PeptideIonization - Bioc460 Practice Problems on Peptide Ionization Properties(solutions on next 2 pages Be able to estimate the approximate net

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Unformatted text preview: Bioc460: Practice Problems on Peptide Ionization Properties (solutions on next 2 pages) Be able to estimate the approximate net charge on a short peptide at any given pH. This requires being given (e.g., on cover sheets of exams) or knowing the approximate pKa P actice Probg ms on ep ide Ionizat on P proteins olut single α pages) values of the rionizable leroups Pin tpeptides iand roperties (s(theions on next -2amino group and From l-carboxyljegroupfor Letherepeptide, eand any ionizable R groups) as well as the earning ob ctives on ctu 4 (Peptid s): single Bα able to estimate the approximate net charge on a short peptide at any given pH. This requires being e chemistry/charge cover sheets oof xams) orgroups in their conjugate aacidof the iconjugate base given (e.g., on properties f e those knowing the approximate pKa v lues and onizable groups forms.in peptides and proteins (the single !-amino group and single !-carboxyl group on the peptide, and any ionizable R groups) as well as the chemistry/charge properties of those groups in their conjugate acid and conjugate base forms. Approximate ("generic") pKa Approximate ("generic") pKa Ionizable Ionizable in peptides & proteins (from in peptides & proteins (from group in peptides group in peptides Berg, Tymoczko & Stryer, Berg, Tymoczko & Stryer, and proteins and proteins Biochemistry, 6th ed., 2007) Biochemistry, 6th ed., 2007) t Prop 8.3 !-carboxyl Practice Problems on 3.eptide Ionization hiol erties (solutions on next 2 pages) P1 side chaen cning objectives for Lect4.re 4 (Peptides): aromatic hydroxyl i ar arboxyl 1 10.9 From l u Be abl imidazole e to estimate the approxim.ate net charge on a -shorno eptide at any given pH. Thi0.8equires being 60 1s r " ami t p given (e.g., on cover sheets of e8.0 s) or knowing tguaapproximate pKa values of the 1onizable groups xam he nidino i 2.5 !-amino in peptides and proteins (the single !-amino group and single !-carboxyl group on the peptide, and a answe z ques ions about p pti ll oniz t on p st pe ties y p oper ie now •Tony ionir able tR groups) asewedeais theacihemirory/rchar,geourneedttoskof those groups in their conjugate cid ahe p onjuga ba theorms. ble groups in pept s a p ote ns), a) t nd questionsf e f 9 i peptide a a ue o • To aanswercKucvurletsespprsaboutonizagengerriionizationideas(e nd arrginiiyoundAppr:dine, atteleasgeneric") pK properties, need oxim oni (" c") p r a b)iztabletr t Aof those iate zable oups (oKin the c Ionizable ne a histioxim a (" t he s of a Ion p tur s e p des pro e group in apprtides ate snBeeptedym& zko t&insr(from group in peptides inBeeptiTym& zko t&insr(from pep oxim i trucrg, iT ) s oc pro e St yer, r t– s table e t he ci r e a) thedpKtoteiansd-base pthe 9ties of the group, how tin group donirotseins proteins)g, acid oc otonatSd yer, it ionizable gro an c)pr a values of Bioper mistry, 6th ed., 2ups) he peptidesz(and the conjugahemeer(pr6th eabove, ) an i p e : I s oche 007 Bi ) i ist l form) + charged, in which case its conjugate base (the unprotonated formocs neutray,, or is td., 2007 he willarboxyl onjugate the examl, in which case its conjugateol ase is – charged? c 3.1 thi b 8.3 !