Design, Construction, arid Applications of a Galactose Selective Electrode Paul J. Taylor” Chemistry Department, Wright State University, Dayton, Ohio 4543 1
Emil Kmetec Biological Chemistry Department, Wright State University, Dayton, Ohio 4543 1
Jay M. Johnson Yellow Springs Instrument Co ., Inc., 1725 Old Springfield Pike, Yellow Springs I Ohio 45387
A rapid (40 s), precise and accurate micromethod for the determlnatlon of galactose In plasma and whole blood is described. The method utilizes an amperometrlc hydrogen peroxide electrode covered by a selective membrane system contalnlng Immobilized galactose oxldase. Optimal response and stabillty of the membrane bound galactose oxldase were lnvestlgated by an evaluatlon of the condltlons for enzyme lmmobillzatlon wlth glutaraldehyde and for long term storage. On the basis of these Investigatlons, the “optimized” enzyme-membrane was deflned as the membrane obtained by lmmobillzatlon wlth 0.29 % by weight, gjlutaraldehyde at pH 5.8. Also, the “optimal” buffer, both In terms of maklng the accurate measurement of galactose arid In terms of useful enzyme-membrane life, was a 0.07 M phosphate buffer, pH 7.3, containing 10 mg % potassium ferrlcyanide and 0.2 mg % cuprlc chlorlde dihydrate. The linear range of the measurement was at least 0 to 500 mg %, and It was shown to extend as high as 3000 mg % , galactose In aqueous solutlon. Of the 39 compounds screened, the only physiologically Important interference found was dihydroxyacetone.
The clinical measurement of circulating galactose levels is important in the preliminary diagnosis of galactosemia and galactose intolerance (1,2). Also, research currently being conducted suggests that galactose may be an important alternative energy source in premature infants and that the metabolism of galactose may impart some degree of regulation to blood glucose levels of diabetic infants (3). The inadequacies of various methods for the measurement of galactose in blood have been reviewed ( 4 ) . At least one of the commercial methods available recommends that a protein precipitation step be done on the plasma or serum sample before it is assayed (5). Some methods require long incubation times (30-120 min) in order to achieve the desired sensitivity (6). Typical required sample volulmes are on the order of 0.5 mL (5). Besides these shortcomings of existing viable methods, some methods have been shown to be unsuitable for the measurement of galactose in plasma and other biological fluids (4, 7). Immobilized enzymes have a variety of applications in analytical chemistry (8). Specifically they have been employed in conjunction with electrochemical sensors to measure such substrates as glucose (9), urea (IO),ethanol (11,12), uric acid (13), glutamine (14), etc. Both potentiometric and amperometric enzyme-electrodes have been reported as have the advantages and disadvantages of each (8). Two of the major disadvantages frequently associated with the amperometric enzyme-electrode have been slow response
and electroactive interferences (8). Typically, slow response has been due to the thickness of the enzyme layer through which substrates and products must diffuse. Electroactive interferences have been a problem particularly when the measurements involve biological fluids (8). Clark reported the first amperometric enzyme-electrode in 1962 (15). Since then Clark has described electrodes for the measurement of glucose, amino acids, ethanol, and cholesterol (16,17). In addition Guilbault and co-workers have described electrodes for the measurement of phosphate, uric acid, glucose, and others (13, 18, 19). There are several electrodeenzyme configurations possible. The enzyme may be either trapped between a membrane and an electrode, free in solution, or immobilized in or on the membrane, Immobilization, if possible, is the most desirable method as it allows prolonged reuse of the enzyme and greater reproducibility. The goals of this study were to immobilize an enzyme, galactose oxidase (E.C. 1.1.3.9), within a membrane, to optimize and evaluate the procedure using the Yellow Springs Instrument Co. Inc., YSI, Model 23A glucose analyzer and to define an optimal buffer system for both the storage of the working membranes and the measurement of galactose in aqueous solution, plasma, and whole blood. The Model 23A employs a specially constructed hydrogen peroxide sensitive electrode covered by a complex membrane containing glucose oxidase (20). Although the Model 23A is marketed as a dedicated glucose analyzer, the only requirement for its use in the measurement of any oxidizable substrate is the production of sufficient HzOz by a specific oxidase to assure response by the hydrogen peroxide sensitive electrode.
EXPERIMENTAL Materials. Arabinose, cellulose acetate resin CA-394-60, cyanoacrylate adhesive 910 EM, dipotassium EDTA, fructose, glucosamine hydrochloride (9870), glucuronolactone, glutathione mercaptosuccinic acid, 1,3-propanediol,raffinose, ribose, and uric acid (98%), were purchased from Eastman. L-Ascorbic acid, cyclohexanone, D-galactose, glutaraldehyde (50%), hydrogen peroxide (3%), lactose, mannitol, potassium ferrocyanide, potassium permanganate, propylene glycol, sodium periodate, sodium phosphate, dibasic sodium phosphate, monobasic monohydrate, sorbitol, sucrose, trichloroaceticacid, and D-XylOSe were obtained from Fischer: catalase (5000 units/g) was a product of Fermco Biochemicals. Cupric chloride dihydrate, potassium ferricyanide, and sodium chloride were purchased from Baker Chemical Company. &Erythrose (85%),D-maltose, and D-trehalose dihydrate were products of Aldrich Chemical Company. Mannose, melibiose dihydrate, galactitol and stachyose were obtained from Nutritional Biochemicals. Fucose, galactosamine hydrochloride, galacturonic acid, and neocuproine were purchased from Calbiochem. Galactose oxidase preparations with activities ranging from 30-130 Units/mg were purchased from Worthington ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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Biochemicals and Sigma. Galactose-1-phosphate was obtained from Sigma also. Methyl-a-D-and methyl-P-D-galactopyranoside were purchased from Pfanstiehl. Polycarbonate membrane material was purchased from Nuclepore Corporation and silicone rubber O-rings were purchased from Bearings, Inc. Membrane Construction. Cellulose Acetate Membrane (US. Patent 3,979,274). Prepare a casting solution by weight consisting of 1part cyclohexanone, 1/24 part Eastman cellulose acetate resin powder 394-60, and ‘123 part isopropanol. Weigh into a flask containing a magnetic stirring bar, and stopper well. Stir at ambient temperature until a clear viscous solution developes (several hours to overnight). Prepare a casting bath in a circular pan that will present about 20 dm2 of surface area when filled with distilled water and equilibrate to room temperature. Clean the surface by dispensing 500 pL of the casting solution onto the water surface with a micropipet. Continually break the perimeter of the membrane that forms until the membrane area equals that of the water surface area. When this membrane cures (about 5 min), carefully pull it from the water with forceps and discard. This will remove dust from the surface of the water. Cast a membrane by dispensing 500 WLof casting solution onto the undisturbed surface of the dust-free water with a quick, even stroke of a micropipet to form a circular pool about 15 cm in diameter. Allow the membrane to cure for 5 to 10 min. If the membrane will not shrink to at least 13 cm, change the water bath and/or make a new casting solution. Discard any membranes that are not between 10 and 13 cm in diameter. After the membrane has cured, it is picked up from the water on a polyethylene sheet. Slip the polyethylene sheet beneath the membrane and lift an edge through the water surface until the edge of the membrane contacts the surface of the polyethylene. Continue pulling the polyethylene from the water drawing the membrane with it and onto it. Allow the membrane to air dry on the polyethylene sheet in a dust-free environment. It should be tightly attached to the polyethylene, smooth, and without cracks or wrinkles; and should have a slight pinkish cast. The resulting membrane is approximately 1 Km thick and contains pores about 6 A in diameter. Immobilization of Galactose Oxidase. Using a microsyringe, 15 WLof 0.50% glutaraldehyde solution, pH 5.8, unless otherwise noted (prepared by dissolving 81 g disodium succinate hexahydrate, 12 g succinic acid, 2 g sodium benzoate, 0.5 g dipotassium EDTA, and 10.0 g of a 50% glutaraldehyde solution in sufficient water to produce 1.0 L of solution) was expelled onto a piece of polyethylene backed cellulose acetate membrane. Another solution, of galactose oxidase (1 mg dissolved in 10 ILLdeionized water) was immediately expelled with a microsyringe onto the cellulose acetate membrane in such a way as to mix with the glutaraldehyde solution. Further mixing of the resulting resin was obtained by alternately drawing the solution into the microsyringe and expelling it back onto the cellulose acetate membrane. In less than 1 min after mixing the solutions, an approximately 5 cm by 8 cm piece of polycarbonate membrane (Nucleopore) material with 300-A pores is placed shiney-side up over the resin and the resin allowed to wick between the cellulose acetate and the polycarbonate membranes. An absorbant tissue was then placed over the enzyme-membrane complex and a large rubber stopper rolled over it while applying light finger pressure. The enzyme-membrane was allowed to air dry for 30 min, then cured at 45 “C for 30 min. Silicone rubber O-rings, 3/8-incho.d., were then glued to the polycarbonate surface using a cyanoacrylate adhesive. After the adhesive dried, the O-ring mounted enzyme-membranes were then punched out and the polyethylene backing removed from the cellulose acetate side of the membrane. They are stored at 4 “C until needed. This procedure makes about 35 membranes. The Model 23A hydrogen peroxide electrode is constructed so that the 3/8-inch0.d. O-ring seats on the tip of the electrode with the cellulose acetate side of the enzyme-membrane flush against the electrode surface. The probe and the seated O-ring membrane are then mounted in the sample chamber so that the membrane O-ring seals around the sample chamber-sensor port. Chamber Buffer Solution. The “buffer solution” used to fill the instrument sample chamber and stabilize the membrane was 0.07 M in phosphate, 0.053 M in sodium chloride, 0.007 M 790
