edited bv GEORGEL. GILBERT Denison University Granville. Ohio 43023
Superoxide Dismutase and the Briggs-Rauscher Reaction
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Cytochrome oxidase
SUBMIT~EDBY
Davld A. Franz Lycming Cothg. Wliiiarnsport. PA 17701
u Superoxide dismulase
Falcon Ferguson and Erwln B w h m a n n IP-PU at lndlanapollr Indlanapolts,IN 46202
The univalent pathway fw the reductton of molecular oxygen and enzymatic defense mechanisms.
The toxicity of oxygen and its univalent reduction species of suoeroxide (.0 9-- ) ... . oeroxide 1.0 q-2 - ).. . and the hvdroxvl radical (OW) have received widespread attention (l,2). i n particular. medical researchers have focused on the mechanism of tiss"e damage mediated by these species, especially during hlood reperfusion following periods of ischemia (3). For chemistry teachers, these oxygen-derived species provide excellent examoles for discussions of molecular-orbital theory, bond order and reactivity, redox potentials, radical reactivity, disproportionation, and enzyme activity. In particular, superoxide dismutase (S.0.D) is known to catalyze the disproportionation of peroxide into oxygen and water (4). In fact, it has heen suggested that cellular enzymatic systems such as S.O.D. and the cytochrome oxidase system were developed under an evolutionary pressure to "detoxify" oxygen (5). These enzymes and their roles are illustrated in the fiewe. The a k o n of S.O.D. in removing superoxide can be demonstrated m i t e easilv with the Rriees-Rauscher (BR) reach mechani& of the BR ieaction is tion (6, 7 ) . ' ~ l t h o u ~the comolex and not fullv unraveled. one orooosd oostulates a nukber of steps inv~ivin~superoxideai an intermediate ( 8 ) . The addition of S.O.1). toan actively oscillatine BR reaction causes immediate cessation of osciilation, accompanied by an effervescence that can be demonstrated to be the release of oxygen. Procedures I . Into a beaker magnetically stirred at moderate speed, pour equal amounts oi the three sulutions necessag' for the HR reaction: A: H1O,(204g of 3UD< H201per 500 mL) H: 0.2 M KlOl and 0.08M H*S04 C: 0.15 M malonic acid, 0.02 M MnSOa, 0.03 %(w/x) starch
' The increased effervescence may be a surfacearea effect. The
addition of 0.5 g of sand to a BR reaction also causes increased effervescence, but the colw oscillations continue. It has been reoorted ~. ~.~ 191 that heatina an S.O.D. tablet at 37 OC in 0.01 N HCI and pepsm wiil denaturetheenzyme. Interestingly. S.O.O. tablet residues from such a treatment were stii etfecl've in quenching a BR reaction, although not quite as readily as fresh taolet material. Whether enzymatic activity remained because of incomplete denaturation is uncertain. Given the complexity of the BR reaction and the heterogeneity of S.O.D. tablets derived from natural sources, it is quite possible that other tablet ingredients, in addition to S.O.D., will auench the oscillations of the reaction. The addition lSiama - ... of - ouiifled -. .-S.O.O. - .- 52515. S.O.D. from bovine liver. 3000 unm. 0.9mg) to a BR react on wiil. alter sufficienttime for m xing, bring about a q-ench~ngof the BR oscillations. Accompanying this will be a frothing that tests positive for oxygen. As a lecture demonstration, however. this is less effective and more expensive than using food store S.O.O. tablets. ~~~
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2. After several oscillations have proceeded, add S.O.D. by crumbling a pellet (0.5g) of the enzyme (available locally at many
health food stores) into the beaker. Note cessation of the oscillating reaction and the increased effervescence (the latter can be picked up by a microphone held over the beaker). 3. Bubbles of froth collect in the shallow vortex caused by magnetic stirring.If aglowing splint is used to pop these bubbles, the splint will burst into flame,showing the release of oxygen. 4. For a comparison demonstration,denature another tablet of the enzyme by crushing and charringa tablet in a porcelain dish held over a Bunsen flame. The addition of enzyme so denatured will fail to quench the oscillations of a BR reaction, although an increased effervescenceis 5. Since S.O.D. is an endogenous edmponent of blood, the addition of s few drops of blood will also quench the BR reaction in dramatic fashion. 1. Biologirol and Clinical Aaporu o/SuprrorideondSupemride Dismuloae; Bannister.
