Electrochemical assay for catecholase activity of mushroom tyrosinase

electrode; R1, 10-turn 10-kfi potentiometer; R2, 1.0004< precision resistor; OA1 and OA3, operational amplifier LM 741; OA2, operational amplifier LM ...
0 downloads 0 Views 420KB Size
Anal. Chem. 1982, 5 4 , 1985-1987

1985

Electrochemical Assay for Catecholase Activity of Mushroom Tyrosinase Gregory P. Power* and Ian M. Rltchle Department of Physlcal innd Inorganic Chemistry, University of Western Australia, Nedlands, Western Australia 6009, Australia

An electrochemical method for assaying enzymes Is described. The technique is based upon the anaiysls of elther the substrate or product concentration by measuring a diffusion limited current at a rotating disk electrode. Also described is the construction of a specially designed electrochemlcai cell of 1 cma capacity. The method is illustrated by determlnlng the enrynnlc actlvity of mushroom tyrosinase In the oxldatlon of 1,2-dIhydroxybenrene. I n this partlcuiar case, It is convenlent to add 1,4-dihydroxybenzene as a mediator and monitor the rate of appearance of p-benzoquinone by the limiting current lmethod. The method is evaluated and shown to give results closely similar to, but of higher preclslon than, the spectrophotometrlc method.

It is well-known that when a current to an electrode is diffusion limited, the magnitude of the current is directly proportional to the Concentration of electroactive species. However, such diffusion currents are only reproducible for certain electrode geometries and hydrodynamic situations. The two most commonly used types of electrodes which have reproducible limiting currents are the dropping mercury electrode (DME) and the disk electrode rotating under conditions of laminar flow (IRDE). Despite this, little use appears to have been made of llimiting current measurements in assaying enzymes. As with all chemical reactions, the rate of an enzyme catalyzed reaction can be followed by monitoring either the rate of disappearance of reactants (substrate) or the rate of appearance of products. One example of the former approach is the work of Ingraham (I),who followed the rate of disappearance of oxygen in a polyphenol oxidase reaction by measuring the oxygen limiting current at a rotating platinum electrode. An example of the latter approach is the study of Kamin and Wilson (2)PV'NIO investigated the activity of glucose oxidase immobilized on a rotating disk, by measuring the current due to the oxidation of a reaction product, H202,at a concentric ring electrode. Not all enzymic reactions can be studied directly in this way since it is not uncommon for both the substrate and products of an enzymic reaction to be electrochemically inert. Under these circumstainces, it is often possible to employ a redox couple as mediator, i.e., a compound which will react rapidly with the substrate (or products) and which is electrically active. Thus Yuan, Kuan and Guilbault (3) have recently assayed creatine kinase isoenzyme MB by using hexacyanoferrate(II1) as a mediator, which reacts with NADPH, a product of the enzyme reaction. The aim of this paper is to describe an inexpensive but convenient and effective assay method based on measuring diffusion currents a t a rotating disk electrode. The utility of the technique is then doimonstrated by using it to assay the enzyme tyrosinase. Tyrosinase is widely distributed in nature and is responsible for the production of melanin pigments by catalyzing the oxidation of tyrosine by alerial oxygen (4).It is commonly used as an indicator of the presence of cancerous growths (5). The substrate toward which tyrosinase shows the greatest activity 0003-2700/82/0354- 1985$0 1.25/0

is 1,2-dihydroxybenzene (catechol), which is oxidized in the presence of oxygen to o-benzoquinone. 1,2-Dihydroxybenzene is, therefore, the best substrate upon which to base an assay procedure for tyrosinase. It is not feasible, though, to monitor directly the appearance of o-benzoquinone. This is because o-benzoquinone is a highly reactive molecule, which undergoes coupling reactions with the 1,2-dihydroxybenzene present to yield a mixture of electrochemically inert products. If, however, we carry out the reaction in the presence of an excess of 1,4-dihydroxybenzene (hydroquinone) as mediator, the o-benzoquinone which is formed by enzymic oxidation is quantitatively reduced back to 1,2-dihydroxybenzene before coupling reactions can occur. The p-benzoquinone generated in this latter reaction is relatively stable and can readily be monitored by observing its diffusion-limited reduction current at an electrode. The overall mediator-enzyme-electron system is depicted schematically in Figure 1.