-c be given for acid neutra • n a n question b)deIchany carboxyl about the chargeof.operties ionizablergroupsust ooxyl ionizable groups 0.9 ake notes si the a ipproximate structures pr 1 4 those of a peptide,omaus jhydr n the a foctic 1 -- m on the sequence. If you need to write out shorthand structures of the groups, do that for practice while c) ithe ole learbasetheroperties aof 6the group,the var"ous ino , but on an exam, tthe conjugatend" the group im daz acid- ning p structures nd p.r0 rties of how i-am groups ionizes: Is he mor0."shortha acid 1e 8 you're ope y ino e ou. 12.5 (!-amou can usform) +acharged, in.s0whicht caseake gconjugate base (the unprotonated form) is protonated e in your nalysis, the l8 s time i will t itsyuanidino neutral,yor restuestsi/onsobbouts: eptide ineutral, inowhich, case eed conjugate base is – charged? St ans qe q the pr a lem p •Toud wuis ion conjugate acid onization pr perties you n its to know 1. a) hat wK d ale the f pproxiimatzanet charge fin pachidesthe nd protieins)eptides at pH 1? at pH 5? at pH W the p oul v b ues oa the 9 oni e ble groups or e ept of (a follow ng p , 7 t pH a ?ure H t 1.5? at H 1 e 9a o b) ? aquestiont pof 1hos the pzabl3?g(You can cn t sult the tpeptide, and hri of dine2005eor t2006 (or on c e ablegin the cove i the at as • In anythe strfuctpKasabout.)e i[onichargersoupsful? iYoheofasa oif alry tnine focusstjust s,ion lsthort peptides Exapproximate stauctures) Why is thi properties e not l ke o be electrophore ng he ionizable vr lues use u'r a m 1 or a - m ding t s f gure ti sequence. groups ndheeaake bao e ipropeoutetsowatrhewhich polowhey wroup moveesr:ight?he ut thinkitngaabout nottonatrges c) -t n ecid- notes onrthe of d group, h e t the g ould ioniz , Is t Bconjuga e cid (pr e cha ed onfpeptideschelrps younto learh casecits rconjugatetieasof(the ionizotonatroupsrin )pirs neutrawhir hsatrhevery orm) + ha ged, i whic n the ha ge proper b s e the unpr able g ed fo m oteins, l, oc i e imconjant itn enzd neutaaa, ytic mech cnismss binding te bractions chaween biomolecules, etc.] port uga e aci yme cr t l l in whi cha ase it , conjugaintease is – bet rged? Studyny question about the charge properties of a peptiprox. nus jusaron ton whole blepgrde at -e-ach ke notes •In a questions/problems: Ap de, foc et ch t ge he ioniza p e ti oups ma pH on the would . I you need to write o net charge t1 uc ures 5 of the 7 do ha oH r 1. t w pH 1. What sequencebe fthe approximate ut shorthand sfor teachof thepgroups, pHt9 t fpr p1ac5iceatH 1e 1? pH r pH H following peptides p hil 3 you're leatlni-ngst7? str-ucrures9? atoperti11.5? atarpH 13? (Youocan exam, the the e able tonnd" ar u A n-CysaA tg and pr pH es of the v ious groups, but n an consult mor t "shor ha the he A. 5? at pH Phe-GsepHyour analpH , the less time it will take you. t ysis you can u in cover Met-ther-Leu-Lys-Asp of Ty 2005 or 2006 Exam 1 for pKa values.) [Why is this useful? You're not SB. dy questions/problems: tu likely to tbeould be the approximashort cpeptideseandoneeding tong peptides at towardt pH 5? atpole 1. Wha w electrophoresing te net harge for ach f the followi figure out pH 1? a which pH C wouldrg-G a p s- 11.5? a ? a p 9ove, right? But 13? (You can connet he table o on cover of helps or 2 to they . 