W. H. and Bannister, J. V., Ed.: Proceedings of *ha Federation d European Biochemical Societies Symposium No. $2; Ei~eviermorth.Holland: New York, 19so. 2. ChemicalondBiochrmicalA~~~~fso/SuperoridsondSupsrozidaDismufore;Bannister, J. V. and Hill, H. A. 0.. Eds.: Proccedinga of fhe Federation of European Biochemical Societies Sympo~iumNo. $2; Eiscvier/Narth-Holland: New York, 1980. 3. Jolly, S. R.: Ksne. W.J.:Bailie, M. 8.: Abram, G.D.; Luechcai, B. R. Ciic. Ras. 1984.84,
217-285. 4. Superoxide and Superoride Dlamulorsa; Michetson, A. M.: McCard, J. M.: Fridouich, I., Ed..: Academic: Nsw York, 1977. 5. Bulkley, G.B. Surgery 1983.94.407411. 6. Brigg~,T. S.;Rauscher. W. C. rl Cham. Edur. 1913.50.496. I. Shakhsahiri, B. 2. Chornieol Demommrions; University of Wireon%in:Madison, W1, 1985:Val. 2, pp 21a266. 8. Noyes. R. M.; Furrow. S. D. J , Am. Chom. Sor. 1982.104.45-48, 9. Kasrs,J.: Kosman,D.J. Chem.Edue. 1983,60,1082.
St. Patrick's Day Special-A Boron Flame Shamrock S u e M l n w BY
Thomas 1.Earles Culpepper Country Hlgh School Culpepper, VA 22701 This demonstration takes only a few minutes but pays great dividends in student interest and makes St. Patrick's Day memorable in chemistry class. Prepare a solution of 10 g of boric acid dissolved in 100 mL of methanol. This will provide a saturated solution. For the presentation I draw a large shamrock with the solution on top of a lab table. I have a large lab table that allows me to draw a shamrock about 2% ft (75 cm) in diameter. Then darken the room. and sav "Haoov St. Patrick's Day3'as you light the stem df the shknrockb;ith a match. A beautiful green flame races around the shamrock and burns Volume 68
Number 1 January 1991
57
for a few seconds. Your students will love it! Be sure t o make plenty of solution because I will guarantee you will have cheers of "can we do i t again?" from your students. If you are no better artist than I, you may want to get one of your talented students to draw the outline of a shamrock in chalk on the table top. Then you can just trace i t with the solution dis~ensedfrom the ~ i ~ e t . For sake bf safety, be sure to stopper your solution, and move it out of the way before you ignite the shamrock. Also. be sure the flames &e entireiy gone before you draw additional shamrocks with the solution. If you are concerned about the surface of your idb tables, you might try burning a small spot of the solution to see whether i t leaves a blemish. Our table tops are made of an
58
Journal of Chemlcal Education
"imitation slate" comnosite with an eooxv coatine. and this demonstration does not hurt them & t i e l e a s t - ~ f t e rall, most of the heat from the burnine alcohol eoes un into the air anyway. If the boric acid residue is a littie difli'cult to clean offwhen vouare finished, trv usirlea little ammonia water to make it more soluble. his is a goGd place t o review neutralization, and even the clean-up can be a learning experience. I normally do this for groups of about 20 students, but there is no reason i t would not work well in a large lecture hall with elevated desks looking down on a demonstration table. This demonstrates a classical qualitative test for boron, but, more importantly, it puts some of the "Oh, neat!" back into chemistry.
