system, that is, a thermal bioanalyzer, is better known as an “enzyme thermistor.” We used the calorimeter for monitoring the enzymatic activity in the assay cycle including regeneration and reactivation. The calorimetric biosensing system is schematically presented in Figure 2. The column packed with immobilized enzyme beads is interchangeable and, therefore, different kinds of metals are readily determined. In addition, other monitoring methods such as amperometry, potentiometry, and spectrophotometry are available by exchanging the thermistor with other transducers, for example, electrodes,photomultipliers, etc. The proposed biosensing methods in microanalysis of heavy metal ions do not require expensive instruments based on atomic absorption spectrophotometryand inductively coupled plasma atomic emission spectrophotometry.Furthermore, this
chela3nt
9substrate lons
I If A k m i n m bath
Figure 2. Flow-calorimetric biosensing system for heavy metal ions based on the apoenzyme reactivation method. Carrier reservoir (buffer solution), pump (flow rate 1.O ml min-’), heat exchanger (thin-walled acid-proof steel tubing: 0.8 mm, i.d.), bath (80Q x 250 mm; 303 K), enzyme column (packed with metalloenzymes immobilized on porous beads), thermistor (attachedto a gold capillary placed in a polymer holder), WB/Amp. (DC-type Wheatstone bridge with a chopper stabilized operational amplifier), Rec. (pen recorder).
Biosensing of Heavy Metals
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novel method does not involve pollution problems due to exhaust fumes. The characteristics of the proposed methods can be summarized as follows: 0
0 0 0
Mild assay conditions High sensitivity High selectivity Reusability of the immobilized enzymes Continuous flow use
0 0
0
0
Compact assay system Feasibility of handling Feasibility of field work Low-priced assay Separability of the metaltrapping and the activitymonitoring process
Procedure
Phenomena
Substrate
response t o Iiolocnzymcs
t Carrior
t ChelatinE t . Carrier
washing aRcnt
t
Subs t r a t c
r c g c n c r a t ion o f apociixymos washing response t o apocnzymcs
t Carricr
washing
Heavy mctal i o n s
p a r t i a l a c t i v a t i o n of apocnzymes
t
Carrier
washing
Substrate
response a t t r i b u t a b l c t o metal i o n s
t
c o o r d i n a t c d i n Clic enzymes Carrier
c
washing
Complctc r c a c t i v d t i o i i of apocnzymcs ( c o n v c r s i o o t o Iiolocnzymcs)
Carrier
washing
Figure 3. Flow chart for the assay procedure.
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466
IV. PROCEDURE A flowchart for the assay procedure is shown in Figure 3. Three rotary valves for injection are connected in series between a damper and a drain valve to avoid their mutual contamination. At first the catalytic activity of the immobilized holoenzymes at the reactor (column or membrane) is measured by injecting its substrate. The change in output is a measure of the activity attributable to the holoenzymes, as schematically illustrated in Figure 4. After exposing the enzymesto the chelating agent, a drastic decrease in the activity is observed. This means that the cofactors, namely heavy metal ions complexed in the catalytic site of the holoenzymes, are mostly removed. The amount of chelating agent required for regenerating the
Scan L
60 min
Y
Figure 4. Schematic presentation of response curves for a reaction cycle.
Biosensing of Heavy Metals
467
apoenzymes varies with the kind of metalloenzyme and with the conditions of the chelating agents (variables such as concentration, pH, volume, etc.). Subsequent injection of a trace amount of the cofactors reactivates the apoenzymes. Thus, the recovery of the activity is determined by adding the substrate. While sufficient cofactors are introduced to the mixture of the cofactor-free and cofactor-bound enzymes, all of the enzymes may be completelyconverted to the holoenzymes. The system is then ready for another assay. A reaction cycle generally takes 40 to 60 minutes, but application of a membrane-type reactor reduces the cycle time (within 30 min).
