Microcalorimetric assay of acetylcholinesterase

195

Biochimica et Biophysica Acta, 6 2 9 ( 1 9 8 0 ) 1 9 5 - - 1 9 8 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA Report BBA 21526

MICROCALORIMETRIC ASSAY OF ACETYLCHOLINESTERASE*

S I M O N R O S E N S T E I N a n d H A R R Y D. B R O W N * *

Rutgers Univer:~ity, New Jersey Agricultural Experiment Station, New Brunswick, NJ 08903 (U.S.A.) (Received J a n u a r y 1 7 t h , 1 9 8 0 )

Key words: Acetylcholinesterase assay; Acetylcholine; Acetylthiocholine; Dithiobisnitrobenzoic acid; (Microcalorimetry )

Summary Comparative assays were made in a spectrophotometer and a microcalorimeter for the reaction between acetylcholinesterase (EC 3.1.1.7) and acetylthiocholine. The rate of light absorbance change and the rate of heat flow were measured from similar and simultaneous reactions in spectrophotometer and microcalorimeter, respectively. At the enzyme activity levels studied, i.e., 0.05--0.15 I.U. in calorimetry and 1--4 I.U. in spectrophotometry, the reaction rates were linear and showed first-order kinetics. A highly significant positive correlation was seen between the two methods (r = 0.997). More importantly, spectrophotometric assay with acetylthiocholine (which utilized a secondary reaction with chromagen, dithiobisnitrobenzoic acid) stood in highly significant positive correlation with calorimetric assays (which did not require a chromagen) either with the same substrate (r = 0.976) or with acetylcholine (r = 0.900). It appears that microcalorimetry can be used in preference to spectrophotometry for enzyme kinetic studies to overcome the complexity of reaction mixture and interference problems and with the advantage of using natural substrates.

The activities of erythrocyte acetylcholinesterase (EC 3.1.1.7) and serum cholinesterases (EC 3.1.1.8 and others) are of clinical significance; low levels of activity may be indicative of a number of disorders, including liver disease and pesticide poisoning. *Paper No. 4283 of the Journal Series, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, NJ. **To whom reprint requests should be addressed.

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One of the c o m m o n assay methods for cholinesterase activity is that of Michel [1], in which the drop in pH of the assay mixture due to acetic acid production is measured. Its disadvantages include the possibility of pH change, due to acid fumes or other airborne contaminants, and the long time interval required. Another method that relies on the pH change is that of Rappaport et al. [2], wherein m-nitrophenol is used as a pH indicator for the spectrophotometric measurement. Though the reaction time in this assay is less than that of Michel's method, it is still dependent upon the pH change and is subject to the above limitations. The method developed by Ellman et ah [3] uses thioester of acetylcholine as the substrate. The enzymic hydrolysis produces acetic acid and thiocholine; the latter reacts with dithiobisnitrobenzoic acid (also known as Ellman's reagent) to form the colored species which is measured spectrophotometrically at 400--420 nm. The reaction is carried o u t in a well-buffered system and the pH remains fairly constant. In a complex system, however, the chromagen may be susceptible to interfering c o m p o u n d s present in the sample [4]. Therefore, it would seem desirable to measure the rate of substrate hydrolysis at constant pH and without reliance on a secondary reaction. The enthalpy of hydrolysis thus offers a direct measurement of the rate of reaction. We present here a comparison between Ellman's spectrophotometric assay and our microcalorimetric assay. A Tian-Calvet conduction-type microcalorimeter was constructed in our laboratory, similar in design to that described by Evans [5]. The direct current o u t p u t was amplified by a Leeds-Northrup amplifier set at a gain of 1000 and recorded on a Leeds-Northrup chart recorder of 0--1 mV full-scale. The instrument was calibrated with a series of Tris-HC1 neutralization reactions [6] ranging from 4.75 to 47.5 mcah The sensitivity of the instrument was thus determined to be 25 mcal per min per pV. For the spectrophotometric assay, a Varian spectrophotometer (model 635) was used, and the o u t p u t was recorded on a Varian chart recorder. A series of spectrophotometric (Fig.l) and calorimetric (Fig.2) assays were run with varying activities of acetylcholinesterase and a linear relationship was seen between the enzyme concentration and rate of the reaction (Fig.3). Unlike with the spectrophotometer, the calorimetric data are noncumulative over the period of reaction time. Therefore, the calorimetric curve remained at a constant level as long as the enzyme was functioning at its maximum capacity (Fig.2). However, the shape of the curve at high enzyme concentration changed after 4--5 min. By doubling the concentration of dithiobisnitrobenzoate in a separate experiment, we found that the decline of the curve was, in fact, due to shortage of dithiobisnitrobenzoate and not due to either enzyme or substrate. For this reason, the heat of reaction at initial mixing was obtained by extrapolation of the curve (Fig.2, broken arrows). It should be pointed out that the dithiobisnitrobenzoate shortage was not a problem in the spectrophotometric assay which involved only the first 2 min of the reaction. The time-course reaction rates obtained from the calorimeter using either acetylthiocholine or acetylcholine were compared statistically with those of the spectrophotometer using acetylthiocholine plus dithiobisnitro-

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benzoate (Fig.4). A highly significant positive correlation was seen with acetylthiocholine and dithiobisnitrobenzoate in both the methods (r = 0.997). Also, highly significant positive correlation was seen in the case of acetylcholine (r = 0.900) as well as acetylthiocholine (r = 0.976) when their spectrophotometric data (without dithiobisnitrobenzoate) were correlated with the calorimetric data of acetylthiocholine plus dithiobisnitrobenzoate.

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Kinetic studies made with a spectrophotometer generally depend on a secondary reaction that yields a measurable colored product. This indirect means suffers from the disadvantages of varying interferences by the chromagen and/or the colored product as the reactions proceed. In our present study, using acetylcholinesterase as a model enzyme for microcalorimetric evaluation, we were able to obtain reaction rates identical to those of spectrophotometer, using the natural substrate and without reliance on a secondary reaction. It appears possible that kinetic studies of similar enzyme systems can be performed on a microcalorimeter without the disadvantages associated with spectrophotometry. This work was supported b y USPHS grant GM 22679 and as a part of Project No. 40102 of the New Jersey Agricultural Experiment Station (Hatch Act Funds}.

References 1 2 3 4 4 6

Michel, H . O . ( 1 9 4 9 ) J. L a b . Clin. Med. 3 4 , 1 5 6 4 - - 1 5 6 8 R a p p a p o r t , F., F i s c b l , J. a n d P i n t o , N. ( 1 9 5 9 ) Clin. C h i m . A c t a 4, 2 7 7 - - 2 8 0 E l l m a n , G . L . , C o u r t n e y , K . D . A n d r e s , V. a n d F e a t h e r s t o n e , R . M . ( 1 9 6 1 ) B i o c h e m . P h a r m a c o l . 7 , 88--95 A u g u s t i n s o n , K. a n d E r i k s s o n , H. ( 1 9 7 4 ) B i o c h e m . J. 1 3 9 , 1 2 3 - - 1 2 7 E v a n s , W.J. ( 1 9 6 9 ) in B i o c h e m i c a l M i c r o c a l o r i m e t r y ( B r o w n , H . D . , e d . ) , p p . 2 5 7 - - 2 7 2 , A c a d e m i c Press, N e w Y o r k Berger, R . L . ( 1 9 6 9 ) in B i o c h e m i c a l M i c r o c a l o r i m e t r y ( B r o w n , I-I.D., ed.), p p . 2 2 1 - - 2 3 4 , A c a d e m i c Press, N e w Y o r k