7Cyst-AHm?ln-tHiHLys-Trp t pHthinking about sult tcharges n thepeptidesthe 2005you006 learn Exam 1 properties of the hy is this usefgroups in nproteins, be electrophoresing short peptides f value W ionizable You'r ikely o theTchargeedior ptKaomplecs.t)edt[owaan d xahicquestiult?heyght jeustot sloveorrtthe whichrhinkting aboutbnetit's rgesin are very important a se n e tha ? cha hend are n't ng ocfigur i out -- r e w m h pole mi would m k f , ightnetBut t ge a one pH, ut cha a on a enzymepcatalyticlmechanisms,the tcharge ns opeheechafge betweenebiomolecules,nsso thich arhavery binding pr to trti s or the tionnzabl rgooupsfeneprotHis,etc.] t's we t interactions a a i umbe r f dif ir nt p e , w ha insoructiptideslookps youatoileaan wha happe t n e ve to he for pr ct ce r t wei'reportng hiere.nzyme catalytic mechanisms, binding interactions between biomolecules, etc.] m doi ant n e You can also make up your own peptide sequencesApppox.ticet chey're mn winteeeptiptidfetat y ach psome for r rac n e; t harge oore hol r se ng i he e have H ionizable side chains, not just non-ionizable R groups, 1ince puch peptpdes7woulpHnly have t11.5 -amino13 sH 5 iH d o 9 pH he ! pH pH s and he--caru-Asn-Coups ror ionizable groups.) ! Glboxyl gr ys-Afg A. P 2. Suppose an enzyme needed a cysteine thiol (sulfhydryl) group for its catalytic activity, and catalysis r quir r- the L hiol B. Met-TyedLeu-Cys -tAsp to act as a nucleophile at the beginning of the catalytic cycle. That would mean the Cys residue had to be in its UNPROTONATED (conjugate base) form. If that particular Cys C. Cres-iArg-iGltn-Hisr-otein Trp a pKa of 7.5, what fraction (per cent, or proportion) of the total enzyme ys due n hat p Lys- had molecules would have the Cys R group in its ACTIVE form at pH 7.0? These aren't that complicated -- an exam question might just ask for the net charge at one pH, but it's Bioc460 o look . Z prac © a pri ha 008 instructive2t008, Drforieglert,ice St wng t2happens to the charge at Penumber ozatifferprntctiHs,psoblhat's, w. 1t a ptide ioni f dion e a p ce r o t ems pha we're doing here. You can also make up your own peptide sequences for practice; they're more interesting if they have some ionizable side chains, not just non-ionizable R groups, since such peptides would only have the !-amino and !-carboxyl groups for ionizable groups.) 2. Suppose an enzyme needed a cysteine thiol (sulfhydryl) group for its catalytic activity, and catalysis required the Cys thiol to act as a nucleophile at the beginning of the catalytic cycle. That would mean These aren't that complicated -- an exam question might just ask for the net charge at one pH, but it's instructive to look for practice at what happens to the charge at a number of different pHs, so that's what we're doing here. You can also make up your own peptide sequences for practice; they're more interesting if they have some ionizable side chains, not just non-ionizable R groups, since such peptides would only have the α-amino and α-carboxyl groups for ionizable groups.) 