Analytica Chimica Acta, 160 (1984) 141-147 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

BQUND COFACTOR/DUAL ENZYME ELECTRODE SYSTEM FOR L-ALANINE

C. P. PAU and G. A. RECHNITZ*

Department of Chemistry, University of Delaware, Newark, DE 19711 (U.S.A.)

(Received 12th November 1983)

SUMMARY

A dual enzyme-bound coenzyme electrode system for quantifying L-alanine is de- scribed. Commercially available dextran-bound NAD was incorporated into an L-alanine dehydrogenase (E.C. 1.4.1.1)/L-lactate dehydrogenase (E.C. 1.1.1.27) enzyme system and held at the surface of a potentiometric ammonia gas sensor. Using this system, L-alanine calibration curves with a slope of 45 mV/decade and 10e5 M detection limit were obtained with a sensor lifetime of at least 10 days. This system is potentially useful for the clinical determination of L-alanine in serum.

Use of immobilized enzymes with electrodes has considerably reduced the cost of determinations involving enzymatic reactions because the expensive enzymes are used repeatedly. However, many such combinations require the addition of cofactors for proper operation and it is often difficult to co- immobilize low-molecular-weight cofactors at an electrode surface along with the primary enzyme or enzymes.

This paper describes the co-immobilization of dextran-bound NAD’ cofactor in a dual ‘enzyme system to construct a detection system for L- alanine that has good selectivity and response characteristics. It is shown that the dual enzyme-bound cofactor system not only shifts the reaction equilibrium in the desired direction, but also provides for in situ regeneration of the active cofactor. Nicotinadeninedinucleotide (NAD) can be effectively immobilized on sepharose [I], glass surfaces [2], polyethyleneimine [3], and dextran [ 41, but only the latter yields high activity and good stability. More than a decade ago, Davies and Mosbach [5] used dextran-bound NAD in conjunction with dehydrogenase enzymes and a cation-sensitive glass electrode to construct a glutamate sensor. That electrode system displayed a very low response slope and narrow concentration range.

In the present work, L-alanine dehydrogenase and L-lactate dehydrogenase are co-immobilized with dextran-bound NAD at the surface of an ammonia gas-sensor to devise an L-alanine sensor with excellent sensitivity, dynamic range, and cost-saving advantages.

In the proposed system, L-alanine is enzymatically oxidized to pyruvate by dextran-NAD:

0003-2670/84/$03.00 0 1984 Elsevier Science Publishers B.V.

142

L-alanine

CH3

dehydrogenase H-COO- + Dextran-NAD+ i- Hz0 ,

H; E.C. 1.4.1.1.

(L-alanine)

CH3

2

--COO- + Dextran-NADH + NH,’ + H’

(pyruvate)

(1)

Dextran-NAD’ is then regenerated in situ using lactate dehydrogenase, e.g.

L-lactate

-COO- + Dextran-NADH + H’ dehydrogenase

\E.c.

(w-ate)

CH3

-r

-COO- + Dextran-NAD’

H

(L-lactate)

(2)

The NH: generated from Reaction 1 is monitored by the ammonia gas sensor. The introduction of the second reaction not only regenerates the dextran-NAD’, but also removes the products of Reaction 1 from the enzyme layer and, thus, shifts the equilibrium to the right. As a result, the use of a poisonous hydrazine trap (to remove pyruvate) and high pH (>9.0) as in the spectrophotometric methods [6] can be avoided. This regeneration scheme is further illustrated in Fig. 1.

For the purpose of comparison and evaluation of the bound coenzyme electrode, a second electrode without the incorporation of dextran-NAD was also prepared and studied. In this case, NAD’ was added to the reaction mixture for each measurement at a final concentration of 2.5 X lo4 M.

EXPERIMENTAL

Apparatus and reagents All potentiometric measurements were made with a Corning model 12

pH/mV meter in conjunction with a Heath/Schlumberger model SR-204 strip-chart recorder. Measurements were made in thermostated cells at 25°C f O.l”C, controlled with a Haake model FS water bath. The ammonia gas sensor was the Orion model 95-10. A solution containing 5 mM ammo- nium chloride in 0.2 M NaCl was used as the internal filling solution for the ammonia sensor.

All biochemical reagents were obtained from Sigma Chemical Co. (St.

143

pyruvate

+

dextran-NADH + n+

L- lactate L-lactate dehydrosenase

Fig. 1. Enzyme cycle and dextran-NAD+ regeneration in the biocatalytic layer.

Fig. 2. Schematic of the bound-cofactors/dual enzyme electrode system: (a) dialysis membrane; (b) biocatalytic layer; (c) NH, gas-permeable membrane; (d) internal filling solution; (e) bottom cap; (f) internal pH sensor; (*) NH, gas product; (0) dextran-NAD; ( l ) L-alanine.