V. BIOSENSING OF HEAVY METAL IONS A. Apoenzyme Beads as Sensing Elements Alkaline Phosphatase as the Recognition Element
Alkaline phosphatase as the recognition element in combination with a couple of monitoring devices was feasible in the most sensitive assay of zinc(I1) and cobalt(I1) ions. The enzyme purified from Escherichia cofi (EC 3.1.3.1., Asahi Chemical Industry Co., Ltd., Ohito-cho, Tagata-gun, Shizuoka-ken, Japan) was immobilized on epoxide acrylic beads (Eupergit C: 100-200 pm particle diameter; 40 nm pore diameter; 180m2g-' surface area; Rohm Pharma, Darmstadt, Germany) and then packed into a small polymer column (0.3 ml). The enzyme is often employed as a labeling reagent for enzyme immunoassay. Hydrolysis of p-nitrophenyl phosphate top-nitrophenol and orthophosphate was monitored for measuring the enzyme activity: p-nitrophenyl phosphate + H,O
+ p-nitrophenol + orthophosphate
(1)
Tris-HC1 100mM buffer @H 8.0, containing 1.OM NaC1) was used and the catalytic activity was calorimetrically determined by injecting 0.1 ml of 100 mM substrate (p-nitrophenylphosphate) (Satoh et al., 1991d). Exposing the column to 5 ml of20 mM 2,6-pyridine dicarboxylate solution (PH 6.0) as the cofactor-complexingagent almost virtually converted the holoenzymes to the apoenzymes. The effect of pH on the chelating agent in the regeneration reaction was tested. No noticeable variation in the level of the regeneration was observed over the pH range of 4.0 to 8.0. The recovery of the activity attributable to the partial reactivation of the apoenzymes was a function of the added amount of zinc(I1) ions. The calibration graph for zinc(I1) ions demonstrated a sigmoidal curve. Zinc(I1) ions ranging from 0.01 to 1.O mM were calorimetrically determined for a 0.5 ml injection. The effect of pH on the reactivation of the apoenzyme was investigated in the weakly acidic pH region (from 4.0 to 6.0 in steps of 0.5). The recovery was unaffected by variations
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468
in pH; this means the proposed biosensing using alkalinephosphatasedoes not need any critical pH adjustment, which is very practical. The column retained much more long-term stability than that of a column packed with immobilized bovine carbonic anhydrase. The reactor was repeated over 120 times during the two months of operation. The spectrophotometric approach to measuring enzyme activity based on detecting changes in absorbance at 405 nm attributable to p-nitrophenol formation provided similar results, except for the highly sensitive assay for zinc(I1) ions in submicromolar levels (Satoh et al., 1990b, c). Potentiometric monitoring of the activity with a flow-through ISFET for detecting pH shifts attributable to orthophosphate released in the hydrolytic reaction was also carried out to determine zinc(I1) ion concentrations in the range of 0.01 to 1.O mM (Satoh et al., 1990d). Cobalt-substituted alkaline phosphatase is also capable of hydrolyzing the orthophosphate ester. Therefore, the same immobilized preparations were applied in the microdetermination of cobalt(I1) ions. Biosensing based on spectrophotometry gave excellent results (Satoh, 1992b),and cobalt(I1) ions ranging in concentration from 1-200 pM were determined. Use of a calorimetric sensing system was possible in the concentration range of 0.04 to 1.O mM. Ascorbate Oxidase as the Recognition Element
Ascorbate oxidase is one of the most typical copper-dependentenzymes involved in the oxidation of L-ascorbate to dehydroascorbate. The reaction is described by 2L-Ascorbate + 0, + 2dehydroascorbate + 2H,O
(2)
Ascorbate oxidase from cucumber (EC 1.10.3.3, Asahi Chemical Industry Co., Ltd.) immobilized onto porous glass beads with controlled pore size (CPG; 5 1.5 nm pore diameter, 120-200 mesh, 44 m'g-', Electronucleonics Inc., Fairfield, NJ, USA) was packed into a column and then mounted in the proposed flow-calorimetric system (Satoh et al., 1987). Since oxidative reactions involving molecular oxygen are usually accompanied by considerable heat generation, highly sensitive and precise biosensing of copper(I1) ions attributable to the significant exothermic reaction was anticipated. Thus, micromolar levels of copper(I1) ions were calorimetrically determined. Regeneration of the apoenzymes was achieved with exposure of 20 mM NJV-diethyldithiocarbamate solution (PH 8.0) to the column. The apoenzymes derived from the ascorbate oxidase was selectively responsive to copper(I1) ions and not to divalent cations in 1 mM level such as Ca(II), Co(II), Mg(II), Ni(II), and zinc(I1). Trace amounts of copper(I1) ions in human blood sera were analyzed by the calorimetric method and compared with those obtained by atomic absorption spectrophotometry. There was satisfactory agreement between these methods. Amperometric monitoring of the enzyme activity with a polarographic oxygen electrode showed a more rapid and sensitive determination of copper(I1) ions. The