EXPERIMENTAL SECTION Reagents and Solutions. Mushroom tyrosinase (E.C. 1.14.18.1) was obtained from Sigma Chemical Co. Its quoted catecholase activity was 240 000 units/mg of solid, where a unit of activity is defined as the amount of enzyme which will cause a change of absorbance of 0.001 per min at pH 6.5 and 25 "C in a 3-mL reaction mix containing (0.167 mM) catechol and (0.070 mM) ascorbic acid. A.R. quality 1,2-benzoquinone and 1,4-benzoquinone were obtained from Fluka Chemicals. These were further purified by vacuum sublimation before use. All other chemicals were of A.R. quality and were used without further purification. Solutions were made up in 0.1 M phosphate buffer, pH 6.5, made with water which had been purified by a Millipore Milli-Q system. The air used was Medical Air supplied by Commonwealth Industrial Gases. Apparatus. The cell assembly is shown schematically in Figure 2. The cell itself is constructed from perspex and has a capacity of 1 cm3. It is thermostated by circulating water from a controlled temperature bath. The built-in reference electrode is a chloridized silver wire immersed in saturated potassium chloride, terminating in a Luggin capillary probe filled with a conducting gel to prevent mixing of the KCl with the cell contents. The counterelectrode is a platinum loop placed symmetrically around the Luggin probe and situated as far as possible from the disk electrode. The cell is also fitted with a stainless steel capillary for bubbling gases through the solution. This serves both to saturate the solution with the desired gas and to aid rapid mixing of aliquots of enzyme or substrate introduced through the septum-covered port to initiate the reaction under study. A Teflon-tipped screw acts as a valve to the drain in the bottom of the cell, which may be thereby emptied and washed out between runs without the cell needing to be taken apart. The apparatus is mounted on a rigid stand and the cell is sealed onto the rotating disk housing with an O-ring seal. The rotating disk, a vitreous carbon rod set in epoxy resin, is driven at a predetermined constant speed by a stepping motor. Electrical contact is made to the disk via the stainless steel shaft by a silver slip-ring and a silver / carbon brush. In order to measure a limiting current, it is necessary to fix the potential of the rotating disk electrode a t some pre0 1982 American Chemical Societv

1986

.

ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 198z

zero current

Flgure 1. Schematic diagram showing the enzymically catalyzed oxidation of 1,Pdihydroxybenzenecoupled to an electrode by the mediator 1,4dihydroxybenzene.

Figure 4. Typical current-time curve obtained during tyrosinase assay: total volume, 1006 pL; 1,2dihydroxybenzene concentration, 0.17 mM; 1,4dihydroxybenzene concentration, 0.07 mM; tyrosinase concentration, 0.36 p g mL-'.

saturated with air, and a stream of air was passed over the solution throughout the assay. With the disk rotating at 850 rpm, and the potentiostat set at -150 mV (with respect to the silver/silver chloride reference electrode), an aliquot of the enzyme solution (solution E) was injected and the resulting current-time curve recorded on a strip chart recorder. Flgure 2. Schematlc cross section of the electrochemical cell and rotatlng disk electrode: 1, rotatlng disk electrode; 2, injection port: 3, cell chamber: 4, water jacket: 5, reference electrode; 6,counterelectrode: 7, Luggin capillary; 8, drain.

RESULTS AND DISCUSSION In the coupled system shown in Figure 1,the rate of oxidation of 1,2-dihydroxybenzene is equal to the rate of production of p-benzoquinone (3, which in turn is proportional to the rate of change of current (I)at the rotating disk. Thus reaction rate = constant X ( d l / d t )

Flgure 3. Circuit diagram of potentiostat for use with electrochemical cell: J1, pair of terminals for measuring potential of working electrode; J2, reference electrode connection; J3, counterelectrode connection; J4, working electrode connection: J5, pair of terminals for measuring a voltage proportional to the current passing through the working electrode; R1, 10-turn 10-kQ potentiometer; 132,1.000-kQ precision resistor: OAl and OA3, operational ampllfier LM 741; OA2, operational amplifler LM 740.

