The Degradation of 14C-Glutamic Acid by L-Glutamic Acid ~ecarboxylase An Undergraduate Biochemistry Experiment Utilizing a Simple Semi-Micro Carbon Dioxide Trap Charles M. Dougherty and Jean Dayan Herbert H. Lehman College (CUNY), Bronx, NY 10468 Biochemical processes often involve a complex series of reactions, catalyzed by enzymes, whose complete elucidation require more than the usual spectrophotometric and physical methods that suffice for simpler reactions. Valuable information about a particular metabolic pathway can often be obtained by the use of radioisotopes (carbon-14, tritium, or phosphorus-32) labeled in specific positions in a precursor molecule. Both the fate and location of such labeled atoms must be determined accurately. This experiment demonstrates, in a relatively simple system, how a particular enzyme may be used to determine the site (or sites) of labeling in its suhstrate. L-Glutamic acid decarboxylase, which has been isolated from a wide variety of sources, rapidly and quantitatively decarhoxylates the alpha carboxyl group of L-glutamic acid to yield gamma-amino hutyric acid (GABA) and carbon dioxide1. The reaction requires pyridoxal phosphate as a coH H0,C-CHA-CH,-C-NH,
I I C0,H HOAC-CH,-CH
-CH,-NH,
+ C0,t
c01
trapping --r, solution
acid and the semi-micro reaction system which is described here. The complete apparatus (See Fig. 1) consists of two 6 X 50 mm Pyrex@culture tubes, (Corning" 9820) connected by ' 8 in. id., %s in. wall approximately 8 cm of Tygona tubing (1 thickness). The tuhe in which the enzyme reaction occurs has been modified by heating a small spot 1.5-2.0 cm below the top and gently blowing a small bubble. The resulting reaction vessel thus resembles the classic Warhurg flask, which has long been used in manometrically following enzyme reactions. The two components of the reaction, enzyme and substrate, are isolated from one another until the apparatus is completely assembled and gas tighL2 The second tuhe contains a COz trapping solution; the trapping of evolved COz is effectively quantitative if the system remains closed for several days. In four years of use, we have found this system to be extremely reliable, convenient to use, inexpensive, and completely dispo~ahle.~
Experimental Approximatelytwo weeks prior to the actual date of the experiment, the instructor should prepare two tubes containing a sample of one of the specifically labeled unknowns for each student (or student pair). 74
Journal of Chemical Education
in buffer
Semi-microreaction apparatus Five microliters of one of the 14C-glutamateunknowns (0.63 Cilpm, 25 pCi/ml) is added with a Hamilton syringe into the bubble of the REACTION TUBE and into the hattorn of the TOTAL ACTIVITY TUBE (a straiaht tube), which serves as a total activity control. The tubes are tLen&red in a dessicator over -phosphorus pentoxide unt~l they are ready to be used. Students are to determine which one of the followina unknowns they have been given from their experimental results upon treatment of their unknown with L-glutamic acid deearhoxylase. Unknowns: (1) L-glutamic acid-l-14C (2) L-glutamic acid-U-14C (3) D,L-gl~tamic-l-'~C (4) 2:l mixture of L-glutamie acid~l-14C:D,L-glutamic acid-lId0 ~ u
GABA
factor and can he followed manometrically (the pressure chance is due to the carbon dioxide which is evolved) or with radioactive isotopes. In order to give students valuable first hand experience in handling radioactive compounds (with a minimum of hazard) and to reinforce concepts which have been presented in our
-Enzyme
(5) 2:l mixture of D,L-glutamie acid~l-14C:L-glutarnic acid-
UV4C Procedure 1. In aelean 6 X 50 mm test tube place 0.10 ml of COztrapping solution and attach the Tygon* tubing. (See Fig. 1.)Carefully introduce 0.10 ml of the enzyme solution (in 0.1 M acetate buffer, pH = 5) into the bottom of the test tube with the bubble on one side. DO NOT allow the enzyme to come in contact with the substrate at this time. While holding the test tube containing the COz trapping solution vertically (!!!I, attach the other end ofthe Tyganatubing to the reaction tube containing enzyme and substrate. Start the reaction by gently vortexing the reaction tube so that the enzyme solution washes the unknown out of the bubble. Keep the reactlon assembly
'
(a)Strasubauch, P. H., and Fischer, E. H., Biochem., 9, 226 (1970); (b) Laswon, A,, and Quinn, A. S., Biochem. J., 105, 483 (1967);(c) Shukuya, R., and Schwert, G. W., J. Biol. Chem., 235, 1649, (1960); (d) Gale, E. F., in "Methods of Biochemical Analysis," Vol. IV (Editor: Glick, D.,),Interscience, New York (1954).p. 285. Note: It is important to keep the amount of carbon dioxide evolved well within the capacity of the trapping solution (about 0.2 g COdml). If this is not done, there is a danger of causing so muchpressure In the system that the Tygofl tubing will pop off releasing radioactive C02. The ouantities of reaaents listed in the exDerimental Drocedure avoid this orobiem. ' The sen- -m cro apparalus can oe aoapleo easily lo an) reaction n rrh.crl CO. 's erolrca an0 lor *h,cn tne appropr ate caroon-14 hoe w compo.nds are ava laore For example. uc narc been aolc to demonstrate that carbon-3and 4 of glucose are liberated as C02 by using respiratory deficient (rho minus or heme minus) yeast mutants which utilize glucose fermentatively as a carbon source.