Biosensing of Heavy Metals
469
assay covered concentrationsranging from 0.5 to 2.0 pM and required about half an hour. L-Ascorbate can be determined by detectingabsorbance at 265 nm, which is used for monitoring the oxidase activity.Thus, copper(I1)ions measured photometrically ranged from 0.1 to 10 pM. A series of these monitoring methods validated the use of immobilized ascorbate oxidase beads as an excellent recognition element for copper(I1) ions. Carbonic Anhydrase as the Recognition Element
Carbonic anhydrase purified from erythrocytes (EC 4.2. 1.1) is known for its remarkably high turnover numbers. The enzyme is a dominant factor in equilibrium between carbon dioxide and bicarbonate in blood as shown in Equation 3, which also expresses esterase activity as seen in Equation 4. CO, + H,O p-nitrophenyl acetate + H,O
-+
H,CO,
(31
3
p-nitrophenol + acetate
(4)
Application of bovine carbonic anhydrase immobilized on the same sort of porous glass beads (CPG) for the specific determination of zinc(I1) and cobalt(I1) ions in combination with flow-calorimetric monitoring turned out to be feasible for the first time (Satoh et al., 1986, 1989b). Injection of 0.5 ml ofp-nitrophenyl acetate into the carrier streams (Tris-HC1 buffer, 0.1 M, pH 8.0) in the calorimetric system resulted in an exothermic response. Since values of changes in enthalpy for ester hydrolysis are normally zero, we considered that the exothermic change resulted from the protonizing heat of acetic acid enzymatically formed in the tris buffer. Exposing the immobilized enzyme beads in a column (packed volume, 0.3 ml) to 5 ml of 10 mM 2,6-pyridine dicarboxylate solution (pH 5.0) as the chelating agent caused regeneration of the apoenzymes. Zinc(I1) ions in the range of 25 to 250 pM could be calorimetrically measured using 0.5 ml injections. Generally, zinc(I1) ions complexed in the active site of zinc enzymes (e.g., hydrolases) are reversibly exchanged with cobalt(I1) ions, and the cobalt-coordinated enzymes still retain their catalytic activity. We also succeeded in assaying submillimolar levels of cobalt(I1) ions (0.05 to 0.2 mM) using cobalt-substituted enzymes (Satoh, 1989b). In this case, less volume of the chelating agent (2.5 ml) was sufficient for regeneration of the enzymes. Galactose Oxidase as the Recognition Element
Galactose oxidase (EC 1.1.3.9) catalyzes oxidation of D-galactose as follows: D-Galactose + 0, 3 D-galacto-hexodialdose + H,O,
(5)
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470
This enzyme isolated from Dactilium dendroidesreadily converted to the apoenzyme under water soluble conditions while it takes a much longer time for reactivation of the apoenzyme. Utilization of the immobilized preparation in combination with the flow-injection technique for monitoring of enzyme activity resolved the problem. Copper(I1) ions were calorimetrically determined in millimolar levels (Satoh, 1990e). Amperometric monitoring with an oxygen electrode and a hydrogen peroxide electrode made the assay more sensitive and rapid (Satoh et al., 1990a). There is room for improvement for a more sensitive response.
B. Apoenzyme Membrane as the Sensing Element Table 1 summarizes successful biosensing of heavy metal ions based on the apoenzyme reactivation method using bead-type immobilized enzymes. Application of the apoenzyme column to flow-injection biosensing demonstrated their long-term stability. Practical use has already been found in clinical and food analyses. In order to obtain a more rapid assay, a contact type of apoenzyme sensor has been developed (Satoh et al., 1992, 1993). Ascorbate oxidase immobilized onto a porous polymer membrane (partially aminated polyacrylonitrile,50 pm thickness, Asahi Chemical Industry Co. Ltd.) was directly attached to a flow-through polarographic oxygen electrode and used as the recognition element for copper(I1) Table 1. Flow-Injection Biosensing of Heavy Metal Ions Based on the Apoenzyme Reactivation Methods
Metal
Zn(I1) Zn(I1) Zn(I1) Zn(I1) Cu(I1) Cu(I1) Cu(I1) CU(I1) Cu(I1) Cu(I1) Co(I1) Co(I1) Co(I1)
Recognition Element
ALP ALP ALP BCA ASOD ASOD ASOD GalOD GalOD GalOD ALP ALP BCA
Monitoring Method
Ranae lmMl
Calonmetry Potentiometry Spectrophotometry Calorimetry Amperometry Calorimetry spectrophotometry Amperometry Amperometry Calorimetry Calorimetry Spectrophotometry Calorimetry
/T /I /P /T 10 /T /P
0.010-1.0 0.010-1.0
0.00014.010 0.0254.25
0.000~.002 0.0014.05
0.00014.010
10
0.1-10.0
/H /T
0.01-10.0 5.0-20.0 O.O&I.O 0.0014.2 0.0054.2
/T /P /T
Nofes: Enzyme:ALP, alkalinephosphatase; ASOD, Ascorbate oxidase; IBCA, Bovine carbonic anhydrase; GalOD,
Galactose oxidase. Method T (thermistor), I (pH-ISFET),P (photomultiplier), 0 (polarographic oxygen electrode), H (hydrogen peroxide electrode).
