Analytica Chimica Acta, 221 (1989) 337-340 Elsevier Science Publishers B.V., Amsterdam -
337 Printed in The Netherlands
FLOW-INJECTION DETERMINATION OF MALATE IMMOBILIZED MALATE DEHYDROGENASE
and ALAN TOWNSHEND*
of Chemistry, University of Hull, Hull HU6 7RX (Great Britain)
(Received 29th November 1988)
Summary. Malate dehydrogenase (MDH) is immobilized chemically on controlled-pore glass and used on-line in a glass minicolumn (25 X 2.5 mm i.d. ). Malate solution passes through the minicolumn of immobilized MDH and the NADH formed is monitored spectrophotometrically from9X10-4downto7X10-“M (36ngin40$)at50samplesh-‘.
Various batch enzymatic methods have been reported for the determination of malate. For example, malate dehydrogenase (MDH) catalyses the oxidation of malate by NAD+: MDH malate + NAD + 6 oxaloacetate + NADH + H+
The concentration of NADH formed, as measured by the increase in absorbance at 340 nm, is proportional to the original malate concentration [ 11. MJate can also be determined by coupling the above reaction with L-glutamate in the presence of glutamate oxaloacetate transaminase. The NADH formed is again detected spectrophotometrically . Guilbault et al.  reported a fluorimetric method which included six enzyme systems for determination of 21 organic acids, including malate. Such batch enzymatic methods are enzyme- and time-consuming. The usefulness of immobilized enzymes for economy and ease of handling is well recognized. This communication describes a simple flow-injection system, based on MDH chemically immobilized on controlled-pore glass (CPG ) for the rapid and reproducible determination of malate, based on Eqn. 1. The NADH formed is monitored spectrophotometrically at 340 nm. Experimental Chemicals. Distilled, deionized water was used throughout. Malate dehydrogenase (L-malate : NAD+ oxidoreductase; E.C. 126.96.36.199,12.5 U mg-’ ), L-malic acid, NAD + (free acid, 98% ), 3-aminopropyltriethoxysilane and controlledpore glass (CPG 240-200,2.4 pm nominal pore size, 120-200 mesh) were ob-
0 1989 Elsevier Science Publishers B.V.
ml Phosphate pH 11.5
Fig. 1. Manifold for malate determination.
detector, 340 nm; W, waste.
tained from Sigma Chemical Co. Glutaraldehyde (50% ) was obtained from BDH. Phosphate buffer (0.1 M) was adjusted to the desired pH (normally 11.5) with 0.1 M sodium hydroxide. Working standard solutions were obtained by appropriate dilution of a stock malate solution (7.5 x 10m3M). Immobilization oflt4DI-I. This was achieved on CPG following the procedure described earlier for alcohol dehydrogenase , except that 40 mg was immobilized on 0.4 g of CPG. The product was packed in a glass column (25 X 2.5 mm i.d. ) . Apparatus and procedures. The components, including the spectrophotometer, injection valve, pump, manifold and reaction coil tubing, were as reported for the determination of acetaldehyde [ 51. The manifold used is shown in Fig. 1. The immobilized enzyme column was maintained at 25 ’ C by a flow of water from a thermostat bath. Results and discussion Optimization studies. An investigation of the effect of flow-rate showed that the peak height absorbance for 4.4 x 10M4malate decreased by 50% as the flowrate was increased from 1.2 to 4.0 ml min-‘. This would be expected because at slower flows the malate solution is in contact with the enzyme for longer periods and there is less dispersion; 2 ml min -l was selected for further work. The influence of pH was investigated with 0.1 M orthophosphate and pyrophosphate buffers. The results are shown in Fig. 2. Maximum activity was found at ca. pH 11.5 compared with pH 9.0 [l] for the soluble enzyme. The peak heights were greater with orthophosphate than with pyrophosphate buffer. Therefore, orthophosphate buffer was chosen for subsequent work. The effect of NAD+ concentration, investigated over the range 0.3 X 10m33.6~ 10e3 M, is shown in Fig. 3a. The absorbance increases with increasing NAD+ concentration until it reaches a maximum at 3 x 10T3 M; 3.6 x 10m3M was selected for further use. It has been reported that the equilibrium can be displaced more in favour of oxaloacetate and NADH if the oxaloacetate is reacted with hydrazine to form the hydrazone [ 11. However, hydrazine in the concentration range 2 x 10V316 x 10e3 M had no effect on the peak height for 3.6 x 10m4 M malate. This might be expected because little time was available for reaction between hy-
Fig. 2. Effect of pH on the peak-height absorbance obtained for 4.4X lo-* M malate: ( X ) 0.1 M orthophosphate buffer; (0 ) 0.1 M pyrophosphate buffer.
0% - ( a )
I 2.0 NAd
Fig. 3. Effect of (a) NAD+ concentration MDH (4.4~10~~ M malate).
and (b) temperature on the activity of the immobilized
drazine and the oxaloacetate produced by the enzymatic reaction inside the column. The effect of temperature on the activity of the immobilized enzyme was examined by passing a flow of water at various temperatures around the column containing the enzyme. The results are shown in Fig. 3b. The enzyme
Fig. 4. Calibration graph and peaks for malate. The number on the peaks are mol 1-i malate.
showed maximum activity at 45”C, but to prolong the life of the enzyme 25’ C was selected for subsequent work. AnalyticaZperformance. When the optimized conditions were used, the calibration graph of peak height vs. malate concentration was linear over the range 7 x 10w6-9 x 10e4 M, with a regression coefficient (21 points) of 0.994. The least-squares calibration equation was peak height absorbance = 219 [ malate] + 0.018. The detection limit (2 x noise) was 7 x 10M6M (36 ng in 40 ~1) and the maximum sample injection rate was 50 h-l. The relative standard deviation for ten injections of 4.4~ 10m4 M malate was 1.5%. The calibration graph and peaks are shown in Fig. 4. The malate dehydrogenase immobilized on CPG showed good stability. The same column was used at 25°C for 1 month without any significant decrease in activity. A.M.A. is grateful to the Iraqi Government (Ministry of Higher Education and Scientific Research ), University of Salahaddin, for financial support.
REFERENCES 1 I. Gutmann and A.W. Wahlefeld, in H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, 2nd edn., Vol. 3, Verlag Chemie, Weinheim, 1974, p. 1585. 2 H. Mollering, in H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, 2nd edn., Vol. 3, Verlag Chemie, Weinheim, 1974, p. 1589. 3 G.G. Guilbault, S.H. Sadar and R. McQueen, Anal. Chim. Acta, 45 (1969) 1. 4 A.M. Almuaibed and A. Townshend, Anal. Chim. Acta, 214 (1988) 161. 5 A.M. Almuaibed and A. Townshend, Anal. Chim. Acta, 198 (1987) 37.