2. Suppose an enzyme needed a cysteine thiol (sulfhydryl) group for its catalytic activity, and catalysis required the Cys thiol to act as a nucleophile at the beginning of the catalytic cycle. That would mean the Cys residue had to be in its UNPROTONATED (conjugate base) form. If that particular Cys residue in that protein had a pKa of 7.5, what fraction (per cent, or proportion) of the total enzyme molecules would have the Cys R group in its ACTIVE form at pH 7.0? SOLUTIONS 1.A. Phe-Glu-Asn-Cys-Arg SOLall ONS List UTIionizable groups: “+” or “–“ indicates the predominant (“majority”) charge state (SS.OLUthanNhalfsn-Cthis charge state); if pH is less than 1 unit away from pKa, so group is morePTe-GlS -A in ys-Arg 1OLUTIIO u A. h ON S List a l i that state as ( myself be if andwrit were s ight on/ unit above or below the not 90% linonizable groupsitI would make hthe pHten notea rfull pHabove/below the sequence): pKa, 11A.“redominantnchargerpredominant ismajority”) charge sta(+)more–han half in this charge state); if . .A. +Phe-Gu--As c-Cyst-he g that pH (“ in parentheses: te ( or ( t ). P” or “–l“ i sn at ys-Ar at h l uA C s A the p Hstiea-lGsonindi -euenioupsg(y mom lpK ake haoup irstnen notesinight tosnaae as i/tbelouldthe sf quence)were a L zn 1 p i s l i s thaabl gr t awa I fr yse f m , so gr ndw i t ot 90% r tha t/ tbove w ow be ie the pH : e List all ionizable groups (I myself ma ke handwritten notes right on/above/below the sequence): a + o un above he ow the K nt “ p or y”) charha ge e hat e t is h p f n the cha +) o – f+”l p r“–“ ndicatessthe pr edompnaa, hema edomin chacge state (more than half in t his cha rge s a te) . f ““ul”orH“–“iitindicateortbelpredomiinant t((“marjjoriitty”) ant rge rstatat (tmorpHhanin alarienthisses: r(ge sttrat(e));; iif Ap s not 9 t c n haon ite i s d woul RO i P at pH weH ppHissleesstthan1 unittaway ffrrom pKa,, so grrouppirox. ne0%hiartge t standaviit ual Gd beUf the pach pre a H i l ss han 1 uni away om pKa so g oup is not 90% in tha t state as it woul d be i f the e H we re a nt ha pH t t H ionullabH uuiitaaboveorrbelpow tthe pK a,, tthe prredomiinapHc1 rge at that pH7is in H rentheses:1.(+) orH–1.. z e nn iona o be ow he pK he p edom nant cha rge a5 thappH is in parenthese1: (+) or ( –) 3 p pa9 pH s 5 p ( ) ffiullpplH funtctbovel g ou l a Approx. n et charge on individual GROU0 at each p H n ! -NH2 (pKa ~8) (N-terminal amino group) Approx. + et charge on individual GROUP at each 0 H + + 0 P p ion abl unctional gr 4.p pH 1 pH 5 pH 7 pH 9 pH 11.5 pH 13 0 – – – – iGluizR lceffboxylonalgrou1) onizab ear uncti (pKa ~oup pH 1 pH 5 pH 7 pH 9 pH–11.5 pH 13 ! N R pK ( ~ ) N te + + + 0 C -NH -SpK pK8 ((8 -t rminal am no g oup) 0 0 0 (–0 ) –0 –0 ! -ysH22((H aa~8a)~N.