selected value. This is achieved by means of a potentiostat in conjunction with the three electrodes in the electrochemical cell. A variety of potentiostats are available commercially, but the simple device shown schematically in Figure 3 is cheaply and easily constructed and well-suited to the purpose. It will run for several weeks on a pair of 9-V radio batteries. The output from the current-to-voltage converter is fed directly to a strip chart recorder for the recording of currenttime curves. Spectrophotometric assays were carried out using a Beckman Acta MIV double-beam recording spectrophotometer. Procedure. The conditions for assay were chosen to be similar to those used in a standard spectrophotometric method (6),except that the reductant, ascorbic acid, was replaced by 1,4-dihydroxybenzene at the same concentration. The following stock solutions were used: (A) 0.10 M potassium phosphate buffer, pH 6.5; (B) 5.0 mM catechol solution; ( C ) 2.1 mM 1,4-dihydroxybenzenesolution; (D) 0.065 mM EDTA solution; (E) 0.06 mg/mL mushroom tyrosinase solution. Solutions B to E were made up freshly before each experiment in solution A. The assay procedure was as follows: Reagents A-D were mixed in the cell in the proportions: 867 pL of A and 33 p L each of solutions B, C, and D. The resulting solution was

The constant in this equation is equal to the reciprocal of the slope of a plot of limiting current at the disk against p benzoquinone concentration in 0.10 M potassium phosphate buffer solution. Using the method of least squares on such a plot, we obtained a constant of 4.18 X lo4 mol m-3 A-l for our disk electrode rotating at 850 rpm. With seven experimental points, the standard error was 0.05 X lo4 mol m-3 A-l and the correlation coefficient 0.9994. The quantity (dI/dt) was determined from graphs of the type shown in Figure 4. It should be noted that the time taken for mixing the enzyme is short, of the order of 1s, and this is followed by a long linear section, the slope of which may be readily determined. The dependence of rate on enzyme concentration for a substrate concentration of 0.17 mM was determined from a series of current-time plots. It was found that a linear relationship between rate and enzyme concentration holds up to an enzyme concentration of at least 0.6 g m-3 (0.6 pg mL-'1. By use of a least-squares method, the slope of this line was found to be 15.0 X lo* A s-l m3 (g of enzyme)-l with a standard error of 0.4 X A s-l m3 (g of enzyme)-'. With 12 experimental points, the correlation coefficient was 0.997. It is now possible to calculate the reaction rate in terms of moles of substrate consumed per second per (grams per cubic meter) (pg mL-') of enzyme. This is given by I(4.18 f 0.05) X 104][(15.0 f 0.4) X lo-'] = mol s-l (g of enzyme)-' (6.2 f 0.2) X The detection limit of this method has not been explored in great detail but is probably set by the direct aerial oxidation of 1,4-dihydroxybenzene, which gives a background current change of nA s-l, which would correspond to about 1mg m-3 of enzyme. Comparison with the Spectrophotometric Method. The spectrophotometric activity is defined in terms of the rate of change of absorbance at 265 nm, the absorbance maximum

1907

Anal. Chem. 1982, 54. 1987-1990

for ascorbate. Using the standard spectrophotometric assay (6), we obtained an absorbance change of (2.3 f 0.3) X s-l averaged over seven measurements. This corresponds to an enzyme activity of (24 f 0.3) X lo5 units (mg of enzyme)-l, as specified earlier. It also agrees with the activity specified by the manufacturer (of 2.4 X lo5 units (mg of enzyme)-'. For comparison of tlhe spectrophotometric assay with the electrochemical assay, the former must also be expressed in terms of moles of substrate consumed per second. This conversion is simply accomplished by multiplying the enzyme activity by the extinction coefficient which we determined from a nine-point plot, of absorbance against ascorbic acid concentration in the ara~aymatrix. The value obtained was (1.92 k 0.02) X lo3 m2 mol-' with a correlation coefficient of 0.9994. Combining this figure with the measured rate of change of absorbance, we obtain for the spectrophotometric rate a value of (6.3 f 0.9) X mol s-l (g of enzyme)-'. It can be seen that the two techniques agree within the limits of experimental error but that the precision of the electrochemical method (5%)is substantially better than the spectrophotometric method (15%). We believe that this is due to the rapid mixing inherent in the design of the electrochemical cell. A maljor advantage of the electrochemical method is that it is independent of the optical properties of the solution and therefore may be used to assay turbid or opaque solutions which could not be assayed spectrophotometrically. The close agreement between the electrochemical assay for tyrosinase using 1,4-dihydroxybenzene as reductant and the standard spectrophotoinietric method using ascorbate as reductant are evidence that tyrosinase per se does not attack 1,4-dihydroxybenzene to any significant extent during the assay. When assaying any given plant or tissue extract for tyrosinase, the possibility that the extract might contain some species which reacts with 1,4-dihydroxybenzene must first be checked by a suitable bilank. The presence in the extract of electroactive species which can be oxidized or reduced at -0.15 V on the carbon electrode is not likely to be a problem since these species will only cause a current jump on injection of the extract and will not contribute to the rate of change of current on which the assay is based.

