Rainer Fried
and Margaret Howse Creighton University Medical School Omaha, Nebraska 68131
A Demonstration of Enzyme Activity for the "kepti~alChymist"
Although many good student laboratory experiments dealing with enzymes and their properties have been published in manuals of biochemistry and biology, there seems to be a need for a simple demonstration, which shows the nature and fundamental properties of enzymes. The present paper describes such an experiment, which is both suitable for classroom demonstration as well as for individual student assignment. The principle of the experiment can be shown with a great number of enzymes; the present method is chosen, because enzyme activity is manifested by color development, the color intensity being proportional to the enzyme concentration. The color can be observed visually, or be measured colorimetrioally. Acetylcholinesterase, (EC. 3.1.1.7) is used as enzyme, with acetyl-thiocholine (I) as substrate, in presence of bisdithionitrobenzoic acid (DTNB, 11) as chromogen.' This convenient method is widely used in research and analytical biochemistry, and is also used frequently as an assay system of insecticides and other compounds, which inhibit cholinesterase. It has been shown that the artificial substrate, acetyl-thiocholine, is equivalent
to the true substrate, acetyl-choline. Brain homogenate or purified enzyme, which is commercially available, can be used equally well. The following reactions take place
"-TNO2 )QT + OA'
coo-
(2)
cooYellow
Experimental Reagents
'ELLMAN,G. L., COURTNEY, K. D., ANDERS,V., Jr., FEATHERSTONE, R. M., Biochem. Phamacol., 7,88 (1961).
AND
acetyl-thiocholine, 7.5 mM, 43 mg/2.0 ml of water (Aldrioh Chemicals, Milwaukee)
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DTNB, (5,5'-dithio-his-2,2'-nitrobenzoioacid;Aldrich) 39 mg/lO ml of 0.1 M Na-phosphate, pH 7.0, containing 15 mg NaHCO. 0.1 M ~ G ~ h o s p h a t pH e , 8.0, contsining 0.001 M NaCl and 0.001 A4 Na-EDTA ("phosphate") physostigmine (eserine) 0.1 m M Procedure
A. Enzyme solution can he prepared by mincing 1 g of fresh or frozen rat brain with 50 ml of 0.1 M "phosphate" in a Waring Blendor for 1 min ("homogenate"). Alternately, acetyl-cholinesterase oan be obtained, for example, from Worthington Biachemicals, Freehold, N. J., or from Sigma Chemical Corporation, St. Louis, Missouri. For use, this enzyme preparation is diluted 1:100 with "phosphate." containing- also 0.1% . . . albumin as st* biliaer. B. 2.0 ml of hamagenate or enzyme solution are placed. into four dialyzing bags. One of these ie placed in a beaker with boiling water for 15 rnin and cooled to room temperature ("boiled ~ - - - - ~ensvmd'i. -~ ~ -, - - ~ - -,C. Prepare five 50 ml-beakers: 2mlDTNB 1mlaeetyla , h , c : 17mlphosphate thiocholine ohwostiemine (d) as above. +0.6 ml . (e) phosphate Flask (a) is used as blank; a didyzing hag containing enzyme is placed into flasks (b), (d), and ( e ) , and the "boiled eneyme" in (c). Physostigmine a competitive inhibitor, is used for comparison in (d). D. Incubate at room temperature (or in 37°C waterbath) with frequent shaking. After 20 min, remove an aliquot, and read color at 412 nm (410420 nm). E. After color has developed, remove the whole dialyzing bags containing the enzyme from the incubation mixture, place into fresh phosphate, shake frequently, and change "outs~de" solution frequently, until the yellow color has been removed. This destaining step may take considerable time; it is shortened by agitation and frequent changes of dialyzing solution. I t is best to carry out thisstep in the odd to improvestability of the enzyme preparation. Dialysis can be stopped when no more color is transferred to the outside compartment. F . When most of the yellow color has disappeared from the brain homogenate, place all dialyzing hags once more into fresh assay mixtures, and incubate snd read color as above. In the repeat assay, the enzyme system is not placed in phosphate buffer bnt in the complete assay system as in flask (b).
Demonstration of Acetyl-Cholinesterose (Brain Homogenate)
Condition
Activity (A4,1/20 min a t 37°C). First assay Second assay
n nn n" nn ibj control 0.8@ 0.64 (c) boiled enzyme 0.00 0.00 physostigmine 0.17 0.12 (d) (el . .. 0.62 Blank (a) subtracted. For details see text. The enzyme was incubated in phosphate buffer during the first assay, and then in the complete system as in (b) in the repeat sqssv -..When the enzyme was mixed directly in the incubation mixture (without dialysis bag), A.,s/20 min was 1.84. . ' "&+.,.". , . , , ", .. .' ., ,, . ",. " ' ." ,',:. ", .,. 1%) blank
+
d
.
.;
..
~
+
+
Results a n d Discussion
It will be observed that color forms readily in the unheated enzyme (Flask b), while no activity is found in the heated enzyme (0). The colored reaction product and other low-molecular weight components of the mixture are removed by dialysis, while the protein remains inside the dialyzing bag. When the dialyzing bags are placed again into fresh incubation mixture, enzyme activity, as shown by color development, once more occurs. Thus the experiment demonstrates that (1) (2) (3) (4)
enzymes exist enayme.9 are non-dialyzable enzymes are heat-labile enzymes can heinhibited by selective reagents ( 5 ) enzyme activity is not last when the eniyme is acting an the subtrate ( 6 ) however, enzymic activity can decrease due to aging and o m be lowered by handling
The latter points are demonstrated by the fact that decrease of activity (in bag b) occurs at the same rate in the enzyme incubated with substrate in the first assay as in the control maintained in phosphate buffer (bag e) under the same conditions as the experimental one.
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Journol o f Chemical Education
Flow-through cuvet: (A1 magnetic stirrer; (81 r o d i o n flask; (C) cuvet [Reprinted by permission of Kontes Gloss Co.)
(Dl photometer.
Representative values of a typical experiment are given in the table. For lecture demonstration, the experiment can be modified by using 5 ml of 1 :50 brain homogenate inside the dialysis bag with an incubation mixture containing 3 rnl of DTNB and 2 ml of acetylthiocholine, completed with phosphate buffer to a total volume of 50 ml. Under these conditions color development can be conveniently assayed by means of a flow-through spectrophotometric tube2 (available from Kontes Glass Co., cat. # I