-3)erminal amiino grroup) + + + 0 0 Gl g 0 – – – – – A u Rcguanidino (pKa ~1)) + + + + +– (0) Glru R -carboxyl((pKa~4.12.5) arboxyl pKa ~4.1 0 – – – – Cys RSH pKa ~83) 0 0 0 ( –) ! -C R-- H (pKa a 38.. ( 0 – – – –– –– Cys OOSH((pK~~.1)3)C-terminal carboxyl group) 0 0 0 ( –) – – APPROXaniidinCHARG12.5 ) + + + + + (0) ++ 2 0 0 –2 –2 –(3 Arrg R--guanEino ((pKa ~12.O)N WHOLE g Rgu . N d T o pKa ~E 5 + + + + 0) ! COOH AT T 3 1) C-t e m na a boxyl g 0 – – – – – P - PT OH pKa~3.1) pH e smiinalf cndividual oup) ! -ECOIDE((pKa~H.IS((C-t(rrum ol ciarrboxyl grroup) 0 – – – – – gPoupOXh.arET ) CHARGE ON WHOLE r PR s' X NE s APPROc . N geTCHARGE ON WHOLE +2 2 0 0 –2 –2 –3 A + 0 0 –2 –2 –3 PEPT DE AT TH S pH sum o nd v dual PEPTIIDEAT THIISpH ((sum off iindiiviidual ggroups'ccharges) roups' harges) 11B. . Met--Tyr-eu-Lys-AspAsp . . B Met Tyr-LLeu-Lysist ionizable gr ListLionizableLeuoups.-Asp Approx. net charge on individual GROUP at each pH 1 . B. Meet-Tyr- groups. sp 1 . B. M t-Tyr-Leu--Lys A LysLsabiloniunceigrral gr. i t e abl g oups ioniizstionifzzablteonoups.oup pH 1 pH 5 pH 7 pH 9 pH 11.5 pH 13 L Approx. net charge on indivi 0 ! -NH2 (pKa ~8) (N-terminal amino group) Approx. + et charge on individual GROUP at each 0 H + + n dual GROU0 at each p H P p ion abl romatic OH grou pH 1 pH 5 pH 7 pH 9 pH–11.5 pH 13 TyrizR laeffunctionalg(pKap~10.9) 0 1 pH 5 pH 7 pH 9 pH 11.5 pH 13 0 0 0 () – ionizab e unctional roup pH ! R (pKa ~ na + + + 0 0 L -NH2" -NH2 8)((N-termi) l amino group) + + + + (0) 00 ! -ysH2-(pKa ~8()pKa-t~10.inal amino group) N N erm8 + + + 0 0 0 Tyr R aromati OH ~4.1) 0 0 0 0 (–) Asp R-carboxylc(pKa (pKa ~10.9) 0 – – – – –– Tyr R -" -NH2icpKH ~10.a )~10.9) aromat ( O a (pK8 0 0 0 0 (0) –) – Lys R + + + + ( ! -COOH (pKa~3.1) (C-terminal carboxyl group) 0 – – – – –0 Lysp R--" -Nboxyl (pKa10.81) H2 (pKa ~ ~4. ) + + + + (– 0) 0 s car – – – – APPROX. NET CHARGE ON WHOLE +0 2 0 0 –1 –3 –3 As-CR-OHbAT a~H.IS (~4.1)um ol cnrboxyl alroup) pPOIcar (oxyl (3 1) pHt(s mina f i a dividu g pKa C- er 0 – – – – – PE T DE pK T ! 0 – – – – – ! -PPpsOch.argeT) CHARtGrE ONl Wrboxyl group) COO'H (pKa~3.1) (C- e mina ca HOLE 02 – – – – grou R X NE s A + 0 0 –1 –3 –– 3 APPROX. NET CHS pH (sum oWndividual +2 0 0 –1 –3 –3 PEPTIDE AT THI ARGE ON f iHOLE PEoups' EhAT THIS pH (sum of individual grPTID c arges) g C. s c - arg- s) 1 .roupC'yshArgeGln-His-Lys-Trp List ionizable groups. Approx. net charge on individual GROUP at each pH C Cys Arg- H Lys Trp 11 .C.. . Cys---Arg-GGlni-sHis--Lys-Trp .. C CysArg-Glln-H is-Lys -Trp 1 ionizst iloniuablteon-oupsoup n p 1 pH 5 p 7 pH 1 pH Li ab e f znc i gral gr. L! -Nionizableegroups. inal amino group) Approx.H et charge on H dividual9GRpH P1.5 each 013 ist ist 2onizabl 8) roupsr.m L H i (pKa ~ g(N-te n in OU0 at pH + + + 0 Approx. He1 charge onpHd7vidual) GROH–11.t5eacpH H3 n in i pH 9 p UP a h– 1 p ion able f ( nc i ~8.l g p 0 t pH 5 CysizR-SH upKtaona3) roup 0 0 (– iArizab-e pKnidino al-geaou12.l ) mino group) on NH2 guu ac~ion N t rrmip 5a pH 1 pH 5 pH 7 pH 9 pH+11.5 p(H 13 ! -g Rl ( fan t 8) ( (pK ~ na + + + 0 0 0 + + + + 0) ! -is HRi-(SH a pKe (N-3)r~6)nal amino group) + + + 0 0 CN - pK ( ~8) ~8. te 0 0 0 ( –) HysR2 midazola (pKa mi + + 0 00 0– 0– Cysg R--SH ani2 ano a3) 10.