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in sodium benzoate, and contained 0.072 g/L of a 5000 Unit/g catalase preparation. Its pH was 7.3. Determination of Enzyme-Membrane Activity. The instruments used were first calibrated to read directly in current using a standard resistor in place of the peroxide electrode. An enzyme-membrane was then installed on the peroxide electrode and mounted in the instrument. Activity was determined by injecting 25 p L of a 200 mg % galactose standard into the instrument. The temperature was maintained at 37 OC in all the studies. The plateau current response displayed was defined as a measure of enzyme-membrane activity. However, the plateau current response thus obtained is a function of several variables only one of which is enzymic activity within the enzymemembrane. Measurements were always done on installed membranes after an equilibration period of 1 h or more and a stable and reproducible response to the 200 mg 7’0 galactose standard was achieved. Evaluations used in this study involved the determination of enzyme-membrane activity at daily intervals under various conditions. Once membranes were installed they were not disturbed, and they were tested under constant conditions until the evaluation was complete, usually a week or more. During this time, system response to peroxide was checked daily by injecting a hydrogen peroxide standard into the instrument and noting the peak current response. This evaluation procedure had no apparent effect on enzyme-membrane activity. Enzyme-membrane activity at time zero was defined as 1.0. Subsequent measurements of enzyme-membrane activity at various time intervals were expressed relative to initial enzyme-membrane activity for each specific membrane. Standards. Galactose standards were prepared in deionized M) as preservative. Standards water containing K2EDTA(2 X made in this way were stable for several months when stored at room temperature. Hydrogen peroxide standards were freshly made from a nominal 3% solution which was titrated periodically against permanganate to determine hydrogen peroxide concentration (21). Studies with Plasma and Whole Blood. Aliquots of a 5000 mg % galactose standard were used to spike either whole blood or plasma samples to the desired galactose concentration. The spiked plasma samples were considered as samples of “unknown” concentration. The samples (EDTA anticoagulant) used were supplied by the Yellow Springs Clinic, Yellow Springs, Ohio. After sample collection, the blood was centrifuged and the plasma was removed with a disposable Pasteur pipet. These plasma samples were immediately pooled and stored frozen until needed. Whole blood samples were not pooled and they were always used when fresh. Individual whole blood samples were first assayed for galactose concentration, as before, and then spiked in the same manner as described above for plasma. Selectivity and Interference Testing Procedure. After the galactose analyzer was calibrated with a 200 mg % galactose standard, various amounts of added substances were analyzed to determine if there was any response by the instrument. To determine whether the added substance had any effect on the enzyme activity of the electrode, 200 mg % standards were again analyzed in the presence clf the substance being tested and any discrepancy from the expecited result was noted. Unless otherwise noted, the buffer used in these studies was “buffer solution” containing 10 mg % K3Fe(CN)6and 0.2 mg % CuC12.2Hz0.
RESULTS A N D DISCUSSION Description of the Model 23A Galactose Analyzer. The peroxide electrode was mounted in the temperature controlled measurement chamber filled with about 350 WLof pre-warmed buffer solution. Standards and samples (25 pL) were injected into the chamber with a YSI precision syringepet (combination syringe and pipet). The chamber content was stirred by an air-driven silicone rubber diaphragm which assured adequate stirring plus rapid equilibration with atmospheric oxygen. A nonlimiting oxygen supply in this system was assured by the silicone rubber membrane which is very permeable to atmospheric oxygen and is in direct contact with the contents of the sample chamber. A temperature compensation probe is also present in the sample chamber. The purpose of this
Table I. Loss of Galactose Oxidase from Enzyme-Membranes after Immobilization with Varying Amounts of Glutaraldehydea Glutaraldehyde (% by wt) Days stored 1.44 0.72 0.29 0.14 0.029 0.014 2 0 0 1.4 9.0 6.0 7.4 4 0 0 1.2 5.6 7 0 0 0 1.8 6.8 a Enzyme-membranes were placed in a “buffer solution” containing also K,EDTA (1.6 X 10-3M)prepared without the addition of catalase. The data represents the enzymic activity in the buffer solution after the time indicated, expressed as a percentage of the total enzymic activity in the enzyme-membrane. probe is to compensate electronically for fluctuations in temperature (37 “C) accompanying sample injection, thereby enhancing speed of system response (22, 23). Principles of Operation. Galactose in the vicinity of the electrode diffuses through the pores in the outer polycarbonate membrane and comes in contact with immobilized galactose oxidase. The following reaction occurs (24,25); galactose
D-galactose + 0, - galactohexodialdose oxidase
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The hydrogen peroxide formed in the membrane diffuses toward the electrode, where it is sensed by the electrode, and toward the sample chamber, where it is destroyed by catalase, resulting in a steady-state hydrogen peroxide concentration a t the electrode surface (23). The plateau current response of the electrode is reached in approximately 40 s. The major factors influencing the steady-state hydrogen peroxide concentration between the membrane and the electrode are substrate concentration in the chamber, permeability of the enzyme-membrane to substrate and hydrogen peroxide and the rate of generation of hydrogen peroxide by the oxidase reaction (enzyme activity) within the enzyme-membrane. Immobilization Studies. Effective Glutaraldehyde Concentration. The effective glutaraldehyde concentration required for the immobilization and stability of galactose oxidase was determined in two ways. First, the enzymemembrane was placed in a phosphate buffer and any enzyme released into the buffer was measured using a coupled assay procedure (26). The results are shown in Table I. The data indicate that immobilization with 0.29% glutaraldehyde represented the lower limit of concentration required for enzyme fixation in the membrane when tested over a period of seven days. At the same time it was found that the initial activity from membrane to membrane prepared at the same glutaraldehyde concentration was less reproducible a t higher concentrations of glutaraldehyde than at lower concentrations, and that enzymemembranes prepared in the presence of 0.029% or less glutaraldehyde were very unstable. Figure 1, for example, illustrates the difference in stability of membranes prepared a t two concentrations of glutaraldehyde. Each point represents an average of four or more determinations, each made on different membranes and different instruments. The main conclusion to be drawn from Figure 1 is that the membranes prepared with 1.44% glutaraldehyde are somewhat less stable than the membranes prepared with 0.29% glutaraldehyde over a 14-day period. After seven days the activity of both types of membranes fell continuously to less than 10% of their original activity. On the basis of these observations, 0.29% glutaraldehyde was chosen as the most effective concentration for immobilization. Effect of Buffer Constituents. The instrument buffer used in these evaluations was “buffer solution”, containing 1.6 x M KZEDTA. Its pH was 7.3. This pH was chosen because the pH optimum of galactose oxidase is reported to be in this
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Figure 2. Effect of pH on immobilization. Stability curves of enzyme-membranes immobilized at pH 5.8 (0),pH 7.3 (A)and 9.0 (0) using 0.29 % by weight glutaraldehyde. Evaluation buffer contained M in K,EDTA “buffer solution” that was also 1.6 X
range (24). EDTA was included in these early evaluation buffers because of its effectiveness in retarding bacterial growth. Galactose oxidase is a copper containing enzyme (27) and preliminary work indicated that the presence of small amounts of EDTA in the buffer mixture did not affect enzyme activity, over a period of several days. Other investigators have reported similar observations (28). Effect of pH. Enzymemembranes were prepared at several pH values and a t the fixed optimal glutaraldehyde concentration of 0.29%. Only small differences in stability resulted when the pH of immobilization was tested over the range 5.8-9.0 (Figure 2). The range in initial enzyme-membrane activity of these membranes was similar. Since most of the data recorded to this point was obtained on enzymemembranes prepared at pH 5.8, this pH was retained for the immobilization of galactose oxidase for the remaining studies. The nature of the reaction of glutaraldehyde with proteins has been widely investigated (29, 30). Originally it was believed that glutaraldehyde formed Schiff bases with proteins (29). Recent evidence suggests that the reaction actually occurs between an oligomer of glutaraldehyde and the protein (29,311. These unsaturated oligomers react with the protein to give a Michael-type adduct (31). Whatever the mechanism of the reaction, a frequently reported characteristic of the reaction of glutaraldehyde with proteins is the occurrence of the reaction over a wide pH range of 5 to 9 (29, 30). The observations made here, support these findings, and the fact that under optimized conditions sufficient cross-linkage occurred during enzyme-membrane preparation to seal the two membranes to each other and to prevent escape of enzyme through the porous outer membrane. Membrane Activity. Enzyme Source. Studies thus far involved only the enzyme preparation isolated from Polyporus circinatus, and commercially available from Worthington Biochemicals Corporation. Another enzyme, isolated from the same genus and species, and available from Sigma BioANALYTICAL CHEMISTRY, VOL. 49,NO. 6,MAY 1977
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Effect of ferricyanide on enzyme-membrane stability. Enzyme-membranes were made by immobilization at pH 5.8 using 0.29 % by weight glutaraldehyde. Evaluation buffers contained: “buffer solution” that was also 1.6 X lo3 M in KgDTA (A)and “buffer solution” that was also 1.6 X lo-, M in K,EDTA and 10 mg % in K3Fe(CN)B (0) Flgure 3.
