A new and rapid colorimetric determination of acetylcholinesterase activity.

A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described. The enzyme activity is measured by following the increase of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. It is based on coupling of these reactions: (enzyme) acetylthiocholine -----+ thiocholine + acetate thiocholine + dithiobisnitrobenzoate -+ yellow color The latter reaction is rapid and the assay is sensitive (i.e. a 10 pl sample of blood is adequate). The use of a recorder has been most helpful, but is not essential. The method has been used to study the enzyme in human erythrocytes and homogenates of rat brain, kidney, lungs, liver and muscle tissue. Kinetic constants determined by this system for erythrocyte cholinesterase are presented. The data obtained with acetylthiocholine as substrate are similar to those with acetylcholine. INTRODUCTION A FEW years ago Bonting and Featherstonel introduced a modification of the Hestrin hydroxamic acid method2 suitable for the determination of cholinesterase levels in small quantities of cells cultured in vitro. This modification was used successfully in several studies involving the control of enzyme levels in cells by manipulating the levels of substrates or closely related compounds present in the medium.3 Several interesting areas of research were indicated by these studies. However, this modified method of enzyme assay, although scaled down to the micro-level, had several disadvantages. Among these was the fact that only a terminal figure could be obtained from the material in one tube of cultured cells. Thus, the time course of the reaction could not be followed without resorting to separate experimental tubes for each time interval desired. The method also had the disadvantage that the color measured *was developed from the remainder of an added substrate-a procedure in which the possibility of error is relatively great when the level of enzyme activity is small. Consideration of the relative merits of various methods which might be useful in studying the time-course of acetylcholinesterase activity in very small tissue samples led us to combine a method reported by Koelle4 with a sulfhydryl reagent studied by Ellman.5 This new method, which is presented here, is extremely sensitive and is applicable to either small amounts of tissue or to low concentrations of enzyme. It makes detailed kinetic studies of acetylcholinesterase activity possible. The progress of the hydrolysis is followed by the measurement of a product of the reaction. 88 A new and rapid calorimetric determination of acetylcholinesterase activity 89 Acetylthiocholine is used as the substrate. This analog of the natural substrate has been used most extensively by Koelle4 for histochemical localization. Other workerss, ’ have used the sulfur analog in the enzyme assay. Their work, in addition to data we shall present, suggests that this compound is a satisfactory substitute for the natural substrate, and differs much less than some of the synthetic substrates frequently used as in assays of phosphatases, trypsin, chymotrypsin, pepsin, etc. The principle of the method is the measurement of the rate of production of thiocholine as acetylthiocholine is hydrolyzed. This is accomplished by the continuous reaction of the thiol with 5 : Sdithiobis-2-nitrobenzoate ion (I)5 (enzyme) H,O + (CH,),&CH,CH,SCOCH, h (CH&CH2CH2S+ + CH,COO+ 2H+ (CH&H$H$+ RSSR + (CH&&CH&H,SSR + RS(I) (II) to produce the yellow anion of 5-thio-2-nitro-benzoic acid (II). The rate of color production is measured at 412 rnp in a photometer. The reaction with the thiol has been shown to be sufficiently rapid so as not to be rate limiting in the measurement of the enzyme, and in the concentrations used does not inhibit the enzymic hydrolysis. By recording the output of the photometer continuously, records of the complete assay can be obtained (Fig. 1). We considered it desirable to establish that this method yields results comparable with other procedures. For this reason, the effects of inhibitors were examined; the kinetic constants were calculated and compared with those obtained by other methods. In addition, we were able to compare assays on blood samples by the ferrichydroxamate methods and the present