Comparison with Oxygen Uptake Method. Tyrosinase may also be assayed by measuring the rate of oxygen uptake. This may be most conveniently achieved by means of an oxygen electrode. However, the oxygen electrode is likely to be less satisfactory than the rotating disk electrode in several respects. The response of the oxygen electrode, which derives its selectivity from a membrane, is intrinsically slower than the rotating disk because of the membrane. Protein fouling of the membrane is also a known problem in enzyme assays using the oxygen electrode. Finally, it is difficult to carry out enzyme assays a t constant oxygen concentration using the oxygen electrode method. If it is also desired to keep the catechol substrate concentration constant, then a reductant such as 1,6dihydroxybenzene must be added. In a previous comparative study using oxygen uptake to follow the kinetics (7),it was found that the apparent rate with 1,Cdihydroxybenzene as reductant was substantially higher (40%) than the corresponding quantity using ascorbate as reductant. The authors of this paper suggest that there should be no intrinsic difference in the enzymic rate and ascribed the measured discrepancy to a difference in the stoichiometry of oxygen absorption. The present results confirm this point and illustrate the desirability of measuring enzyme kinetics by several different methods. ACKNOWLEDGMENT G.P.P. wishes to thank the University of Western Australia for a General Development Grant Fellowship. LITERATURE CITED (1) Ingraham, L. Anal. Chem. 1956, 28, 1177-1179. (2) Kamin, R. A.: Wilson, G. S . Anal. Chem. 1980, 52, 1196-1205. (3) Yuan, C.-L.; Kuan, S, S.; Guilbault, G. G. Anal. Chem. 1981, 53, 190- 193. (4) Bohinski, R. C. "Modern Concepts in Biochemistry"; Allyn and Bacon: Boston, MA, 1979. (5) . . Vielkind, U.: Schiaae, W.; Anders. F. A . Krebsforsch. Klin. Onkol. 1917, 90, 285. (6) "Catechol Oxidase (E.C. 1.14.18.1)"; Sigma Chemical Company Informatlon Bulletin: April 1979. (7) Kertesz, D.; Zlto, R. Blochlm. Blophys. Acta 1962, 64, 153.

RECEIVED for review August 21, 1981. Resubmitted June 7, 1982. Accepted June 14, 1982.

Amperometric Enzymic Determination of Triglycerides in Serum Handanl Wlnartasaputra, Shla S. Kuan,' and George G. Gullbault" Department of Chemistry, llniverslty of New Orleans, New Orleans, Louisiana 70 148

An amperatnetrlc three-electrode system has been developed for the assay of trlglycerldes. Two enzymes used in analysis, namely, glycerol dehydrogenase and diaphorase, are immoblllred on a collagen membrane. Serum samples are Incubated wlth a mlcroblal lllpase In phosphate buffer containing 2,6dlchlorophenollndophenol(DCPIP). The resulting glycerol Is oxldlred by NAD+ In the presence of glycerol dehydrogenase; the NADH produced is then oxldlred by DCPIP. The anodlc current generated by oxldatlon of the reduced form of the DCPIP at the surface of the worklng electrode at a constant of 0.300 V Is measured. Serum trlglycerides ranglng from 10 to 500 nng/dL can be easlly assayed In less than 15 mln by uslng only 25 pL of sample.

A fully enzymatic determination of triglyceride in biological samples has been reported mostly by spectroscopic methods (1-5). Electrochemical methods for measurement of enzymatic reactions have likewise been applied successfully (6-8). The fully enzymatic biamperometric determination of glycerol and triglycerides with open tubular carbon electrodes has been reported (9). This particular method employed soluble enzymes. This paper proposed the use of a three-electrode amperometric method and dual-immobilized enzymes on a collagen membrane method for determination of triglycerides in serum samples. The use of soluble enzymes in the reaction is also studied. The reactions of the proposed method are

'Food and Drug Admin.iutration, 4298 Elysian Fields Av., New Orleans, LA 70148.

triglycerides

lipase

glycerol

0003-2700/82/0354-1987$01.25/00 1982 American Chemical Society

+ free fatty acids

(1)