R1 1 MCB 450 REVIEW FOR EXAM 1 EXAM 1 Thursday Feb 12 On material covered in Lectures 1 through 7 For room assignments please see course web site R1 2 Information Tips 1 Questions like homework questions in difficulty and format all M C 2 The exam will have 35 40 questions 3 A copy of last semester s exam has been posted 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 5 You will be give a table of pKas Use those in your calculations unless the question gives you another pKa value 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 7 Look at the Learning Objectives slides at the ends of each lecture R1 3 Things that have come up 1 Amino acids structures properties titration 2 Isoelectric point of a peptide 3 Buffer questions 4 Ion exchange chromatography 5 Carbohydrates R1 4 Grouping amino acids by relationships similarities Simple THE R GROUPS ON THESE 14 ALL START WITH CH2 Carboxyl group Amide Asp Asn Glu Gln Ser Cys Met Phe Tyr Trp Leu Lys Arg His R1 5 Hydroxyl group Contains S Aromatic R1 6 Branched chain hydrophobic Imino acid V Basic Y s side haped chain Y V sha chain e d i s ped Things to know about amino acids R1 7 KNOW THE 3 LETTER ABBREVIATIONS Grouping by structural relationships of R group Simple Gly Ala Branched chain hydrophobic Imino acid Leu Ile Val NON POLAR Pro except Y OH W indole N Aromatic Phe Tyr Trp Amide Asn Gln Hydroxyl group Ser Thr Tyr Carboxyl group Asp Glu vely CHARGED pH 7 Basic Arg Lys His vely CHARGED pH 7 Contain S Cys Met POLAR UNCHARGED POLAR UNCHARGED NON POLAR R1 8 Things to know about amino acids Ionizable side chains Asp Glu carboxyl group Arg Lys His guanidinium amino imidazole Tyr hydroxyl Cys sulfhydryl R1 9 Conservative changes in amino acids in different proteins Considered similar for construction of a sequence alignment Acidic side chain D E Basic K R H Aromatic F Y W Hydrophobic I L V M F A Hydroxyl group S T Amide N Q some polarity KNOW THE SINGLE LETTER ABBREVIATIONS R1 10 Things to know about amino acids Covalent modifications of side chains O linked sugars Ser Thr N linked sugars Asn Phosphorylation Ser Thr Tyr Acetylation Lys Methylation Arg N H C O CH3 R1 11 Titration of glycine net charge 1 0 H 1 H fully protonated THE pI IS THE AVERAGE OF pKa1 and pKa2 ISOELECTRIC POINT net charge 0 9 0 Removal of COOH proton essentially complete Removal of NH3 proton has just begun 5 55 2 1 R1 12 Titrations of free amino acids Generalization Amino acids with an R group that does not ionize have similar titration curves to Gly just the NH2 and COOH titrate R1 13 Titration of glutamic acid net charge 1 0 1 H pK2 9 0 pKas governing formation of the 1 and 1 forms govern formation of uncharged glutamate and pI 2 1 4 1 3 1 2 H pKR 4 1 pK1 2 1 2 H Determining the isoelectric point of a simple peptide 1 R1 14 1 Need to identify the amino acids with ionizable groups amino 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 NH3 NH2 0 carboxyl Asp Glu carboxyl COOH 0 COO His imidazole N H N 0 Lys amino NH3 NH2 0 Tyr side chain OH OH 0 O Arg guanidino N H2 NH2 amino R1 15 Determining the isoelectric point of a simple peptide 1 straightforward if the pKa s of the ionizable groups are well separated 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 16 What s the pI of Met Asp Gly His Met Gly side chains don t ionize so ignore His pKa 6 0 Asp pKa 4 1 H H H N N N O O O start with the form of the peptide with all its ionizable groups protonated i e most vely charged form Asp pKa 4 1 COOH terminal amino pKa 8 0 H 3N His pKa 6 0 N H COOH terminal carboxyl pKa 3 1 What s the pI of Met Asp Gly His R1 17 COOH N H H 3N COOH COOH N H H 3N pKa 4 1 COO N H 3N COOH 2N 1 N H COO COO 2 pKa 3 1 COO H 3N pKa 8 0 Net charge 0 pKa 6 0 COO 1 COO 2 N THESE TWO IONIZATION STEPS GOVERN FORMATION OF THE ZERO CHARGE ISOELECTRIC FORM OF THE PEPTIDE pI 4 1 6 0 2 5 05 Equations R1 18 pH log10 H Ka H A HA pKa log10Ka pH pKa log10 pKa pH log10 A HA A HA R1 19 Problem a What is the concentra on of a lac c acid bu er pKa 3 9 that contains 2 701 M of CH3CH OH COOH and 0 901 M of CH3CH OH COO Give your answer using 2 decimal points Bu er HA A 2 701 M 0 901M ANSWER Final concentra on of lac c acid bu er 3 60 M b What is the pH Give your answer using 1 decimal point pH pKa log10 A HA H H 3 9 log10 0 901 M 2 701 M 3 9 log10 0 34 3 9 0 5 ANSWER pH 3 4 Problem R1 20 Q 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 pH pKa log10 A HA H H pKa 7 group 7 0 7 0 log10 A HA 0 An log 0 A HA 1 ra o 1 part A and 1 part HA therefore A is 1 part out of 2 parts total 1 2 x 100 Answer 50 00 pKa 8 group 7 0 8 0 log10 A HA 1 An log 1 A HA 0 1 ra o 1 part …
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