Laboratory Practical Exams in the Biochemistry Lab Course John F. Robyt and Bernard J. White Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011
Laboratory practical exams are common in biology courses and have been sueeested for the aeneral chemistry The exercises in the anaiytical chemistry lab courses are usually a series of qualitative and/or quantitative unknowns, essentially making the whole course a series of practical exams.3 In the biochemistry lab course, however, it has not been traditional to give practical exams. We have developed practical-lab exams for use in our biochemistry course, incorporating the same kinds of student experiences and evaluations that are found in biology and chemistry In our senior-level biochemistry laboratory class, the students perform and learn a number of qualitative and quantitative procedures for the determination of biochemical materials. and we eive a number of unknowns, for example, the determination of the amount of protein, the determination of rhe structure of a di- or tripeptide, and the determination of the structure of a carbohydrate di- or tri-saccharide. 'I'he students learn four ~uantitativemethods for the determinntion of proteins: spe~trophotometricabsorption a t 280 nm, ~ determine the biuret, Folin-Lowry, and B r a d f ~ r d .They presence of carbohydrate in a sample by the Molisch test? perform Benedict's test for reducing sugars, and separate and identify mono-, di-, and trisaccharides by thin-layer chromatography.. They separate and identify peptides and amino acids hv T1.C and test for them on theT1.C by using fluorescamine.4 They isolate an enzyme, assay it, and determine various kinetic parameters, such as specific activity, K,,,, and kcat.They also isolate RNA and DNA and perform qualitative and quantitative tests, such as absorption in the UV, thediphenylamine test for DNA, and the orcinol test for RNA.4 They isolate lipids from egg yolks and saponify the fatty acids and qualitatively and quantitatively determine the fatty acid esters by gas chromatography,4 and they perform the Liebermann-Burchard test for the presence of cholesterol. We have developed lab practical exams based on the lahoratory experiences that the students bave had during the semester. We usuallvallow two 3-h laboratory periods at the end of the semester ;or the exams. The stud& are permitted t o use their lah text and laboratorv notes taken during the semester. One of our successful exams is the determination of the components of various unknown mixtures of carbohydrate, protein. amino acids, nucleic acids, and cholesterol. The students are told that tbey could have these materials or mixtures of them in their unknown and are asked t o determine what is in their unknown. If they have carbohydrate,
they determine what specific types of carbohydrates, for example, D-glucose, D-fructose, lactose, maltose, raffinose, ete. If thev find urotein.. thev . are asked to determine the amount ojprotein present, and if tbey bave nucleic acids, thevare askedto determine if i t is RNAor DNA. If thev have amFno acids, they are asked to determine which one! are uresent. Examples of thevarious mixtures that we have used are: RNA and DNA; carhohydrates and protein; protein and DNA or RNA; carhohydrates and amino acids; carbohydrates and DNA; amino acids and RNA or DNA; and mixtures containing three types of compounds, such as carbohydrate, protein, and amino acids or carbohydrate, protein, and DNA. The specific types of carbohydrates and amino acids can also be varied. Both reducing and nonreducing carbohydrates and mono- di-, and trisaccharides can be used. The use of L-tvrosine. L-uhenvlalanine. and L-trwtophan could give a faise prokin'test by absorbing a t 28i)nm, but. if one of the other auantitative nrotein methods, such as ~olin-~owr~ ~radfoidmethods, or is used, the results would he negative, indicating that the unknown did not contain protein. The student would have to deduce what was absorbing a t 280 nm and then confirm of the presence of the 280nm-absorbing amino acids(s) by TLC analysis. The table gives the composition of the unknowns that have been used. The components of the unknowns are dissolved in water or buffer and are given to the students as 10mL solutions. The students are told to conserve the unknown, and, if more is required, points will he deducted from their score. The students perform tests on their unknowns and report the names of the tests performed, the observations made, and the conclusions drawn on an exam reportform (see Figure) that is turned in a t the conclusion of the exam. Another examination that we use involves enzymes. The
Composlllon of Blochemlcal Unknowns for Lab-Practical Exam In Blochemlstrya No.