vertical to prevent mixing the components of the two tubes. Even though the decarhoxylation reaction is complete in a few minutes, the COz trapping is not complete for several hours. Place the reaction assembly (tubes vertical !) in a 150-ml beaker and stare in the designated area until the next laharatory period. Cover the top of the other test tube with Parafilm'and place i t in your storage beaker. 2. After a one week equilibration period, disconnect the Tygone tubing (wear gloves) from the assembly. Add 0.10 ml of 0.1 M acetate buffer (pH = 5) to the TRAPPING TUBE, then add 0.2 ml of Aquasol, mix, and corefully transfer the solution to a scintillation vial. Wash the trapping tube with four 0.2 ml portions of scintillation fluid and transfer the washings to the vial. Add 10 rnl of sein$llation fluid to the vial, cap, label, and store until you are ready to count all three samples. 3. To the REACTION TUBE, add 0.10 mlof the CO2 trapping solution and 0.2 ml of scintillation fluid, mix, and then transfer the solution to a second scintillation vial. Rinse the reaction tuhe with four 0.2-ml portions of scintillation fluid as above. After adding the washings to the vial, add 10 ml of scintillation fluid, cap, and label vial. 4. The TOTAL ACTIVITY TUBE (the second tube containing the unknown sample) will be used to determine the total amount of radioactivity of the unknown. To this tuhe, add 0.10 mlof pH 5 buffer and, aftergentle vortexing to helpdissolve the glutamic acid, add 0.10 ml of COz trapping solutioh. Transfer the contents of the tube to a suitably labeled scintillation vial, and wash the tube with 5 portions of scintillation fluid (0.2 ml each) and add the washings to the scintillation vial. Finally, add 10 ml of scintillation fluid to the vial and cap R.. P . . .~ .-"T P I Y . ~
5. Measure the radioactivity of the three vials and of a background blank and anunquenched carbon-14 standard in the ..
.. .
rections of your instructor.
Calculations After subtracting background counts and converting counts per minute (CPM) to disintegrations per minute (DPM), students should calculate the percentage of radioactivity recovered in C02 and GABA both on the basis of total radioactivity recovered and on the initial radioactivity of sample. They should then decide which method gives a more valid estimate of the percent distribution of the unknown s a m ~ l eStudents . should also be asked to calculate the oercent of radibactivitv in COI and GABA for each of the ~, o s r i.. h l ennknowns trcrncmorrln: thnr rhc rnlyrnt. I, rprvlll, ~.,r1111 I . - Y . ~ t ~.,.~~m d ji. Over fuur year,, 31udmr's r c ~ ~ l t , .v, . ~ l v u l ~ m 1 4 [ I N l ~ . i s ,I I, tnl re. covered radioactivity, agreed with the theoretical values &3%-4%. ~
~~
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Solutions and Reagents 0.1 M Sodium acetate buffer (pH = 5 ) containing 0.1 M NaCI L~Glutamicacid decarboxylase (E. coli) (Worthington Biochemical Corporation) 20 mg/ml in acetate buffer COz trapping solution: phenethylamine (scintillation grade): methanol: water; 2:1:0.25 (by volume) Aquasol Scintillation Fluid (New England Nuclear)
Acknowledgment \Ye would like tu thank I h . I . w n K r t m ? n t r tllr his helpl'.~l iugpesrions in t h e drvt.lopmrnt 01 this rsprritnent.
An alternative method of handling the total radioactivity of the sample is to supply the students with two "reaction tubes" with the unknown in the bubble of each. One tube is used to set up the complete apparahls a s described; the second is used to set up a control apparatus using exactly the same reagents except substituting a boiled enzyme solution for the active enzyme. The reaction tube of this second set up becomes the TOTAL ACTIVITY TUBE. This modification allows students to gain a better feeling for controls and introduces the concept of quenching by protein (negligible in this case).
Volume 59
Number 1
January 1982
75
