EXAM 1 Thursday, Feb 12 MCB 450 On material covered in Lectures 1 through 7 REVIEW FOR EXAM 1 For room assignments, please see course web site R1-11. Questions like homework questions in difficulty and format (all M-C) Information & Tips 4. Some Qs may require calculations in order to identify correct answer option. You will need a scientific calculator for the exam No graphing or programmable calculators allowed 6. If you get stuck on a Q or are not sure of the answer, move on and do the ones you can - then go back to the ones that caused problems. R1-2 3. A copy of last semester's exam has been posted 7. Look at the "Learning Objectives" slides at the ends of each lecture 2. The exam will have ~ 35-40 questions 5. You will be give a table of pKas: Use those in your calculations, unless the question gives you another pKa value1. Amino acids: structures, properties, titration 2. Isoelectric point of a peptide 3. Buffer questions 4. Ion exchange chromatography 5. Carbohydrates Things that have come up R1-3Simple: Amide: Carboxyl group: Grouping amino acids by relationships & similarities Asp, Asn, Glu, Gln Ser Cys, Met Phe, Tyr, Trp Leu Lys, Arg, His THE R-GROUPS ON THESE 14 αα ALL START WITH CH2 R1-4Hydroxyl group: Contains S: Aromatic: R1-5Branched chain hydrophobic: Basic: Imino acid: R1-6 V Y Y-shaped side chain: V-shaped side chain:Grouping αα by structural relationships (of R-group) Simple: Gly, Ala Branched chain hydrophobic: Leu, Ile, Val Imino acid: Pro Aromatic: Phe, Tyr, Trp Amide: Asn, Gln Hydroxyl group: Ser, Thr, Tyr Carboxyl group: Asp, Glu Basic: Arg, Lys, His Contain S: Cys Met NON-POLAR POLAR, UNCHARGED POLAR, UNCHARGED NON-POLAR -vely CHARGED @ pH 7 +vely CHARGED @ pH 7 (except Y-OH, W indole N) Things to know about amino acids R1-7 KNOW THE 3-LETTER ABBREVIATIONSIonizable side chains Asp, Glu carboxyl group Arg guanidinium Lys amino His imidazole Tyr hydroxyl Cys sulfhydryl Things to know about amino acids R1-8Conservative changes in amino acids in different proteins Acidic side chain D, E Basic: K, R, (H) Aromatic: F, Y*, W* *some polarity Hydrophobic: I, L, V, M, F (A) Hydroxyl group: S, T Amide: N, Q Considered "similar" for construction of a sequence alignment: R1-9 KNOW THE SINGLE-LETTER ABBREVIATIONSCovalent modifications of side chains O-linked sugars: Ser, Thr N-linked sugars: Asn Phosphorylation: Ser, Thr, Tyr Acetylation: Lys Methylation: Arg Things to know about amino acids N H O CH3 C R1-10H+ H+ fully protonated: net charge: +1 0 -1 Titration of glycine ISOELECTRIC POINT net charge = 0 Removal of COOH proton essentially complete, Removal of NH3+ proton has just begun THE pI IS THE AVERAGE OF pKa1 and pKa2! 9.0! 2.1 !5.55!R1-11Titrations of free amino acids Generalization: Amino acids with an R-group that does not ionize…. …..have similar titration curves to Gly (just the α-NH2 and α-COOH titrate) R1-12net charge: +1 0 -1 -2 H+ H+ H+ Titration of glutamic acid R1-13 pKR=!4.1 ! pK2=!9.0 ! pK1=!2.1 !pKas governing formation of the +1 and -1 forms govern formation of uncharged glutamate, and pI = 2.1 + 4.1 2 = 3.11. Need to identify the amino acids with ionizable groups: α-amino, α-carboxyl, side chain 2. Need to be able to find their pKas in the table provided 3. Need to know what charge the ionizable group will have when protonated or deprotonated: " " """"""protonated deprotonated α-amino: NH3+ (+) NH2 (0) α-carboxyl, Asp/Glu carboxyl: COOH (0) COO- (-) His imidazole : N+H (+) N (0) Lys ε-amino: NH3+ (+) NH2 (0) Tyr side-chain OH: OH (0) O- (-) Arg guanidino N+H2 (+) NH2 (-) R1-14 Determining the isoelectric point of a simple peptide-11. straightforward if the pKa's of the ionizable groups are well-separated Determining the isoelectric point of a simple peptide 2. identify the two ionization steps that govern formation of the form of the peptide with no net charge (i.e. the neutral form with 0 charge) 3. = ionization steps in which… the +1 charged form of the peptide is converted to the 0 charge form, and… the 0 charge form is converted to the –1 charged form. 4. the pI is the average of the pKas that govern these two ionization steps 5. a simple way to do this is to start with the form of the peptide with all its ionizable groups protonated (i.e. most +vely-charged form): 6. then see what happens as the pH is raised: the ionizable groups will become deprotonated in the order of ascending pKa R1-15What's the pI of Met-Asp-Gly-His? N!O!H!N!O!H!N!O!H!Met & Gly side chains don't ionize, so ignore Asp pKa 4.1 His pKa 6.0 H3N+ COOH COOH N+H Asp pKa 4.1 His pKa 6.0 terminal α-amino pKa 8.0 terminal α-carboxyl pKa 3.1 start with the form of the peptide with all its ionizable groups protonated (i.e. most +vely-charged form): R1-16pI = (4.1 + 6.0)/2 = 5.05 H3N+ COOH H3N+ COO- H3N+ COO- H3N+ COO- H2N COOH COOH COO- COO- COO- COO- N+H N+H N+H N N +2 +1 0 -1 -2 pKa 3.1 pKa 6.0 pKa 8.0 pKa 4.1 Net charge THESE TWO IONIZATION STEPS GOVERN FORMATION OF THE ZERO-CHARGE (= ISOELECTRIC) FORM OF THE PEPTIDE: What's the pI of Met-Asp-Gly-His? R1-17Equations R1-18 pH = -log10 [H+] [H+] [A-] [HA] Ka = pKa = -log10Ka pH = pKa + log10 [A-] [HA] pKa = pH - log10 [A-] [HA]a)!What!is!the!concentra-on!of!a!lac-c!acid!buffer!(pKa!=!3.9)!that!contains!2.701!M!of!CH3CH(OH)COOH!and!0.901!M!of!CH3CH(OH)COOC?!Give!your!answer!using!2!decimal!points!.!![Buffer] !!=![HA]!!!!!!!!+!!!!![AC]!!!=!2.701!M!!!+!!!!!0.901M!!ANSWER:!!Final!concentra-on!of!lac-c!acid!buffer!=!3.60!M!!b)!What!is!the!pH?!Give!your!answer!using!1!decimal!point.!!pH!=!pKa!+!log10![AC]/[HA]!!!=!3.9!+!log10!!(0.901!M/2.701!M)!!!=!3.9!+!log10!(0.34)!!=!3.9!+!(C0.5)!!ANSWER:!pH!=!3.4!!H.H.!R1-19 ProblemQ.!A!molecule!has!pKa!values!of!7,!8,!and!9.!At!pH!equals!7.0,!es-mate!roughly!what!percentage!of!each!of!the!three!groups!is!inthe!deprotonated!state.!Use!2!decimal!points.!!
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