chemicals, was tested also. Membranes were also made with a carboxymethylcellulose ion-exchange purified lypholysate of the Worthington preparation (27). There are two conclusions which can be drawn from the data obtained. The use of the CM-cellulose preparation, in enzyme-membrane construction does not necessarily offer any advantages, at least in terms of enzyme-membrane stability, over the use of the commercial preparations. Membranes made with the two commercial preparations exhibited essentially the same stability during storage and use in the instrument. Initial enzyme-membrane activity ranged from 10-18 nA. The minimum enzyme membrane activity required for optimal use in the instrument is 8 nA. While commercial preparations varied in specific activity, a constant weight of enzyme was used in the membrane preparation. Useful membrane life of these optimized enzyme-membranes ranged from eight to ten days. Permeability of Membrane. Special membranes were constructed to study the permeability properties of the enzyme-membrane system. These enzyme-membranes were constructed as before except that the more porous polycarbonate membrane replaced the cellulose acetate membrane in order to permit the penetration of externally injected ferrocyanide and its reaction directly at the electrode surface. Studies on these special membranes indicated that the response, and presumably the permeability of the special membrane, did increase somewhat over the first 2 or 3 days but then remained relatively constant. This could explain the slight increase in activity observed over the first 2 or 3 days in the stability curve (upper curve Figure 3) for the optimized enzyme-membrane. Permeability to hydrogen peroxide and galactose are increasing and therefore steady-state hydrogen peroxide concentration is increasing concomitant to the increase of current output. It appeared that permeability did not change after the third day, and that the system response to hydrogen peroxide remained constant. It was concluded that the loss in system response to galactose after the third day, was due to losses in enzyme activity which must occur within the enzyme-membrane. Effect of Ferricyanide. Using the “buffer solution”, preliminary testing was done with human plasma. When samples were spiked with a galactose standard, it was frequently observed that less than 100% of the added galactose was indicated by the electrode. Addition of ferricyanide to the sample chamber before the addition of plasma markedly reduced the discrepancy. Without ferricyanide, the depression in membrane activity produced errors ranging from 30 to 80% depending upon the plasma sample. With a sample chamber concentration of 3 mg % ferricyanide, the error was still substantial (10-15%) but with a concentration of 10 mg % ferricyanide the error observed was in general less than 5%. 792
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Flgure 4. Effect of EDTA and cupric chloride on enzyme-membrane
stability in the presence of ferricyanide. Enzyme-membranes were made by immobilization at pH 5.8 using 0.29% by weight glutaraklehyde. Evaluation buffers contained; “buffer solution” that was also 1.6 X lo-, M in K,EDTA and 10 mg YO K,Fe(CN)6 (0),“buffer solution” that was also 10 mg % in K,Fe(CN), (A), and “buffer solution” that was also 10 mg % K,Fe(CN), and 0.2 mg % in CuCI,-2H2O ( 0 ) Evidently, the activity of the enzyme within the membrane was depressed by reducing substances in the plasma. The oxidizing agent, potassium ferricyanide, in the buffer apparently nullified the effects of the reducing substances in plasma. It also had the additional benefit that enzymemembrane activity was enhanced by a factor of approximately three in the presence of ferricyanide. In general, oxidizing agents enhance membrane activity and reducing agents decrease membrane activity as indicated in the Selectivity and Interferences section. This effect of oxidizing agents upon the activity of soluble galactose oxidase has been reported (32). The mechanism of this effect is not clear but may involve the bound copper in the enzyme (32). The enzyme is only active when copper is in a higher oxidation state (32-34). There is disagreement as to whether this higher oxidation state is copper(I1) or copper(II1) (32, 34). Oxidizing agents, such as NaI03, K2Cr207,NaI04, K3Co(NO& and NazMo04.2Hz0,were tried b,oth singly and in combination, and, in general, behaved as did K,Fe(CN)6. However, none were as compatible and stable as ferricyanide. If the reduced forms of the oxidizing agents are allowed to reach the peroxide electrode, they will be oxidized and thus give a positive interference. The cellulose acetate filter membrane effectively blocks potassium ferricyanide from reaching the electrode. Effect of EDTA. Including ferricyanide in the buffer eliminated the error in the plasma analysis but resulted in a rapid loss in activity as shown in Figure 3 (lower curve). EDTA was removed from the buffer in an attempt to improve stability. The results are shown in Figure 4, curve A. Each point represents an average of at least three determinations, each done on a different membrane and instrument. It is evident that enzyme stability in the buffer containing 10 mg % ferricyanide, in the absence of EDTA, is much improved, in contrast to what had already been observed for the soluble enzyme in buffer containing added EDTA. Effect o f Added Copper. The behavior of the enzymemembranes shown in Figure 4 suggests that the copper associated with galactose oxidase may be more susceptible to attack by EDTA in the presence of ferricyanide. Since a gradual loss of copper, and/or inactivation of the copperenzyme site, was suspected, a trace of copper in the form of cupric chloride dihydrate was added to the buffer (Figure 4, curve B). In this buffer (“buffer solution” that was also 10 mg % in ferricyanide and 0.2 mg % in CuC12-2H20), all of the membranes tested were more active after 25 days’ storage and use than they were initially. Figure 5 illustrates the results obtained when these buffer constituents, cupric chloride dihydrate and K2EDTA, were tested in “buffer solution”
Table 11. Selectivity and Interferences Level Effect of tested, Relative substance Substance tested mg % responsea on slopeb Arabinose 200 0 1.0 Dihydroxyacetone 50 5.30 1.0 D-Erythrose 120 1.0 0.07 N-Ethyl maleimide 0 0.96 1000 0 1.0 Fructose 200 0 1.0 Fucose 200 10 0.86 1.0 Galactosamine hydrochloride Galactitol 200 0 1.0 0 1.0 Galactose- 1-phosphate 40 200 0 1.0 Galacturonic acid 0 200 1.0 Glucosamine hydrochloride Glucose 1000 0 1.0 Glucuronolactone 1.0 200 0 Glycerol 1.0 500 0.067 Lactose 1.0 200 0.15 0 1.0 D-Maltose 200 0 Mannitol 200 1.0 Mannose 200 1.0 0 Melibiose 200 0.53 1.0 1.41 Me th yl-a - D-galacto0.93 200 pyranoside monohydrate 1.41 0.93 Met h yl-p-D- galac to200 pyranoside 0 10 1.0 Neocuproine 0 1.0 1,3-Propanediol 1000 0 200 1.0 Propylene glycol 100 0.33 1.0 Raffinose 200 0 1.0 Ribose 0 1.0 200 Sorbitol 0 1000 1.0 Sorbose 0.59 1.0 100 Stachyose 200 0 1.0 Sucrose D-Trehalose dihydrate 1.0 0 100 Trichloroacetic acid 100 0 1.0 D-Xylose 0 1.0 200 a Response of system toward the substance tested relRelative to an equal molar concentration of galactose. ative system response when standard galactose is assayed in the presence of the substance tested. without ferricyanide. The earlier observation that EDTA has no effect on the stability of the enzyme in this buffer was confirmed. As with the buffers containing 10 mg % ferricyanide, membranes were much more stable in buffer that also contained cupric chloride dihydrate than they were in the other combinations depicted in Figure 5. Thus, an optimal buffer solution could be defined as the “buffer solution” containing ferricyanide and copper. Explanation. A plausable explanation for this conclusion can be made. If the copper atom in each molecule of resting