Louis, MO). L-Alanine dehydrogenase (E.C. 1.4.1.1) was a suspension in 2.4 M ammonium sulfate. L-Lactate dehydrogenase (E.C. 1.1.1.27, type XI: from rabbit muscle) was a lyophilized powder. Dextran-NAD (catalog number N3383) contained 48 pmol of P-NAD per gram of solid. All solutions were prepared with distilled, deionized water. Buffer solutions (pH 8.0) were prepared by dissolving 0.2 moles of Tris in 1.0 1 of water containing 2 g of disodium dihydrogenethylenediaminetetraacetic acid dihydrate (EDTA) and then adjusting the pH with 0.2 M HCl.

Procedure The dual enzyme/coenzyme combination was incorporated into the elec-

trode by placing 15 ~1 of L-alanine dehydrogenase (3.3 units), 0.1 mg of L-lactate dehydrogenase (90 units) and 2 mg of dextran-NAD directly on the gas-permeable membrane of the ammonia sensor and mixing well. Excess of water in the mixture was then removed by vacuum. A smooth thin film of solid was left on the membrane. A dialysis membrane (Technicon type C), precut to the size of the permeable membrane, was then placed on top of the solid film. The sensor was assembled as recommended by the manufacturer and soaked in 0.01 M phosphate buffer of pH 6 (containing 5 mM EDTA) for 24 h in order to remove the ammonium sulfate present in the L-alanine dehydrogenase suspension. The phosphate buffer solution was renewed several times. This procedure yields a smooth biocatalytic layer of minimum thickness on the electrode system. A schematic of the resulting electrode is shown in Fig. 2. A second electrode system was prepared similarly but with- out dextran-bound NAD.

For potentiometric measurements, 10 ml of Tris buffer (pH 8.0) was

144

added to the glass cell thermostated at 25.0 f O.l”C. The solution was stirred with a small teflon-coated stirrer at a constant speed. After a constant base- line potential had been reached, 10-100 ~1 of the stock substrate solutions of known concentrations was injected into the buffer solution and the potential changes were recorded. In the studies of potentiometric response to L-cysteine, L-serine, L-isoleucine, L-aspartic acid, and L-valine, pyruvic acid (final concentration of 10m3 M) was added to the buffer for the regener- ation of the active dextran-bound NAD.

Recovery studies were conducted with a synthetic serum containing 3.23 X 10~--6.06 X lo4 M L-alanine. The composition of the synthetic serum is listed in Table 1. The concentration of each component of the “serum” was chosen to match its normal level reported in human serum [ 71. The final pH of the “serum” was 7.46. The electrode system was immersed into 9.0 ml of Tris buffer of pH 8 at 25°C. When a stable base-line potential had been reached, 1 ml of the synthetic serum, pre-incubated to 25”C, was injected into the buffer and the potential changes were measured. A calibra- tion curve constructed on the same day was used to calculate the L-alanine concentration.

RESULTS AND DISCUSSION

The potentiometric response of the sensor system at pH 8 and 25°C is shown in Fig. 3. It can be seen that a calibration slope of -45.1 f 0.9 mV/ decade (standard error of 0.35, correlation coefficient of 0.9992) for L-

alanine is obtained over the range of 2 X 10-‘-l X 10e3 M. This improved sensitivity over the glutamate electrode (slope of 10-20 mV/decade) re- ported by Davies and Mosbach [5] may be a result of using a higher concen- tration of the dextran-bound cofactor, better enzyme activity, and the more advanced internal sensing electrode. In preliminary studies, it was found that a minimum of 2 mg of dextran-NAD was required to achieve the reported sensitivity and that further increases in the amount did not improve the response of the electrode. The amount of dextran bound NAD used in this study (expressed as moles of nucleotide per unit activity of the deaminating

TABLE 1

Composition of simulated serum

Compounda Cont. (M) Compounda Cont. (M) Compounda Cont. (M)

Arginine (HCl) 2.3 X lo-’ Lysine (HCl) 2.6 x lo- Threonine 2 x 1o-5 Aspartic acid 9x 1o-5 Methionine 3 x lo-” Valine 6.8x lo+ Glutamic acid 8.9 X lo+ Phenylalanine 1.6 x lo4 NaHCO, 2.5 x lo-’ Histidine (HCl) 1.2 x lo4 Tyrosine 8 x lo+ NaCl 1.0 x 10-l Isoleucine 1.0 x lo4 Tryptophan 7x lo* Alanine 3.23-6.06 x lo4

a All amino acids are (L) isomers.

