200401M2 - SCIENCE sncrm ” l/fl ESCSAQMFHMERLM zouummm...

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Unformatted text preview: SCIENCE sncrm ”' l/fl/ ESCSAQMFHMERLM zouummm AVE N HATERLIJI] on rm as: Chem 237 Midterm #2!) March 10, 2004 Name: ————— I.D.# _ Answer the questions on the paper provided. If you need more paper or clarification of a question, be sure to ask. 1. You have isolated and analyzed a peptide, with the following results. i) the composition of the peptidei ("Z Ala 1 lie 2 Tyr 1 Aig 1 Met 1 ii) the N-terminal amino acid is Arg iii) trypsin cleaves the peptide to generate a single amino acid and a pentapeptide iv) chymotrypsin cleaves the peptide to generate two tripeptides v) CNBr does not cleave the peptide at all. P' "1"" vi) carboxypeptidase analysis of the intact peptide was not clear, but you could tell that the two amino acids detected were Ala and Met.l 3 a. What procedure would you use to generate the data in part (i)? What are the conditions needed for this procedure? b. What conditions are needed to determine iii, iv and v? What does (vi) tell you? (1. Suggest a primary structure for the peptide. Use all the information above. 11. How could you use Edman Degradation to simplify this entire exercise? 1+. i ' "“d In"; " .r - Elli/r: " E "3-“ MW. L L.' "~ i 1 “ (E _.r-9l~rl VAA Ip..,‘lf‘x W1. p;/\$r .“ ‘--‘f , ._- -l_IAI':.;'-. I'.... l “13-. :.-. ..'..n’_‘ \__,/ 1-.» we. M -r"‘“'- 3 It; that: or 1:71 ).-. m q-«.a-...--.,_»a @h‘"; 'i'fl'lr; Riv. --"r\r-¢;t'i "ll; "- L/?.r. H11 1: N" 6 inf.” L'1?.'n'-r'l ;_,'- .\ Mufti/L I2“; dWIéa 2. Suppose, in true fairy-tale fashion, you were locked in a laboratory with purified gym and any equipment and reagents you needed1 and were told to lb determine a Km and Vmax values for the enzyme by daWn. a. In gene-ad, how would you go about doing this? What data would you plot (two plots)? From these plots, how would you determine the Km and Vmax values for the enzyme? 1). You manage to get the Km and Vmu values by dawn. But then you are told that another student, using the same reagents and enzyme preparation as you, had gotten the same Km value as you, but their Vmax value is significantiy larger. _E W‘L How did this discrepancy occur? Without re-doing the eXperiment, how might 0‘ ”W" you show that both sets of data are correct? DO c" {WI-‘9 I: out T'f 054“.” "19.5-er C» Jr‘ . r?) rec arch “We \(C‘Jtif‘s Ln} (-5! ', . rep Cir-cm ”5-. “4 E fiDCluCI-i‘ '1 1‘\‘N-€- ,5’3‘” PQC‘\H E31 _' l -.'1 ' :0 "5&1 u _ m LI "cf“; a. ' :J‘: “I: 5‘ lr-"itft " ”:12! "I! t ( EFF”; 2" ._'_b. b) T)":-';V‘E’f3€mr'-I 3. Isolated B'subunits of HbA (normal hemoglobin) can be reassembled into 34 tetramers using a specific set of conditions (you do not need to know these conditions to do the problem). These 84 tetramers have 21 P50 value of 6 torr and show 110 cooperativity. The isolated u'subunits of HbA have 21 pl of 7.4, and the [0 n7, B‘subunits have a 131 of 6.9. a. Assuming that you have purified HbA and have a method to selectively break the quaternary interactions between the a and [3 subunits, how might you separate the B'subunits from the u'subunits, using one of the separation techniques we have talked about in 013353 Be sure to include any pertinent details about the method you use (resin type, pH, salt concentration, etc). b. Draw an 02 binding curve for the 64 tetramers; be sure to label the axes properly, and include an Mb and Hb curve for reference. Why did you draw the curl/re the way you did? 5.— Suppose that there is a person in the world with no n‘genes, and whose blood had only 84 tetramers. What physiological consequences would this condition have for that individual? q\ Jro japan le. 9-; at} F .. fl. w -'l"-§_ _ ”PL (when enlarge rill-fir)!“- _ c - . 1' - Q l~~_ .1. r!” ‘ -.--. J: ,«43 "J 5, l. 1-; r-‘l ? r- l_ m 6.13:. ‘Q KO F l f 74» 1" ll” ‘ r -0 ll' l’ " \ u, l ’ r ~ 5dr a a “a ,‘1 1 k, 1 [ 1‘ I n l r l . I C C _| I J I 1‘1 . ll'. ‘ I Q ) l I C _) n 1 - “\ .‘; hf - '2" n? .‘:-_.’\‘ {MA/\- I “1,11: LT kiln-$1“) 3'3"? ‘2“ (‘41,: Whale. J-UT-I‘v‘i ‘1'- flfg J rs-J‘. r l‘ 11.3 X_ :l. x I - i a -\ . ‘ \ I I. \flfl‘pi'a ’ (r r I ' U; ll” i (A Cr ’ WI!“ :(‘1 (C W Short Answer Questions—answer- #4 and #5 4. Choose one of the following techniques. How does it work and why? all size exclusion chromatography _ 6 b. affinity chromatography " — l c. isoelectric focusing. 5. Choose one of the following phenomenav-IHOW and why does it ork? , a. the Bohr effect ' Z‘F' b. the effect of BPG Answer (2’th of The following: 6. When you determine an amino acid composition, you do not find any Gln or Asn in the final analysis, but you do find Gln and Asn in data from Edman Degradation. Why? 7. In terms of interactions with the enzyme, what is the difference between a competitive inhibitor and a noncompetitive JMJ'bJ'tor? Graphically, how would H'- - distinguish between the two? I 8. Given the SDS-PAGE analysis shown, what characteristics can you determine about the protein in lane A? Why? Given the anion exchange 6011mm profile shown, what characteristics can you determine about the protein in peak B? Why? (U. n Ogl— pR = 71' / 3 III:- 3 I: Poole Cone, E'fiflEEN‘S‘E 13.» 12-!) “I". {In EI: m WEI-‘1 :Ein ._...rremn: 211’: 13' 'F-FEEH' ‘3 fig -. . - "- . 1.1!}..11111': -.'1:1. .1 E '_ "E. '. ..I. ”EFT; 20.1"..- E: 11 .d IE“ _. Egrciw'olla‘l' 3411' ‘6. 3219 1311121111 Inc: hm} 306W flwwifiéwfi"‘ms ”mg. 3%“ 41$ 13.1.3 1.- :01: 3.15:1: in naA has 1.1;..- £9321 _. . um; 3:36 Milan ' ' . 913E.” Minimum '1 E 11 . H m bfiyfiofi‘mufi HMMMfiflQfi'Mnanwm1111mm;£21 "E [email protected] % wfifimwm fl_ll.££igifi1fl‘1'13’fim§h"-'Sl§:flfi§fisrfbmu _. h11.1'1.-I.111I5'I.'!.11.'I. Had-“5.11.- I} -M 211% ”13.1% "fihi': uty. $13ch am .1: gym wand: mzlw awaits a. "1'3“: HUMP-BEE IE ”la. 6 . ._H—q—b. ...
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