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enzyme spends time in both the reduced, inactive state and also the oxidized, active state, then in a neutral redox environment, the time-ratio of copper reduced/copper oxidized is a constant. However, if the enzyme is stored in either an oxidizing or reducing environment, then this ratio over time will be either decreased or increased, respectively. Since copper(I1) forms a stronger complex with EDTA than does copper(1) (35), complexation and/or removal of copper from the enzyme is more likely to occur in the oxidized, active state than the reduced, inactive state simply because in an oxidizing environment the enzyme-copper spends more time in the copper(I1) state. In general, it is true that copper(I1) complexes are more stable than copper(1) complexes because of the lack of crystal field stabilization energy in d” copper(1) complexes (36). This predicts, that in an oxidizing environment, susceptibility of the enzyme-bound copper to complexation by any available ligand, e.g., EDTA, is enhanced. Both the data and the proposed explanation suggest that even in the absence of EDTA, the enzyme is more susceptible to inactivation due to loss of copper from the active site in an oxidizing environment than in a neutral redox environment. Selectivity and Interferences. Tables I1 and I11 list some of the substances that might interfere in this system. The column labeled “Relative Response” is the response of the galactose oxidase electrode to the substance listed expressed relative to the response for an equal molar galactose solution. There are two possible ways in which a substance may interfere with the measurement. It may give a positive reading when injected into the instrument or it may change the slope of the response to galactose. The latter constitutes just as serious an interference as the former and is represented in the tables by a number in the column headed “Effect of Substance on Slope”. This number is a multiplier for the slope in the presence of the interference. An entry of “zero” in the
Table 111. Effects of Oxidizing and Reducing Substances Level tested, mg %
Effect of substance on s1opeb.C 0.33
Relative responsea
Effect of substance on s1opecpd 0.99 0.97
Substance tested Ascorbic acid 4 0 Glutathione 35 0 ... Mercaptosuccinic acid 100 0 0.32 0.81 Potassium ferricyanide 100 0 3.0 1.03 Potassium ferrocyanide 100 0 0.20 0.95 Sodium periodate 100 0 ... 0 Uric acid 5 0 ... 1.0 a Response of system toward the substance tested relative to an equal molar concentration of galactose. Experiment performed in “buffer solution” in the absence of K,Fe(CN), and CuC1,.2H20. Relative system response when standard galactose is assayed in the presence of the substance tested. Experiment performed in “buffer solution” that was also 10 mg % in K,Fe(CN), and 0.2 mg % in CuC1,.2H,O. ~~
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column labeled “Relative Response” and a “1.0”in the column labled “Effect of Substance on Slope” is indicative of no interference. Interfering substances include D-erythrose, galactosamine, glycerin, lactose, melibiose, methyl-a-Draffinose, galactopyranoside, methyl-P-D-galactopyranoside, stachyose, mercaptosuccinic acid, sodium periodate, and dihydroxyacetone. The apparent interference of many of these substances may simply be due to the presence of galactose as an impurity in the reagent being tested or more likely, the substances are substrates for galactose oxidase. Of the thirty-nine substances screened, the only potentially physiologically significant interfering species was dihydroxyacetone. The last column in Table I11 reflects the improvement due to the use of the redox buffer and added cupric ion. Measurements of Aqueous Galactose Standards. The measurement of aqueous galactose standards in the region 0-500 mg 70was linear. The slope of the least squares fit of with a y-intercept of -2.56 X and the data is 5.00 X a correlation coefficient of 0.999. Increasing the concentration to 3000 mg 70also produced a linear calibration curve. The slope of the least squares fit was 5.49 X with a y-intercept of -0.16 and a correlation coefficient of 0.998. All data points for both curves are the average of at least three determinations, each on a different membrane and instrument. Measurement of Galactose in Plasma and Whole Blood. Plasma and whole blood samples, shown to be free of galactose, were spiked with galactose to known levels between 5 and 400 mg 70.Both types of samples produced linear calibration curves. The least squares fit of the plasma data had a slope of 0.980, a y-intercept of 1.76 mg 70,and a correlation coefficient of 0,999. Similarly, whole blood yielded a slope of 1.05, a y-intercept of -1.81 mg %, and a correlation coefficient of 0.999 when subjected to least squares treatment. Each data point represented the average of a t least three determinations, each done on a different membrane and instrument. The pooled estimate of the standard deviation on the plasma and whole blood was 2.1 and 2.7 mg %, respectively.
LITERATURE CITED (1) R. C. Harris, Department of Pediatrics, Columbia University, New York, N.Y., Personal Communlcation, 1975. (2) T. Hersh, Nutrition News, 35, 5 (1972). (3) J. W. Sparks, A. Lynch, R. A. Chez, and W. H. Glinsman, Pediat. Res., 10, 51 (1976).
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S.Frings, J . Electfoanal. Chem., 7, 398 (1964). (5) Gaiactostat Instruction Sheet, Worthlngton Biochemical Corporation, Freehold, N. J., 1974. (6) G. G. Guiibauh, P. J. Brignac, Jr., and M. Juneau, Anal. Chem.,40, 1256 (1968). (7) H. L. Pardue and C. S. Frings, Anal. Chem. 36, 2477 (1964). (8) L. D. Bowers and P. W. Carr, Anal. Chem., 48, 544 (1976). (9) S. J. Updike and G. P. Hicks, Nature (London),214, 986 (1967). (10) G. G. Guiibauit and F. R. Shu, Anal. Chem., 44, 2161 (1972). (11) G. G. Gullbault and G. T. Lubrano, Anal. Chim. Acta, 69, 189 (1974). (12) L. C. Clark, Jr., in “Biotechnoi and Bioeng. Symp.,” No. 3, C. B. Wingard, Ed., Wiley, New York, N.Y., 1972, p 377. (13) J. Nanjo and G. G. Guilbault, Anal. Chem., 46, 1769 (1974). (14) G. G. Guilbault and F. R. Shu, Anal. Chim. Acta, 56, 333 (1971). (15) L. C. Clark, Jr., and C. Lyons, Ann. N.Y. Acad. Sci., 102, 29 (1962). (16) L. C. Clark, Jr., presented at the Proc. Internat. Union Physiological Sciences, Munich, Germany, July 1971. (17) L. C. Clark, Jr., and C. R. Emory in “Ion and Enzyme Electrodes in Biology and Medicine”, M. Kessier, L. C. Clark, Jr., D. W. Lubbers, I.A. Silver, and W. Simon, Ed., Park Press, Baltimore, Md., 1976, p 161. (18) G. G. Guiibauit and G. Nagy, Anal. Chlm. Acta, 78, 69 (1975). (19) G. G. Guiibault and G. T. Lubrano, Anal. Chim. Acta, 60, 254 (1972). (20) L. C. Clark, Jr., US. Patent 3439455 (1970). (21) I. M. Kolthoff, E. B. Sandell, E. J. Meehan, and S. Bruckenstein, “Quantitative Chemical Analysis”, 4th ed., The Macmillan Company, London, England, 1969, p 834. (22) Application Notes, YSI Model 25 Oxidase Meter and YSI-Clark 2510 Oxidase Probe, Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio, 1976. (23) Instiuction Manual, YSI Model 23-A Glucose Analyzer, Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio, 1976. (24) J. A. D. Cooper, W. Smith, M. Bacila, and H. Medina, J. Biol. Chem., 234, 445 (1959). (25) L. Cleveland, R. E. Coffman, P. Coon, and L. Davis, Biochemistry, 14, 1108 (1975). (26) D. Amaral, F. Kelly-Faicoz, and B. L. Horecker, Methods fnzymol., 9, 87 (1966). (27) D. J. Kosman, M. J. Ettinger, R. E. Weiner, and E. J. Massaro, Arch. Biochem. Biophys., 185, 456 (1974). (28) D. Amaral. L. Bernsteln, D. Morse, and B. L. Horecker, J . Biol. Chem., 236 2281 (1963). (29) F. A. Quiocho in “Insoiubilized Enzymes”, M. Salmona, C. Saronio, and S. Garottlni, Ed., Raven Press, New York, N.Y., 1974, p 113. (30) 0. Zaborsky, “Immobilized Enzymes”, The Chemical Rubber Company, Cleveland, Ohio, 1973, p 66. (31) F. M. Richards and J. R. Knowles, J . Mol. Biol., 37, 231 (1968). (32) G. R. Dyrkacz, R. D. Libby, and G. A. Hamilton, J . Am. Chem. SOC.,96, 626 (1976). (33) F. Kellv-Faicoz. H. Greenbera. and B. L. Horecker. J . Bid. Chem.. 240. 2966 (1965). (34) D. J. Kosrnan, R. D. Bereman, M. J. Ettlnger, and R. S. Giordano, Bimhem. Biophys. Res. Commun., 54, 856 (1973). (35) F. A. Cotton and G. Wilkinson, “Advanced Inorganic Chemlstry”, 2nd. ed.. Wllev. New York. N.Y.. 1966. D 895. (36) J. E. H u G y , “Inorganic Chemistry: knciples of Structure and Reactivity”, Harper and Row, New York, N.Y., 1972, p 300. (4) H. L. Pardue and C.
