Electrode for Measuring Urease Enzyme Activity Joseph G . Montalvo, Sr. Diaision of Polymer and Physical Chemistry, Gulf South Research Institute, P. 0. Box 26500, New Orleans, La. 70126 SEVERAL ELECTROCHEMICAL procedures have been developed for the determination of the enzyme urease (1-3). A review of the use of electrochemical methods to measure enzymes has been presented (4). Relatively large amounts of the substrate are consumed in all of the electrochemical techniques reported for enzyme analysis which follow the general enzymatic reaction S
E -+
Products
where the substrate S undergoes enzymolysis by the enzyme E to form Products. This is because the enzymatic reaction is homogeneous throughout the test solution. A continuous or semicontinuous electrochemical routine analysis for an enzyme requires significant quantities of substrate. Such large quantities of substrate could represent a large expenditure in some cases, such as the dehydrogenase systems which require expensive substrates. If, however, the enzyme analysis could be performed by heterogeneous catalysis at an electrode surface, very small amounts of substrate should be required. The amount required would be limited by the area of the electrode. In this paper is reported the development of an electrode for analysis of the enzyme urease uia catalytic decomposition of the substrate urea at the electrode surface. The electrode performed well, indicating that development of electrodes utilizing more expensive substrates would be in order. EXPERIMENTAL
Preparation of Electrode. The electrode was made by coupling the urea substrate to the active surface of a cationic electrode responsive to ammonium ion. The electrode was covered with a layer of cellophane trapping a thin layer of concentrated urea solution between the electrode and the membrane. The urea diffuses out of the cellophane membrane and reacts with the enzyme urease to produce ammonium ion at the membrane surface. The resulting ammonium ion activity gradient causes diffusion of the ion back to the electrode, where it is sensed. The urea concentration over the electrode surface was maintained constant by gravity flow from a small reservoir of urea. This type of electrode is called here an enzyme electrode because it is used to measure an enzyme. Coupling of an electrode sensor with an enzyme has been reported in the literature (5-8). This type of electrode should be called a substrate electrode because it is used to measure substrate concentration. This terminology (1) W. C. Purdy, G. D. Christian, and E. C. Knoblock, Northeast Section, American Aksociation for Clinical Chemists, 16th National Meeting, Boston, Mass., Aug. 17-20, 1964. (2) S.A. Katz, ANAL.CHEM., 36,2500(1964). (31 . , G. G. Guilbault. K. Smith, and J. G. Montalvo, Jr., ibid., 41, 600 (1969). (41 6.G. Guilbault, ibid.,40,459R (1968). ( 5 ) L. C. Clark, Jr., and C. Lyons, Aim. N . Y. Acad. Sei., 102, 29 (1962). (6) S. J. Updike and G. P. Hicks, Nature, 214,936(1967). (7) G.G. Guilbault and J. G. Montalvo, Jr., J . Amer. Chem. SOC., 91, 2164 (1969). (8) G. G. Guilbault and J. G. Montalvo, Jr., Anal. Letters, 2, 283 (1969).