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ions (Satoh et al., 1992a). Use of the system mounted with the apoenzymemembrane sensor resulted in a more rapid assay (26 min). Furthermore, microdetermination of dual heavy metal ions was tried with hybrid enzymes immobilized onto the same kind of membrane (Satoh, 1993). Alkaline phosphatase and ascorbate oxidase were coimmobilized onto the polymer membrane and tightly attached to a polarographic oxygen electrode. Ascorbate oxidase functioned not only as the recognition element for copper(I1) ions but also as an indicator enzyme for amperometric monitoring of alkaline phosphatase activity on the same membrane. For sensing zinc(I1) ions, the following coupled reactions were amperometrically monitored: L-Ascorbyl-2 phosphate + H,O
-+ L-ascorbate + orthophosphate
(6)
2 L-Ascorbate + 0, + 2 dehydroascorbate + 2 H,O
(7) The apoenzyme membrane was regenerated by pumping cofactor-complexing agents for removing each cofactor, namely copper(I1) and zinc(I1) ion from the catalytic site of alkaline phosphatase or ascorbate oxidase. Thus, zinc(I1) ions in 2 to 200 pM concentrations and copper(I1) ions in 2 to 100 pM concentrations were determined through activation of each of the immobilized apoenzymes on the same supporting membrane.
VI. CONCLUSION Flow-injection microassay of heavy metal ions such as cobalt(II), copper(II), and zinc(I1) ions based on apoenzyme reactivation methods was found feasible with immobilized metalloenzymes. Regardless of the shape of the immobilized metalloenzymes, that is, beads or membrane, this method of unique monitoring is now widely applied in several analytical areas. Further developmental studies towards establishing the generality and versatility of these analytical techniques involving immobilized apoenzymes in flow systems are currently in progress.
REFERENCES Danielsson, B. (1990). Calorimetric biosensors. J. Biotechnol. 15, 187-200. Danielsson, B. & Mosbach, K. (1988). Enzyme thermistors. In: Methods in Enzymology (Mosbach, K., Ed.), Vol. 137, pp. 181-197. Academic Press, New York. Mattiasson, B., Nilsson, H., & Olsson, B. (1979). An apoenzyme electrode. J. Appl. Biochem. I , 377-3 84. Risinger, L., Ogren. L., & Johansson, G. (1983). Determination ofzinc(I1) ions with a reactor containing immobilized carboxypeptidase A in a flow system. Anal. Chim. Acta 154,251-257. Satoh, I. (1989a). Biosensing using calorimetric devices. In: Chemical Sensor Technology (Seiyama, T., Ed.), Vol. 2, pp. 26%282. Kodansha Ltd., Tokyo, Japan. Satoh, I. (1989b). Continuous biosensing of heavy metal ions with use of immobilized enzyme-reactors as recognition elements. In: Proceedings of the MRS International Meeting on Advanced Materi-
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als, Vol. 14 (Biosensors, Karube, I., Ed.), pp. 4550. Material Research Society, Pittsburgh, Pennsylvania, USA. Satoh, I. (19904. Apoenzyme reactivation microassay for zinc(1I) ions with flow-through transducers. In: Proceedings of the 3rd International Meeting on Chemical Sensors, pp. P/lO&P/l07. The Organizing Committee of the 3rd International Meeting on Chemical Sensors, Cleveland, OH, USA. Satoh, I. (1990e). Calorimetric biosensing of heavy metal ions with the reactors containing the immobilized apoenzymes. AM. N.Y. Acad. Sci. 613,401404. Satoh, I. (1991d). An apoenzyme thermistor microanalysis for zinc(I1) ions with use of an immobilized alkaline phosphatase reactor in a flow system. Biosensors and