RECEIVED for review January 13,1977. Accepted February 17, 1977.
Emil Kmetec Biological Chemistry Department, Wright State University, Dayton, Ohio 4543 1
Jay M. Johnson Yellow Springs Instrument Co ., Inc., 1725 Old Springfield Pike, Yellow Springs I Ohio 45387
A rapid (40 s), precise and accurate micromethod for the determlnatlon of galactose In plasma and whole blood is described. The method utilizes an amperometrlc hydrogen peroxide electrode covered by a selective membrane system contalnlng Immobilized galactose oxldase. Optimal response and stabillty of the membrane bound galactose oxldase were lnvestlgated by an evaluatlon of the condltlons for enzyme lmmobillzatlon wlth glutaraldehyde and for long term storage. On the basis of these Investigatlons, the “optimized” enzyme-membrane was deflned as the membrane obtained by lmmobillzatlon wlth 0.29 % by weight, gjlutaraldehyde at pH 5.8. Also, the “optimal” buffer, both In terms of maklng the accurate measurement of galactose arid In terms of useful enzyme-membrane life, was a 0.07 M phosphate buffer, pH 7.3, containing 10 mg % potassium ferrlcyanide and 0.2 mg % cuprlc chlorlde dihydrate. The linear range of the measurement was at least 0 to 500 mg %, and It was shown to extend as high as 3000 mg % , galactose In aqueous solutlon. Of the 39 compounds screened, the only physiologically Important interference found was dihydroxyacetone.
The clinical measurement of circulating galactose levels is important in the preliminary diagnosis of galactosemia and galactose intolerance (1,2). Also, research currently being conducted suggests that galactose may be an important alternative energy source in premature infants and that the metabolism of galactose may impart some degree of regulation to blood glucose levels of diabetic infants (3). The inadequacies of various methods for the measurement of galactose in blood have been reviewed ( 4 ) . At least one of the commercial methods available recommends that a protein precipitation step be done on the plasma or serum sample before it is assayed (5). Some methods require long incubation times (30-120 min) in order to achieve the desired sensitivity (6). Typical required sample volulmes are on the order of 0.5 mL (5). Besides these shortcomings of existing viable methods, some methods have been shown to be unsuitable for the measurement of galactose in plasma and other biological fluids (4, 7). Immobilized enzymes have a variety of applications in analytical chemistry (8). Specifically they have been employed in conjunction with electrochemical sensors to measure such substrates as glucose (9), urea (IO),ethanol (11,12), uric acid (13), glutamine (14), etc. Both potentiometric and amperometric enzyme-electrodes have been reported as have the advantages and disadvantages of each (8). Two of the major disadvantages frequently associated with the amperometric enzyme-electrode have been slow response
and electroactive interferences (8). Typically, slow response has been due to the thickness of the enzyme layer through which substrates and products must diffuse. Electroactive interferences have been a problem particularly when the measurements involve biological fluids (8). Clark reported the first amperometric enzyme-electrode in 1962 (15). Since then Clark has described electrodes for the measurement of glucose, amino acids, ethanol, and cholesterol (16,17). In addition Guilbault and co-workers have described electrodes for the measurement of phosphate, uric acid, glucose, and others (13, 18, 19). There are several electrodeenzyme configurations possible. The enzyme may be either trapped between a membrane and an electrode, free in solution, or immobilized in or on the membrane, Immobilization, if possible, is the most desirable method as it allows prolonged reuse of the enzyme and greater reproducibility. The goals of this study were to immobilize an enzyme, galactose oxidase (E.C. 1.1.3.9), within a membrane, to optimize and evaluate the procedure using the Yellow Springs Instrument Co. Inc., YSI, Model 23A glucose analyzer and to define an optimal buffer system for both the storage of the working membranes and the measurement of galactose in aqueous solution, plasma, and whole blood. The Model 23A employs a specially constructed hydrogen peroxide sensitive electrode covered by a complex membrane containing glucose oxidase (20). Although the Model 23A is marketed as a dedicated glucose analyzer, the only requirement for its use in the measurement of any oxidizable substrate is the production of sufficient HzOz by a specific oxidase to assure response by the hydrogen peroxide sensitive electrode.