should be far less confusing than naming the electrode according to the coupled material. The electrode assembly consisted of the glass bulb of a Beckman 39137 cationic electrode covered with a 15-p cellophane membrane. (The cellophane film was developed in this laboratory by Dr. Elias Klein and coworkers.) A 150-p nylon netting (J. C. Penny and Co.) spacer was placed between the glass bulb and cellophane film. An “0” ring was used to anchor the electrode coverings firmly against the glass bulb. Two micro polyethylene tubings, 7 cm long and 500 p in outside diameter, were inserted under the 0 ring and placed between the glass bulb and the nylon netting. The tubings extended 3 mm over the glass sensing bulb and were placed on opposite sides of the bulb. Excess membrane and netting malerial were cut flush with the 0 ring; both coverings were sealed to the glass electrode with Cutex nail hardener. The exposed ends of the tubings were held in place along the stem of the electrode. One tube was connected cia a needle to a 2-cc syringe mounted against the cationic electrode. Procedure. After, preparation, the electrode was soaked in Tris (hydroxy-methyl) aminomethane buffer, O.lM, pH 7.0 for one day. The syringe was filled with 0.5M urea in Tris buffer and the space between the electrode and the membrane was filled and flushed by applying positive pressure to the syringe. Excess urea solution flowed out the open end of the exit micro tubing and was discarded. After complete flushing, the syringe was filled with urea solution to the 1-cc mark to establish a fixed hydrodynamic head. A Corning research pH meter equipped with a saturated calomel electrode was used to make the potential measurements. The recorder terminals of the pH meter were connected to a Sargent mV recorder. All measurements were carried out in a thermostated cell at 25 0.1 “C. Standard enzyme solutions were prepared by suitable dilution of a stock solution with Tris buffer. The Teflon (Du Pont) bar stirring the urease test solution was turned off when the solution reached the required temperature. The test solution was allowed to become quiescent before placing the electrodes into the solution. A steady state potential was obtained in about 4 to 7 minutes. After determination of the enzyme concentration or activity of a urease solution, fresh substrate ‘solution was introduced over the electrode surface by forcing 40 pl of substrate solution to pass through the substrate layer. Both the substrate and reference electrodes were then placed in an electrode washer (miniature pipet washer) and the potential was monitored until a low level of ion concentration was obtained in the substrate film over the electrode. The washing process removed ionic contaminants (which were sensed by the electrode) in the urea substrate film and removed urease from the electrode surfaces.
*
RESULTS AND DISCUSSION
At steady-state conditions, the electrode response depended on the urea concentration, its flow rate through the membrane, and urease activity. The electrode response varied logarithmically with enzyme activity over the range studied, 0.05-1.06 Sumner units/mg. Also, concentration of the reaction product (NHd+) at the electrode surface under steady-state conditions produced a linear plot with enzyme activity. These linear functions show that the response of the electrode to enzyme activity was first order. Under the conditions of the
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experiment (the initial urea concentration is greater than the urease concentration) the urea-urease system appears to fit the general kinetic expression which predicts that the reaction is first-order in enzyme concentration even though the substrate concentration may be suboptimal (9). Since the cellophane membrane used in making the electrode is permeable to urea but not to the enzyme urease, the enzymatic hydrolysis of urea can occur only after urea diffuses through the cellophane. As is expected, decreased sensitivity is obtained in a stirred solution, since the NH4+ ion gradient is dissipated. Determination of urease activity with the urease enzyme electrode has several advantages over the potentiometric method of Katz (2) and Guilbault and coworkers (3) and various colorimetric methods (10-12). Extremely small (9) K. J. Laidler, “Chemical Kinetics,” McGraw-Hill, New York, 1950, p 274. (10) C. C. Chin and G. Gorin, Anal. Biochem., 17,60 (1965). (11) G. Gorin, E. Fuchs, L. G. Butler, S . L. Cbopra, and R. T. Hersh, Biochem., 1,911 (1962). (12) H. T. Fister, “Manual of Standardized Procedure for Spectrophotometric Chemistry,” Method N-15a, Standard Scientific Supply Co., N. Y., 1950.
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amounts of substrate are required in an analysis, only about 50 11. Furthermore, the amount of substrate required is independent of the volume of test solution. A more elegant enzyme electrode for urease analysis is currently under investigation. Most really expensive substrates have a much higher molecular weight than urea so that a study to determine whether larger substrates would diffuse adequately through the cellophane membrane and allow the electrode to perform properly will be undertaken. Electrodes made by coupling to a consumable reagent may have wide applications because a reagent in a chemical reaction (catalytic or noncatalytic) coupled to an appropriate electrode sensor (potential, current, or conductive) might be used to determine another reagent of the reaction in solution. Steadystate conditions should be obtainable even though more than one reactant in a reaction can diffuse across the membrane barrier. Studies of this nature are currently under investigation in this laboratory. RECEIVED for review July 3, 1969. Accepted August 28, 1969, The financial assistance of the National Institutes of Health (Grant Number 1 SO1 FR05672-01) is gratefully acknowledged.
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