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Silberman, R.; Day, S.; Jeffers. P.: Klanderman. K.; Phillips, M. G.; Zipp, A. J. Chem. Educ. 1975, 64.622. Jones, M. M. J. Chem. Educ. 1 9 7 7 , 5 4 , 178. MacNevin, W. M. J. Chem. Educ. 1 9 6 1 , 3 8 , 144. RobvL , ..I.F.: ~ White. 6. J. Biochemical Technioues: Theow and Practice: ~rook&ole, ~onterey,CA, 1987. White, 6. J.; Robyt, J. F. J. Chem. Educ. 1988, 65, 164 ~
600
Journal of Chemical Education
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Composition
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Composition
0.75 mg/mL pmtein. His. Oai 75 pg/mL RNA 8 1 mg/mL protein 0.5 mg/mL Oal. His. DNA 35 pg/mLRNA 8 0.25 mg/mL protein 1 mg/mL Fru. Suc, His. Leu 0.75 mg/mL protein 0.6 mg/mL DNA 8 protein 0.5 mg/mL protein, Gal, Mai 1 mg/mL Gal, Raf, His, Giy 1 mg/mL m i . Mai. Raf
11
50 pg/mL RNA 8 1 mg/mL Leu. Pro. Fru 2 mg/mL protein 0.25 mg/mL protein 8 0.5 mg/mL Fru, Raf 60 pg/mL RNA 8 0.1 mg/mL Leu. Phe 0.4 mg/mi protein 0.75 mg/mL protein, Gic. Suc 35 pg/mL RNA, DNA 50 pg/mL RNA, His Gly 35 pg/mL RNA 8 0.5 mg/mL orottein. Leu 0.5 mg/mL Fru. Suc. Lac
12 13 14 15 16 17 18 19 20
e m i n used was serum albumin. DNA used war oali mymus (Sigma Type I). RNA used war yean (sigma Type VI). Abbreviations used for the carbohydrates and amino BCI~S are those in common usage.
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n"cl.,s .Cld. M a D'OtL.". af.",r which Ol these type. ' o .O.poYnd. .r. p'L.e't *n your -om M.3 ( 1 , I t Yo" h.vs v s n o .Cid.. *t*.lnL *DL n u w r pr=.cnt .nd t h d r a s < probsble iaen?lty: 1 2 1 i t you h."a c.rbahy*r.te, drtL",na t M n--r pr=.cm m a rb.lr .om* P7sb.blr ld..rlts; ( 3 ) If Y o u h " C 11pm.. *c*cr.,nr tnc tw. I tr3glYce~%a*, W S P ~ D u p i d , or cn.*c.*~r~~: I * ) ~t mu m.v=nnClLtc .CMS, dctcr.ln. 1r If 1. -1. DNA. or D t h ma rnsn dct.r.lr,* I t . ~~"c.ntr.tLon; ,,I I* you h.m .-tl". d.v.r.>r.= conc.ntr.f'an Ilpm..
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and molecular weieht. Thev then have to prepare the assav reagents and perfoim the &antitative anaiyses necessary tb obtain the kinetic parametere of the enzvme. We select enzymes that are reidily available and hive relatively easy assav procedures, such as a-amylase, glucose oxidase, invertase; phenol oxidke, and dehydrogenases that use the coenzyme NAD+ or NADPf. The report for this exam is in the form of a journal manuscript: there is an introduction describing the reaction that the enzyme catalyzes and the properties of the enzyme; there are the exprimentalprocedures used in determining the enzyme activity and kinetic Darameters:. there are the-results obtained f& the enzvme 'activity (expressed in international unitslml), the specific activitv of -rotei in). the data for determinine the . (unitslme . K,, kc.,, and the computatio