EXPERIMENTAL Materials. Arabinose, cellulose acetate resin CA-394-60, cyanoacrylate adhesive 910 EM, dipotassium EDTA, fructose, glucosamine hydrochloride (9870), glucuronolactone, glutathione mercaptosuccinic acid, 1,3-propanediol,raffinose, ribose, and uric acid (98%), were purchased from Eastman. L-Ascorbic acid, cyclohexanone, D-galactose, glutaraldehyde (50%), hydrogen peroxide (3%), lactose, mannitol, potassium ferrocyanide, potassium permanganate, propylene glycol, sodium periodate, sodium phosphate, dibasic sodium phosphate, monobasic monohydrate, sorbitol, sucrose, trichloroaceticacid, and D-XylOSe were obtained from Fischer: catalase (5000 units/g) was a product of Fermco Biochemicals. Cupric chloride dihydrate, potassium ferricyanide, and sodium chloride were purchased from Baker Chemical Company. &Erythrose (85%),D-maltose, and D-trehalose dihydrate were products of Aldrich Chemical Company. Mannose, melibiose dihydrate, galactitol and stachyose were obtained from Nutritional Biochemicals. Fucose, galactosamine hydrochloride, galacturonic acid, and neocuproine were purchased from Calbiochem. Galactose oxidase preparations with activities ranging from 30-130 Units/mg were purchased from Worthington ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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Biochemicals and Sigma. Galactose-1-phosphate was obtained from Sigma also. Methyl-a-D-and methyl-P-D-galactopyranoside were purchased from Pfanstiehl. Polycarbonate membrane material was purchased from Nuclepore Corporation and silicone rubber O-rings were purchased from Bearings, Inc. Membrane Construction. Cellulose Acetate Membrane (US. Patent 3,979,274). Prepare a casting solution by weight consisting of 1part cyclohexanone, 1/24 part Eastman cellulose acetate resin powder 394-60, and ‘123 part isopropanol. Weigh into a flask containing a magnetic stirring bar, and stopper well. Stir at ambient temperature until a clear viscous solution developes (several hours to overnight). Prepare a casting bath in a circular pan that will present about 20 dm2 of surface area when filled with distilled water and equilibrate to room temperature. Clean the surface by dispensing 500 pL of the casting solution onto the water surface with a micropipet. Continually break the perimeter of the membrane that forms until the membrane area equals that of the water surface area. When this membrane cures (about 5 min), carefully pull it from the water with forceps and discard. This will remove dust from the surface of the water. Cast a membrane by dispensing 500 WLof casting solution onto the undisturbed surface of the dust-free water with a quick, even stroke of a micropipet to form a circular pool about 15 cm in diameter. Allow the membrane to cure for 5 to 10 min. If the membrane will not shrink to at least 13 cm, change the water bath and/or make a new casting solution. Discard any membranes that are not between 10 and 13 cm in diameter. After the membrane has cured, it is picked up from the water on a polyethylene sheet. Slip the polyethylene sheet beneath the membrane and lift an edge through the water surface until the edge of the membrane contacts the surface of the polyethylene. Continue pulling the polyethylene from the water drawing the membrane with it and onto it. Allow the membrane to air dry on the polyethylene sheet in a dust-free environment. It should be tightly attached to the polyethylene, smooth, and without cracks or wrinkles; and should have a slight pinkish cast. The resulting membrane is approximately 1 Km thick and contains pores about 6 A in diameter. Immobilization of Galactose Oxidase. Using a microsyringe, 15 WLof 0.50% glutaraldehyde solution, pH 5.8, unless otherwise noted (prepared by dissolving 81 g disodium succinate hexahydrate, 12 g succinic acid, 2 g sodium benzoate, 0.5 g dipotassium EDTA, and 10.0 g of a 50% glutaraldehyde solution in sufficient water to produce 1.0 L of solution) was expelled onto a piece of polyethylene backed cellulose acetate membrane. Another solution, of galactose oxidase (1 mg dissolved in 10 ILLdeionized water) was immediately expelled with a microsyringe onto the cellulose acetate membrane in such a way as to mix with the glutaraldehyde solution. Further mixing of the resulting resin was obtained by alternately drawing the solution into the microsyringe and expelling it back onto the cellulose acetate membrane. In less than 1 min after mixing the solutions, an approximately 5 cm by 8 cm piece of polycarbonate membrane (Nucleopore) material with 300-A pores is placed shiney-side up over the resin and the resin allowed to wick between the cellulose acetate and the polycarbonate membranes. An absorbant tissue was then placed over the enzyme-membrane complex and a large rubber stopper rolled over it while applying light finger pressure. The enzyme-membrane was allowed to air dry for 30 min, then cured at 45 “C for 30 min. Silicone rubber O-rings, 3/8-incho.d., were then glued to the polycarbonate surface using a cyanoacrylate adhesive. After the adhesive dried, the O-ring mounted enzyme-membranes were then punched out and the polyethylene backing removed from the cellulose acetate side of the membrane. They are stored at 4 “C until needed. This procedure makes about 35 membranes. The Model 23A hydrogen peroxide electrode is constructed so that the 3/8-inch0.d. O-ring seats on the tip of the electrode with the cellulose acetate side of the enzyme-membrane flush against the electrode surface. The probe and the seated O-ring membrane are then mounted in the sample chamber so that the membrane O-ring seals around the sample chamber-sensor port. Chamber Buffer Solution. The “buffer solution” used to fill the instrument sample chamber and stabilize the membrane was 0.07 M in phosphate, 0.053 M in sodium chloride, 0.007 M 790
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
in sodium benzoate, and contained 0.072 g/L of a 5000 Unit/g catalase preparation. Its pH was 7.3. Determination of Enzyme-Membrane Activity. The instruments used were first calibrated to read directly in current using a standard resistor in place of the peroxide electrode. An enzyme-membrane was then installed on the peroxide electrode and mounted in the instrument. Activity was determined by injecting 25 p L of a 200 mg % galactose standard into the instrument. The temperature was maintained at 37 OC in all the studies. The plateau current response displayed was defined as a measure of enzyme-membrane activity. However, the plateau current response thus obtained is a function of several variables only one of which is enzymic activity within the enzymemembrane. Measurements were always done on installed membranes after an equilibration period of 1 h or more and a stable and reproducible response to the 200 mg 7’0 galactose standard was achieved. Evaluations used in this study involved the determination of enzyme-membrane activity at daily intervals under various conditions. Once membranes were installed they were not disturbed, and they were tested under constant conditions until the evaluation was complete, usually a week or more. During this time, system response to peroxide was checked daily by injecting a hydrogen peroxide standard into the instrument and noting the peak current response. This evaluation procedure had no apparent effect on enzyme-membrane activity. Enzyme-membrane activity at time zero was defined as 1.0. Subsequent measurements of enzyme-membrane activity at various time intervals were expressed relative to initial enzyme-membrane activity for each specific membrane. Standards. Galactose standards were prepared in deionized M) as preservative. Standards water containing K2EDTA(2 X made in this way were stable for several months when stored at room temperature. Hydrogen peroxide standards were freshly made from a nominal 3% solution which was titrated periodically against permanganate to determine hydrogen peroxide concentration (21). Studies with Plasma and Whole Blood. Aliquots of a 5000 mg % galactose standard were used to spike either whole blood or plasma samples to the desired galactose concentration. The spiked plasma samples were considered as samples of “unknown” concentration. The samples (EDTA anticoagulant) used were supplied by the Yellow Springs Clinic, Yellow Springs, Ohio. After sample collection, the blood was centrifuged and the plasma was removed with a disposable Pasteur pipet. These plasma samples were immediately pooled and stored frozen until needed. Whole blood samples were not pooled and they were always used when fresh. Individual whole blood samples were first assayed for galactose concentration, as before, and then spiked in the same manner as described above for plasma. Selectivity and Interference Testing Procedure. After the galactose analyzer was calibrated with a 200 mg % galactose standard, various amounts of added substances were analyzed to determine if there was any response by the instrument. To determine whether the added substance had any effect on the enzyme activity of the electrode, 200 mg % standards were again analyzed in the presence clf the substance being tested and any discrepancy from the expecited result was noted. Unless otherwise noted, the buffer used in these studies was “buffer solution” containing 10 mg % K3Fe(CN)6and 0.2 mg % CuC12.2Hz0.
