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RECEIVEDfor review June 21,1989. Accepted November 22, 1989. This research was supported by a cooperative research agreement with the Dow Chemical Company, U.S.A.
Optimization of Multienzyme Flow Reactors for Determination of Acetylcholine P. Chandrani Gunaratna and George S. Wilson*
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045
Immobillzed enzyme reactors have been used wlth highperformance liquid chromatography (HPLC) and electrochemical detection to detect acetylcholine and choline In brain tissue samples. Acetylcholine and choline eluting from the LC column are introduced into a reactor containing immobilized acetylcholinesterase, which hydrolyzes acetylcholine to choline. The product is converted by a second enzyme, choline oxidase, to hydrogen peroxide, which Is determined amperometrically. Several novel Immobilization techniques including lmmobillzation through enzyme-specific antlbodles were used to Immobilize these enzymes to retain maximum activity. Improved detection limits were observed when the enzymes were Immobilized through the avidin-biotin linkage. Better sensitivity and detection limit were obtained when both enzymes were hnmobillzed together on the same support through the avldln-biotin linkage than when they were separately immobillzed and used in two columns. The POstcolumn system was applied to brain tissue extracts.
INTRODUCTION Acetylcholine (Ach), the main neurotransmitter in the cholinergic neurotransmitter system, is found in mammalian brain tissues. Determination of Ach and Ch levels in the brain is very important due to the significant role it plays in neuropsychiatric diseases ( I ) . Increasingly sensitive assay techniques are required to measure the low concentrations of Ach and Ch in the brain.
* To whom correspondence should be addressed.
Several physicochemical methods are available for the determination of Ach. These methods include GC-MS (2) and radiometric assays (3). However, these techniques are associated with time-consuming, complicated procedures and require expensive equipment. Recently, immobilized enzyme reactors (IMERs) have been used with high-performance liquid chromatography (HPLC) and electrochemical (EC) or chemiluminescence detection to assay Ach (4-7). These methods are now gaining popularity. Use of IMERs utilizing HPLC with EC detection is based on the separation of Ach from brain tissue extracts by reverse-phase HPLC, passing the effluent through acetylcholinesterase (AchE) and choline oxidase (ChO) immobilized reactors, followed by the amperometric detection of hydrogen peroxide produced by the following reaction scheme: Ach
AchE
Ch
choline (Ch)
ChO
betaine
+ CH3COO-
+ 2H202
However, the IMERs are not without problems. One major limitation is the loss of enzyme activity in the immobilization step due to either the binding of the enzyme to the support through or near its active site or binding in an orientation such that the active site is inaccessible. Performance of the IMER depends on the properties of the enzyme immobilized. For example, in our system AchE is more durable than ChO and can be immobilized without losing activity. ChO is very sensitive to its environment, and any change in the immediate environment surrounding the enzyme can cause a significant loss in activity. Various methods of immobilization have been used in our studies to retain the maximum activity of the enzymes. These
0003-2700/90/0362-0402$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 4, FEBRUARY 15, 1990
0-C-
f" I
+
O 'I
H'
m
N
enzyme
->
N H ~
NH2 spacer
r N
/
-
