c. doubling the volume of the assay by adding buffer (assume you are using a spectrophotometric assay)

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1 FST 123 1st Midterm Examination May 1, 2012 Name Key 1. We have discussed enzyme kinetics under the limiting conditions of very high and very low substrate concentrations. Under each of these conditions ([S] o << K m and [S] o >> K m ), indicate the effect on the measured velocity of an enzyme-catalyzed reaction (i.e., in mm/min) that would be caused by the following modifications of the assay (12 pts.): a. doubling [E] b. doubling [S] o c. doubling the volume of the assay by adding buffer (assume you are using a spectrophotometric assay) 2. Bovine serum albumin (BSA) is a protein found in mammalian blood and milk. It is rmally thought of as a momer with a molecular weight of 66,500 Da and an isoelectric point of 4.7. However, it forms moderately stable dimers if the ionic strength is t too high, and these dimmers can sometimes be separated from the momer. Which of the techniques listed below might be expected to separate the BSA momer from the BSA dimer? (You may assume that the dimer is held together tightly) (8 pts.) Technique gel filtration chromatography ion exchange chromatog. on DEAE cellulose ion exchange chromatog. on CM cellulose Native Polyacrylamide gel electrophoresis SDS Polyacrylamide gel electrophoresis Isoelectric focusing Will BSA adsorb to: DEAE cellulose, at ph 8.2 CM cellulose, at ph 6.2 Yes,, maybe

2 3. a. Complete the following purification table for a single-step purification. (12 pts.) Fraction Volume Activity Protein Total Activity Specific Activity Recovery Purification (ml) (u/ml) (mg/ml) (U) (U/mg) (%) (fold) Crude Extract Final Fraction % 66.7 b. Describe at least two distinctly different experimental methods you could use to determine whether or t this protein was purified to homogeneity by this procedure (i.e., you have a solution of enzyme how do you find out if the enzyme is pure?). (8 pts) Possible answers (4 pts each for any two correct answers): SDS gel, look for bands corresponding to impurities Native PAGE, look for bands corresponding to impurities, but you should do it at two ph values or two %T IEF Gel, look for bands corresponding to impurities Specific activity (give 2 or 3 points, because it s hard to tell what the specific activity SHOULD be. Symmetrical behavior on a chromatography column (I don t like this one, but it s in the book and I put it in the lecture) 4. Oxalic acid is a dicarboxylic acid with pk a1 = 1.27 and pk a2 = A buffer is made by dissolving g of disodium oxalate (Na 2 C 2 O 4, MW 134 g/mole) and grams of sodium hydrogen oxalate (NaHC 2 O 4, MW 112 g/mole) in 100 ml of water. a. What is the ph of the buffer? We re using Na 2 C 2 O 4 and NaHC 2 O 4, so it s the second pk that s relevant g/134g/mole = 6.29 x 10-3 moles of Na 2 C 2 O 4 ; 6.29 x 10-3 moles/0.1 L = 6.29 x 10-2 M 0.415g/112g/mole = 3.71 x 10-3 moles of NaHC 2 O 4 ; 6.29 x 10-3 moles/0.1 L = 3.71 x 10-2 M ph = pk a + log [C O 2" 2 4 ] 6.29 = log = = 4.50 [HC 2 O " 4 ] 3.71 b. What is the concentration of the buffer? 6.29 x 10-2 M x 10-2 M = 0.1 M c. What is the ionic strength of the buffer? The species & concentrations are Buffer Conc Counterion Counterion conc component 2- C 2 O 4 - HC 2 O x 10-2 M 2Na x 10-1 M 3.71 x 10-2 M Na x 10-2 M sum 1.63 x 10-1 M I = 1 {( 6.29 "10#2)(#2) 2 + ( 3.71"10 #2 )(#1) 2 + ( 1.63 "10 #1 )( 1) 2 } = 0.226M 2 5. Subtilisin, a protease from Bacillis subtilus, is a serine protease that catalyzes the reaction Protein + H 2 O Peptide 1 + Peptide 2 2

3 The mechanism involves a covalent intermediate in which Peptide 1 (the peptide donating the carboxyl group to the peptide bond that is hydrolyzed) is covalently bound to the enzyme. Only one Cleland diagram describes the kinetic mechanism of this reaction. Sketch it, using the line below: Protein P 2 H 2 O P 1 OH E E Protein=E-P 1 P 2 E-P 1 E-P 1 H 2 O =E P 1 OH E 6. Briefly describe the principle behind measurement of protein concentration using each of the following methods (i.e, how does each method work). (12 pts.) a. The Biuret method Dipeptide unit of protein makes a colored complex with Cu(II) in alkaline solution. b. The BCA (bicinechoninic acid) method Cu ++ reacts with several groups ion the protein in alkaline solution to make Cu +. BCA is added, and the BCA-Cu + complex is colored c. The Kjeldahl method The protein is digested by heating in acid (+ catalyst), and the ammonia produced is determined by distillation & titration d. The Bradford method Dye binds to the positively charged groups on a protein in acidic solution, and changes color when bound. 7. Briefly define the following terms as they relate to enzymology (12 pts.) tertiary structure the overall 3D fold of a protein isoelectric point ph at which a protein has net charge. (must say ph for full credit) specific activity the number of activity units divided by the amount of protein in mg. Ramachandran diagram Plot showing the allowed and t-allowed combinations of the torsion angles φ and ψ determined by steric interations. (must say steric interations for full credit) 3

4 Turver number molecules (or moles) of substrate concerted to product per mole (or molecule) of enzyme per unit time = V max E T reaction order For a rate law d[a] dt n i. A description of molecularity would be okay, also = k[a] na [B] nb [C] nc... the reaction order is the sum of the 8A student performed an experiment to determine the kinetic parameters of an enzyme she had just purified. She measured initial velocities, at different values of [S] 0, and keeping all other parameters constant. She obtained the following data and double reciprocal plot: Experimental Data [S] 0 V 0 (mm) (µm/min) Double Reciprocal Plot Calculated parameters slope 1/V-intercept 1/[S]-intercept x x a. What is the K M of this enzyme for this substrate under these conditions? Be sure to give its units. (5 pts.) K M = -1/x-intercept = -1/(-12.5) = 0.08 mm 4

5 b. What is the V max of this enzyme for this substrate under these conditions? Be sure to give its units. (5 pts.) V max = 1/y-intercept = 1/3.125 x 10-3 = 320 µm/min c. If the assay (1.0 ml) contained 0.1 mg of pure enzyme with a molecular weight of 50,000 Da, what is the turver number of the enzyme under these conditions? Be sure to give its units. (5 pts.) if V max = 320 µm/min, divide it by E T in µm. 0.1 mg/50000 mg/mmole = 2 x 10-6 mmoles = 2 x 10-3 µmoles 2 x 10-3 µmoles/1 ml = 2 x 10-3 mm =2 µm (320 µm/min)/ 2µM = 160 min -1 5