Bioelectronics6(4), 375-379. Satoh, I. (1991a). Flow-injection calorimetry of heavy metal ions using apoenzyme-reactors. Netsusokutei (Calorimetry and Thermal Analysis) (in Japanese) 18(2), 89-96. Satoh, I. (1991e). Flow-injection microdetermination of heavy metal ions using a column packed with immobilized apoenzyme beads. J. Flow Injection Anal. 8(2), 111-126. Satoh, I. (1993). Amperometric biosensing of heavy metal ions using a hybrid type of apoenzyme membrane in flow streams. Sensors and Actuators, in press. Satoh, I. (1992b). Use of immobilized alkaline phosphatase as an analytical tool for flow-injection biosensing of zinc(I1) and cobalt(I1) ions. AM. N.Y. Acad. Sci. 672,240-244. Satoh, I., Abe, R., & Nambu, T. (1988). Bioelectrochemical sensing of copper(I1) ions using an immobilized apoenzyme column. Denki Kagaku 56(12), 10451049. Satoh, I. & Aoki, Y. (1990d). Biosensing of zinc(I1) ions using an apoenzyme reactor and an ISFET detector inflow streams. DenkiKagaku58(12), 1114-1118. Satoh, I., Ikeda, K., & Watanabe, N. (1986). Microanalysis of zinc@) ion by using an apoenzyme thermistor. In: Proceedings of the 6th Sensor Symposium (Takahashi, K., Ed.), pp. 203206. Institute of Electrical Engineers of Japan, Tokyo, Japan. Satoh, I., Itoh, H., & Anzai, H. (1992a). Flow-injection amperometric biosensing of copper (11) ions using a contact-type of an apoenzyme sensor. In: Proceedings of the 2nd World Congress on Biosensors, pp. 1 8 H 9 0 . Elsevier Advanced Technology, Oxford. UK. Satoh, I., Kasahara, T., & Goi, N. (199Oa). Amperometric biosensing of copper(I1) ions with use of an immobilized apoenzyme reactor. Sensors and Actuators BI, 499-503. Satoh, I., Kimura, S., & Nambu, T. (1987). Biosensing of copper(I1) ions with an apoenzymethermistor containing immobilized metalloenzymes in flow system. In: Digest of Technical Papers, The 4th International Conference on Solid-state Sensors and Actuators (Transducers '87, Matsuo, T., Ed.), pp. 789-790. Institute of Electrical Engineers of Japan, Tokyo, Japan. Satoh, I. & Masumura, T. (1990b). Flow-injection biosensing of zinc(I1) ions with use ofan immobilized alkaline phosphatase reactor. In: Technical Digest of the 9th Sensor Symposium (Sasaki, A., Ed.), pp. 197-200. Institute of Electrical Engineers of Japan, Tokyo, Japan. Satoh, I. & Nambu, T. (1991b). Flow-injection photometric biosensing of copper(I1) ions with use of an immobilized ascorbate oxidase column. In: Technical Digest of the 10th Sensor Symposium (Nakamura, T., Ed.), pp. 77-80. Institute of Electrical Engineers of Japan, Tokyo, Japan. Satoh, I. & Yamada, Y. (1991~).Flow-injection biosensing of cobalt(I1) ions with use of an immobilized alkaline phosphatase reactor in a flow system. In: Digest of Technical Papers of the 6th International Conferenceon Solid-state Sensorsand Actuators (Transducers '91, Chang, S.-C., Ed.), pp. 699-702. The Institute of Electrical and Electronics Engineers, Inc. Piscataway, NJ, USA. Townshend, A. & Vaugham, A. (1970). Application of enzyme-catalysed reactions in trace analysis - V Determination of zinc and calcium by their activation of the apoenzyme of calf-intestinal alkaline phosphatase. Talanta 17,289-298. Vallee, B.L. (1980). Zinc and other active metals as probes of local conformation and htnction of enzymes. Carlsberg Res. Commun., 15,423-441. Wagner, F.W. (1988). Preparation of metal-free enzymes. In: Methods in Enzymology (Riordan, J.F. & Vallee, B.L., Eds.), Vol. 158 A, pp. 21-32. Academic Press, San Diego.