RESULTS A N D DISCUSSION Description of the Model 23A Galactose Analyzer. The peroxide electrode was mounted in the temperature controlled measurement chamber filled with about 350 WLof pre-warmed buffer solution. Standards and samples (25 pL) were injected into the chamber with a YSI precision syringepet (combination syringe and pipet). The chamber content was stirred by an air-driven silicone rubber diaphragm which assured adequate stirring plus rapid equilibration with atmospheric oxygen. A nonlimiting oxygen supply in this system was assured by the silicone rubber membrane which is very permeable to atmospheric oxygen and is in direct contact with the contents of the sample chamber. A temperature compensation probe is also present in the sample chamber. The purpose of this
Table I. Loss of Galactose Oxidase from Enzyme-Membranes after Immobilization with Varying Amounts of Glutaraldehydea Glutaraldehyde (% by wt) Days stored 1.44 0.72 0.29 0.14 0.029 0.014 2 0 0 1.4 9.0 6.0 7.4 4 0 0 1.2 5.6 7 0 0 0 1.8 6.8 a Enzyme-membranes were placed in a “buffer solution” containing also K,EDTA (1.6 X 10-3M)prepared without the addition of catalase. The data represents the enzymic activity in the buffer solution after the time indicated, expressed as a percentage of the total enzymic activity in the enzyme-membrane. probe is to compensate electronically for fluctuations in temperature (37 “C) accompanying sample injection, thereby enhancing speed of system response (22, 23). Principles of Operation. Galactose in the vicinity of the electrode diffuses through the pores in the outer polycarbonate membrane and comes in contact with immobilized galactose oxidase. The following reaction occurs (24,25); galactose
D-galactose + 0, - galactohexodialdose oxidase
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The hydrogen peroxide formed in the membrane diffuses toward the electrode, where it is sensed by the electrode, and toward the sample chamber, where it is destroyed by catalase, resulting in a steady-state hydrogen peroxide concentration a t the electrode surface (23). The plateau current response of the electrode is reached in approximately 40 s. The major factors influencing the steady-state hydrogen peroxide concentration between the membrane and the electrode are substrate concentration in the chamber, permeability of the enzyme-membrane to substrate and hydrogen peroxide and the rate of generation of hydrogen peroxide by the oxidase reaction (enzyme activity) within the enzyme-membrane. Immobilization Studies. Effective Glutaraldehyde Concentration. The effective glutaraldehyde concentration required for the immobilization and stability of galactose oxidase was determined in two ways. First, the enzymemembrane was placed in a phosphate buffer and any enzyme released into the buffer was measured using a coupled assay procedure (26). The results are shown in Table I. The data indicate that immobilization with 0.29% glutaraldehyde represented the lower limit of concentration required for enzyme fixation in the membrane when tested over a period of seven days. At the same time it was found that the initial activity from membrane to membrane prepared at the same glutaraldehyde concentration was less reproducible a t higher concentrations of glutaraldehyde than at lower concentrations, and that enzymemembranes prepared in the presence of 0.029% or less glutaraldehyde were very unstable. Figure 1, for example, illustrates the difference in stability of membranes prepared a t two concentrations of glutaraldehyde. Each point represents an average of four or more determinations, each made on different membranes and different instruments. The main conclusion to be drawn from Figure 1 is that the membranes prepared with 1.44% glutaraldehyde are somewhat less stable than the membranes prepared with 0.29% glutaraldehyde over a 14-day period. After seven days the activity of both types of membranes fell continuously to less than 10% of their original activity. On the basis of these observations, 0.29% glutaraldehyde was chosen as the most effective concentration for immobilization. Effect of Buffer Constituents. The instrument buffer used in these evaluations was “buffer solution”, containing 1.6 x M KZEDTA. Its pH was 7.3. This pH was chosen because the pH optimum of galactose oxidase is reported to be in this
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Figure 2. Effect of pH on immobilization. Stability curves of enzyme-membranes immobilized at pH 5.8 (0),pH 7.3 (A)and 9.0 (0) using 0.29 % by weight glutaraldehyde. Evaluation buffer contained M in K,EDTA “buffer solution” that was also 1.6 X
range (24). EDTA was included in these early evaluation buffers because of its effectiveness in retarding bacterial growth. Galactose oxidase is a copper containing enzyme (27) and preliminary work indicated that the presence of small amounts of EDTA in the buffer mixture did not affect enzyme activity, over a period of several days. Other investigators have reported similar observations (28). Effect of pH. Enzymemembranes were prepared at several pH values and a t the fixed optimal glutaraldehyde concentration of 0.29%. Only small differences in stability resulted when the pH of immobilization was tested over the range 5.8-9.0 (Figure 2). The range in initial enzyme-membrane activity of these membranes was similar. Since most of the data recorded to this point was obtained on enzymemembranes prepared at pH 5.8, this pH was retained for the immobilization of galactose oxidase for the remaining studies. The nature of the reaction of glutaraldehyde with proteins has been widely investigated (29, 30). Originally it was believed that glutaraldehyde formed Schiff bases with proteins (29). Recent evidence suggests that the reaction actually occurs between an oligomer of glutaraldehyde and the protein (29,311. These unsaturated oligomers react with the protein to give a Michael-type adduct (31). Whatever the mechanism of the reaction, a frequently reported characteristic of the reaction of glutaraldehyde with proteins is the occurrence of the reaction over a wide pH range of 5 to 9 (29, 30). The observations made here, support these findings, and the fact that under optimized conditions sufficient cross-linkage occurred during enzyme-membrane preparation to seal the two membranes to each other and to prevent escape of enzyme through the porous outer membrane. Membrane Activity. Enzyme Source. Studies thus far involved only the enzyme preparation isolated from Polyporus circinatus, and commercially available from Worthington Biochemicals Corporation. Another enzyme, isolated from the same genus and species, and available from Sigma BioANALYTICAL CHEMISTRY, VOL. 49,NO. 6,MAY 1977
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Effect of ferricyanide on enzyme-membrane stability. Enzyme-membranes were made by immobilization at pH 5.8 using 0.29 % by weight glutaraldehyde. Evaluation buffers contained: “buffer solution” that was also 1.6 X lo3 M in KgDTA (A)and “buffer solution” that was also 1.6 X lo-, M in K,EDTA and 10 mg % in K3Fe(CN)B (0) Flgure 3.
chemicals, was tested also. Membranes were also made with a carboxymethylcellulose ion-exchange purified lypholysate of the Worthington preparation (27). There are two conclusions which can be drawn from the data obtained. The use of the CM-cellulose preparation, in enzyme-membrane construction does not necessarily offer any advantages, at least in terms of enzyme-membrane stability, over the use of the commercial preparations. Membranes made with the two commercial preparations exhibited essentially the same stability during storage and use in the instrument. Initial enzyme-membrane activity ranged from 10-18 nA. The minimum enzyme membrane activity required for optimal use in the instrument is 8 nA. While commercial preparations varied in specific activity, a constant weight of enzyme was used in the membrane preparation. Useful membrane life of these optimized enzyme-membranes ranged from eight to ten days. Permeability of Membrane. Special membranes were constructed to study the permeability properties of the enzyme-membrane system. These enzyme-membranes were constructed as before except that the more porous polycarbonate membrane replaced the cellulose acetate membrane in order to permit the penetration of externally injected ferrocyanide and its reaction directly at the electrode surface. Studies on these special membranes indicated that the response, and presumably the permeability of the special membrane, did increase somewhat over the first 2 or 3 days but then remained relatively constant. This could explain the slight increase in activity observed over the first 2 or 3 days in the stability curve (upper curve Figure 3) for the optimized enzyme-membrane. Permeability to hydrogen peroxide and galactose are increasing and therefore steady-state hydrogen peroxide concentration is increasing concomitant to the increase of current output. It appeared that permeability did not change after the third day, and that the system response to hydrogen peroxide remained constant. It was concluded that the loss in system response to galactose after the third day, was due to losses in enzyme activity which must occur within the enzyme-membrane. Effect of Ferricyanide. Using the “buffer solution”, preliminary testing was done with human plasma. When samples were spiked with a galactose standard, it was frequently observed that less than 100% of the added galactose was indicated by the electrode. Addition of ferricyanide to the sample chamber before the addition of plasma markedly reduced the discrepancy. Without ferricyanide, the depression in membrane activity produced errors ranging from 30 to 80% depending upon the plasma sample. With a sample chamber concentration of 3 mg % ferricyanide, the error was still substantial (10-15%) but with a concentration of 10 mg % ferricyanide the error observed was in general less than 5%. 792
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Flgure 4. Effect of EDTA and cupric chloride on enzyme-membrane
