Protocolo De Colinesterasa

Biochemical Pharmacology,1961, Vol. 7, pp 88-95. PergamonPressLtd., Printedin Great Britain.

A NEW AND RAPID COLORIMETRIC OF ACETYLCHOLINESTERASE
and
Department
ROBERT M. FEATHERSTONE

DETERMINATION ACTIVITY
JR.

GEORGE L. ELLMAN, K. DIANE COURTNEY, VALENTINOANDRES,

of Pharmacology, University of California Medical Center, San Francisco, California
(Received

14 November 1960)Abstract-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

-----+thiocholine -+

+ acetate yellow color

+ dithiobisnitrobenzoate

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 constantsdetermined 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. Thismodification 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 thefact 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 erroris 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 isapplicable 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 ofthe 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 ofphosphatases, 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+ RS(II)

+ 2H+

(CH&H$H$-

+ RSSR (I)

+...