145

1 -b,,[.ub.tr.t.] (I)

50

40

; 30 E

: 2(

IC

0

2 Tlnm

Fig. 3. Potentiometric response of the electrode system in Tris buffer of pH 8.0, 25°C. Substrate: (a) L-alanine; (b) L-serine. Dotted region shows the maximum response to L-

cysteine, r_.-aspartic acid, L-isoleucine, and L-valine.

Fig. 4. Actual potentiometric response of the bound-cofactor electrode system at differ- ent pH: (a) 7.5; (b) 8.0; (c) 8.5; (d) 9.0. In all instances, 20 ~1 of 0.016 M L-alanine was injected into 10 ml of Tris buffer. Arrows indicate where injection takes place.

enzyme) was at least one order of magnitude greater than that used in the earlier glutamate electrode.

The limit of detection, defined as the concentration of L-alanine obtained when extrapolating the linear region of the standard curve to the base-line potential is lO+ M, which is near the detection limit of the ammonia sensor itself. This detection limit is at least one order of magnitude below the nor- mal alanine concentration in human serum [6, 71. Consequently, serum samples can be substantially diluted with buffer to minimize variation in sample pH and osmolarity.

A steady-state potential is usually reached within 7 min and sustained for at least 10 min. The recovery time (i.e., time for return to baseline) varied with substrate concentration, but generally ranged between 15 and 20 min. Both response and recovery times are comparable with other similar systems.

The detection system was very stable. The calibration slopes did not change significantly over a period of nine days, as shown in Table 2. The potentiometric response and long-term stability of the electrode system were similar to those of the second electrode (not shown) in which free NAD was used instead of dextran-bound cofactor, indicating that the use of the immobilized cofactor does not sacrifice performance.

The effect of pH on response (AE vs. time) is shown in Fig. 4. Initial

146

TABLE 2

Time stability of the bound cofactor/dual enzyme electrode system

Day Calibration slopea (mV/decade)

2 5 6 7 8 9 46.1 45.9 43.6 43.0 44.0 43.0

aTaken in the linear portion of the L-alanine calibration curve (2 X 1O-5-l X lo* M).

slopes of the response curves are 12, 20, 23.5, and 25 mV min? at pH 7.5, 8.0, 8.5, and 9.0, respectively. These slopes reflect the kinetics of the enzy- matic deamination of L-alanine in the biocatalytic layer. The increase of the initial slopes from pH 7.5 to 9.0 agreed with the reported kinetic parameters [ 81. In this study, pH 8.0 was chosen to maximize the stability of the en- zymes.

It has been reported that L-serine, L-cysteine, L-aspartic acid, L-valine, and L-isoleucine are slowly deaminated by L-alanine dehydrogenase [8] . The potentiometric responses to those amino acids are also shown in Fig. 3. Because the enzymatic deamination of these amino acids does not produce pyruvic acid, contrary to the case of L-alanine, 10m3 M pyruvic acid was added to the samples in order to regenerate the active dextran-NAD. This pyruvic acid concentration was chosen to correspond to the maximum con- centration produced in the biocatalytic layer in the L-alanine experiments so that the potentiometric responses to the various amino acids can be com- pared. It can be seen from Fig. 3 that none of the aforementioned amino acids, except L-serine, yielded significant response up to 3 mM. Moreover, the normal L-serine in human serum was reported to be below 3 X lo4 M [ 71 while the normal L-alanine was above 3.5 X lo4 M [ 6, 81. Thus, a 1: 10 dilution of a sample containing 3.5 X lo4 M of L-alanine would yield a potentiometric response within the linear range of the standard curve while a 1:lO dilution of a 3 X lo4 M L-serine would yield an insignificant re- sponse.

TABLE 3

L-Alanine recovery from synthetic serum

Substrate addeda Substrate found (lo+ M) (lOA M)

Recovery (%)

3.23 4.04 5.05 6.06

3.21 f 0.02b 99.4 4.18 LO.03 103.5 5.12 * 0.04 102.4 6.15 + 0.15 101.5

Average 101.5

aBy werght. bStandard deviation of 3 replicates.

141

Results of the recovery studies are summarized in I’,able 3. An average CC 101.5% recovery was obtained with a maximum error of 3.5%. These 1 ~sul;s demonstrate that the bound cofactor/dual-enzyme electrode system co Ild I>e useful for the determination of L-alanine in serum. All of the enzgmes and cofactors are reusable, so that appreciable cost saving is possible.

We gratefully acknowledge the support of the National Institutes of Health Grant GM-25308.

REFERENCES

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80 (1977) 1. 7 T. Gerritson, M. L. Rehberg and H. A. Waisman, Anal. Biochem., 11(1965) 460. 8 A. Yoshida and E. Freese, Biochim. Biophys. Acta, 96 (1965) 248.