stability in the presence of ferricyanide. Enzyme-membranes were made by immobilization at pH 5.8 using 0.29% by weight glutaraklehyde. Evaluation buffers contained; “buffer solution” that was also 1.6 X lo-, M in K,EDTA and 10 mg YO K,Fe(CN)6 (0),“buffer solution” that was also 10 mg % in K,Fe(CN), (A), and “buffer solution” that was also 10 mg % K,Fe(CN), and 0.2 mg % in CuCI,-2H2O ( 0 ) Evidently, the activity of the enzyme within the membrane was depressed by reducing substances in the plasma. The oxidizing agent, potassium ferricyanide, in the buffer apparently nullified the effects of the reducing substances in plasma. It also had the additional benefit that enzymemembrane activity was enhanced by a factor of approximately three in the presence of ferricyanide. In general, oxidizing agents enhance membrane activity and reducing agents decrease membrane activity as indicated in the Selectivity and Interferences section. This effect of oxidizing agents upon the activity of soluble galactose oxidase has been reported (32). The mechanism of this effect is not clear but may involve the bound copper in the enzyme (32). The enzyme is only active when copper is in a higher oxidation state (32-34). There is disagreement as to whether this higher oxidation state is copper(I1) or copper(II1) (32, 34). Oxidizing agents, such as NaI03, K2Cr207,NaI04, K3Co(NO& and NazMo04.2Hz0,were tried b,oth singly and in combination, and, in general, behaved as did K,Fe(CN)6. However, none were as compatible and stable as ferricyanide. If the reduced forms of the oxidizing agents are allowed to reach the peroxide electrode, they will be oxidized and thus give a positive interference. The cellulose acetate filter membrane effectively blocks potassium ferricyanide from reaching the electrode. Effect of EDTA. Including ferricyanide in the buffer eliminated the error in the plasma analysis but resulted in a rapid loss in activity as shown in Figure 3 (lower curve). EDTA was removed from the buffer in an attempt to improve stability. The results are shown in Figure 4, curve A. Each point represents an average of at least three determinations, each done on a different membrane and instrument. It is evident that enzyme stability in the buffer containing 10 mg % ferricyanide, in the absence of EDTA, is much improved, in contrast to what had already been observed for the soluble enzyme in buffer containing added EDTA. Effect o f Added Copper. The behavior of the enzymemembranes shown in Figure 4 suggests that the copper associated with galactose oxidase may be more susceptible to attack by EDTA in the presence of ferricyanide. Since a gradual loss of copper, and/or inactivation of the copperenzyme site, was suspected, a trace of copper in the form of cupric chloride dihydrate was added to the buffer (Figure 4, curve B). In this buffer (“buffer solution” that was also 10 mg % in ferricyanide and 0.2 mg % in CuC12-2H20), all of the membranes tested were more active after 25 days’ storage and use than they were initially. Figure 5 illustrates the results obtained when these buffer constituents, cupric chloride dihydrate and K2EDTA, were tested in “buffer solution”
Table 11. Selectivity and Interferences Level Effect of tested, Relative substance Substance tested mg % responsea on slopeb Arabinose 200 0 1.0 Dihydroxyacetone 50 5.30 1.0 D-Erythrose 120 1.0 0.07 N-Ethyl maleimide 0 0.96 1000 0 1.0 Fructose 200 0 1.0 Fucose 200 10 0.86 1.0 Galactosamine hydrochloride Galactitol 200 0 1.0 0 1.0 Galactose- 1-phosphate 40 200 0 1.0 Galacturonic acid 0 200 1.0 Glucosamine hydrochloride Glucose 1000 0 1.0 Glucuronolactone 1.0 200 0 Glycerol 1.0 500 0.067 Lactose 1.0 200 0.15 0 1.0 D-Maltose 200 0 Mannitol 200 1.0 Mannose 200 1.0 0 Melibiose 200 0.53 1.0 1.41 Me th yl-a - D-galacto0.93 200 pyranoside monohydrate 1.41 0.93 Met h yl-p-D- galac to200 pyranoside 0 10 1.0 Neocuproine 0 1.0 1,3-Propanediol 1000 0 200 1.0 Propylene glycol 100 0.33 1.0 Raffinose 200 0 1.0 Ribose 0 1.0 200 Sorbitol 0 1000 1.0 Sorbose 0.59 1.0 100 Stachyose 200 0 1.0 Sucrose D-Trehalose dihydrate 1.0 0 100 Trichloroacetic acid 100 0 1.0 D-Xylose 0 1.0 200 a Response of system toward the substance tested relRelative to an equal molar concentration of galactose. ative system response when standard galactose is assayed in the presence of the substance tested. without ferricyanide. The earlier observation that EDTA has no effect on the stability of the enzyme in this buffer was confirmed. As with the buffers containing 10 mg % ferricyanide, membranes were much more stable in buffer that also contained cupric chloride dihydrate than they were in the other combinations depicted in Figure 5. Thus, an optimal buffer solution could be defined as the “buffer solution” containing ferricyanide and copper. Explanation. A plausable explanation for this conclusion can be made. If the copper atom in each molecule of resting
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37.C
Flgure 5. Effect of EDTA and cupric chloride on enzyme-membrane stability in the absence of ferricyanide. Enzyme-membranes were made by immobilization at 5.8 using 0.29% by weight, glutaraldehyde. Evaluation buffers contained; “buffer solution” alone (A), “buffer solution” that was also 0.2 mg % in CuCI,.2H20 (0),and “buffer solution” that was also 1.6 X M in K,EDTA (0)
enzyme spends time in both the reduced, inactive state and also the oxidized, active state, then in a neutral redox environment, the time-ratio of copper reduced/copper oxidized is a constant. However, if the enzyme is stored in either an oxidizing or reducing environment, then this ratio over time will be either decreased or increased, respectively. Since copper(I1) forms a stronger complex with EDTA than does copper(1) (35), complexation and/or removal of copper from the enzyme is more likely to occur in the oxidized, active state than the reduced, inactive state simply because in an oxidizing environment the enzyme-copper spends more time in the copper(I1) state. In general, it is true that copper(I1) complexes are more stable than copper(1) complexes because of the lack of crystal field stabilization energy in d” copper(1) complexes (36). This predicts, that in an oxidizing environment, susceptibility of the enzyme-bound copper to complexation by any available ligand, e.g., EDTA, is enhanced. Both the data and the proposed explanation suggest that even in the absence of EDTA, the enzyme is more susceptible to inactivation due to loss of copper from the active site in an oxidizing environment than in a neutral redox environment. Selectivity and Interferences. Tables I1 and I11 list some of the substances that might interfere in this system. The column labeled “Relative Response” is the response of the galactose oxidase electrode to the substance listed expressed relative to the response for an equal molar galactose solution. There are two possible ways in which a substance may interfere with the measurement. It may give a positive reading when injected into the instrument or it may change the slope of the response to galactose. The latter constitutes just as serious an interference as the former and is represented in the tables by a number in the column headed “Effect of Substance on Slope”. This number is a multiplier for the slope in the presence of the interference. An entry of “zero” in the
Table 111. Effects of Oxidizing and Reducing Substances Level tested, mg %
Effect of substance on s1opeb.C 0.33
Relative responsea
Effect of substance on s1opecpd 0.99 0.97
Substance tested Ascorbic acid 4 0 Glutathione 35 0 ... Mercaptosuccinic acid 100 0 0.32 0.81 Potassium ferricyanide 100 0 3.0 1.03 Potassium ferrocyanide 100 0 0.20 0.95 Sodium periodate 100 0 ... 0 Uric acid 5 0 ... 1.0 a Response of system toward the substance tested relative to an equal molar concentration of galactose. Experiment performed in “buffer solution” in the absence of K,Fe(CN), and CuC1,.2H20. Relative system response when standard galactose is assayed in the presence of the substance tested. Experiment performed in “buffer solution” that was also 10 mg % in K,Fe(CN), and 0.2 mg % in CuC1,.2H,O. ~~
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column labeled “Relative Response” and a “1.0”in the column labled “Effect of Substance on Slope” is indicative of no interference. Interfering substances include D-erythrose, galactosamine, glycerin, lactose, melibiose, methyl-a-Draffinose, galactopyranoside, methyl-P-D-galactopyranoside, stachyose, mercaptosuccinic acid, sodium periodate, and dihydroxyacetone. The apparent interference of many of these substances may simply be due to the presence of galactose as an impurity in the reagent being tested or more likely, the substances are substrates for galactose oxidase. Of the thirty-nine substances screened, the only potentially physiologically significant interfering species was dihydroxyacetone. The last column in Table I11 reflects the improvement due to the use of the redox buffer and added cupric ion. Measurements of Aqueous Galactose Standards. The measurement of aqueous galactose standards in the region 0-500 mg 70was linear. The slope of the least squares fit of with a y-intercept of -2.56 X and the data is 5.00 X a correlation coefficient of 0.999. Increasing the concentration to 3000 mg 70also produced a linear calibration curve. The slope of the least squares fit was 5.49 X with a y-intercept of -0.16 and a correlation coefficient of 0.998. All data points for both curves are the average of at least three determinations, each on a different membrane and instrument. Measurement of Galactose in Plasma and Whole Blood. Plasma and whole blood samples, shown to be free of galactose, were spiked with galactose to known levels between 5 and 400 mg 70.Both types of samples produced linear calibration curves. The least squares fit of the plasma data had a slope of 0.980, a y-intercept of 1.76 mg 70,and a correlation coefficient of 0,999. Similarly, whole blood yielded a slope of 1.05, a y-intercept of -1.81 mg %, and a correlation coefficient of 0.999 when subjected to least squares treatment. Each data point represented the average of a t least three determinations, each done on a different membrane and instrument. The pooled estimate of the standard deviation on the plasma and whole blood was 2.1 and 2.7 mg %, respectively.
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RECEIVED for review January 13,1977. Accepted February 17, 1977.