one. METHODS The reaction rates were recorded with a Beckman DU spectrophotometer equipped with a Beckman adapter and a Minneapolis-Honeywell recorder. The general method used was to place buffer in the photocell and add concentrated solutions of reagents by means of micropipettes. The mixture was stirred by continued blowing through the pipettes while moving them around the bottom of the photometer cells. In this way, reagents were added, mixed, and the cover of the cell compartment replaced within 10-15 sec. Our photometer required 3040 set to become stabilized to new light conditions. Thus, there was about 1 min when the readings were due to a combination of factors (e.g. bubbles rising through the solutions, sulfhydryl material in the “enzyme”, etc.) which were unrelated to the desired measurements. Subsequent readings were strictly dependent on the absorption of the solution under consideration and even rapid changes were followed by the recorder faithfully, as evidenced by the reproducibility of time-transmission curves (Fig. 1). 90 GEORGE L. ELLMAN et. al. Solutions Buffer. Phosphate, 0.1 M, pH 8.0. Substrate. Acetylthiocholine iodide, * O-075 M (21.67 mg/ml). This solution was used successfully for lo-15 days if kept refrigerated. Reagent. Dithiobisnitrobenzoic acid (DTNB) 0.01 M of the 5 : 5-dithiobis-2nitrobenzoic acid7 prepared as described previously,5 39.6 mg were dissolved in 10 ml pH 7.0 phosphate buffer (O-1 M) and 15 mg of sodium bicarbonate were added. The reagent was made up in buffer of pH 7 in which it was more stable than in that of pH 8. Enzyme. Bovine erythrocyte cholinesterase (Nutritional Biochem. Corp., 20,000 units) was dissolved in 20 ml of 1% gelatin. This solution was diluted 1 : 200 with water for use, yielding a solution of 5 units/ml. General method A typical run used : 3.0 ml pH 8.0 buffer, 20.0 ~1 substrate, 100.0 ~1 DTNB (reagent), 50.0 ~1 enzyme. The results of several runs are shown in Fig. 1. The blank for such a run consists of buffer, substrate, and DTNB solutions. The absorbances: were read from the strip charts and plotted on a rectangular graph paper, the best line drawn through the points and the slope measured. In a run such as that described above, the linear portion of the curve describing the hydrolysis was observed during the first 15-20 min of the reaction; the slope is the rate in absorbance units/min. At this pH level, there is an appreciable non-enzymic hydrolysis of the substrate, and for long runs it was necessary to correct for this. The rate of non-enzymic hydrolysis of acetylthiocholine at 25” was 0.0016 absorbance units per min. The procedures have been extended to micro-size. A run comparable to those in Fig. 1 was made in a micro-cell (total solution volume was O-317 ml). The rate was 0*102/min, the same as that determined in the larger cuvettes. Since the extinction coefficient of the yellow anion (II) is known5 the rates can be converted to absolute units, viz. : A absorbance/min rate (moles/l. per min) = 1.36 x lo4 In dealing with cell extracts or suspensions, a blank consisting of extract or suspension, DTNB, and buffer may be required to correct for release of thiol material from the cells and the absorbance of the other materials in the suspension. Method for blood A fairly stable suspension was formed from whole blood or washed human erythrocytes. Since the acetylcholinesterase is on the cell membrane, hemolysis was not necessary. The assay of blood was carried out as follows: (1) A suspension of the blood cells4 in phosphate buffer (pH 8.0, 0.1 M) was prepared. The most practical dilution was 1 : 600 (e.g. 10~1 blood into 6 ml buffer). (2) Exactly 3.0 ml of the suspension were pipetted into a cuvette. * California Corporation for Biochemical Research, Los Angeles, California. t This is now available from the Aldrich Chemical Co.. 2369 No. 29th. Milwaukee 10, Wisconsin. $ Strip charts printed in absorbance units are available from Minneapolis-Honeywell Corporation, Chart 5871. 4 Red cell counts were performed by the clinical laboratory. FI G . 1. P ho to gr ap h of s tr ip ch ar t re co rd of t w o id en tic al as sa ys . A t th e ar ro w , ph ys os tig m in e sa lic yl at e (f in al c on ce nt ra tio n, 3 x IO -’ M ) w as a dd ed to a t hi rd re pl ic at e.