~12.5) 0 0 0 (+) – – Ar R gu pK i ~8. ~ + + + + (– Lys R -" -N(H d(pK (pKa 8) + + + + (0) 00) AriCO--iguianidano(.pK(C-~erm5nal carboxyl group) + + + + + (0) H s R m ( az i e 1) a 6) + + 0 0 ! -g R OH dpKol~3 (pKa~t 12. i ) 0 – – – –0 –0 HiPPRiOXNNETpKa AR6)8) ON WHOLE 0 0 0 L s - -" . H2 ( (pK ~ ~ G + + + (0) 0 AysRRmi-dazole CH a 10. E ++ 4 ++ 3 ++ 2 00 –1 –2 LysCRI"H (pKapKIS~(C-t()rum ol cnrboxyl alroup) + + + + (– 0) 0 PEPO-D-E AT (~H.a pH 8s mina f i a dividu g ! - T O NH2 T 3 1) 10. e 0 – – – – ! -PPpsOch.argeT) CHARtGrE ONl Wrboxyl group) 0 – – – grCOOH (pKa~3.1) (C- e mina ca HOLE A ou R ' X NE s +4 +3 +2 0 –1 –– 2 APPROX. NET CHS pH (sum oWndividual +4 +3 +2 0 –1 –2 PEPTIDE AT THI ARGE ON f iHOLE PEoups' EhAT THIS pH (sum of individual grPTID c arges) groups' charges) 2. Suppose an enzyme needed a cysteine thiol (sulfhydryl) group for its catalytic activity, and catalysis required the Cys thiol to act as a nucleophile at the beginning of the catalytic cycle. That would mean the Cys residue had to be in its UNPROTONATED (conjugate base) form. If that particular Cys residue in that protein had a pKa of 7.5, what fraction (per cent, or proportion) of the total enzyme molecules would have the Cys Bioc460 in its ACTIVE form at pH Peptide ionization practice problems, p. 2 R group 2008, Dr. Ziegler, © Spring 20087.0? Bioc460 2008, Dr. Ziegler, © Spring 2008 Bioc460 2008, Dr. Ziegler, © Spring 2008 Peptide ionization practice problems, p. 2 Peptide ionization practice problems, p. 2 an enzyme needed a cysteine thiol (sulfhydryl) group for its catalytic activity, and catalysis the Cys thiol to act as a nucleophile at the beginning of the catalytic cycle. That would mean residue had to be in its UNPROTONATED (conjugate base) form. If that particular Cys n that protein had a pKa of 7.5, what fraction (per cent, or proportion) of the total enzyme es would have the Whenever a iquestionTasks fabout t either.0the ratio of base/acid (or acid/base), or the Cys R group n its AC IVE orm a pH 7 ? proportion of a functional group that's in one charge state or the other, you know you'll question asks about etheeHenderson-oHbase/acid (Equation,ase), or relates oportion of a pH, pKa, and need ith r the ratio f asselbalch or acid/b which the pr 3 parameters: roup that's in onbase/acid state or thit's thlogarithmic relationship). the Henderthe pH (given: pH = 7.0) e charge ratio (but e o a er, you know you'll need You know sonEquation, which relates 3 parameters: pH, pKa, and base/acid ratio (but it's a and now th f this ven: p (GIVEN n the p don't use Cys-SH (GI pKa relationship). You kthe pKaeopH (giCys-SHH = 7.0) aasd7.5, soKa of this the "generic"VEN value). Solve n't use the "genefor "base/aciduratio, olve fconvert /acid ratio, andper centt to fractiobase form, as in the ric pKa val e). S and or base to fraction or conver that's in n or t's in base form, example atxamend at the end of general ted general chemistry review as in the e the ple of the posted the pos chemistry review PDF. [base ] [ acid] pH = pKa + log " [base ] R ! S ! % $ = # [ acid] R ! SH & log [base ] = pH ! pKa = 7.0 ! 7.5 = !0.5 [acid] [ base] 0.316 R ! S! = 10!0.5 = = [acid ] 1 R ! SH 0.316 0.316 Fraction base = = = 0.24 1 + 0.316 1.316 cent, 24% of the Cys thiol would be in the form of the good nucleophile, the conjugate o - as e thiolate anion, Rr S–. a per cent, 24% of the Cys thiol would be in the form of the good nucleophile, the conjugate base, i.e., the thiolate anion, R-S–. 8, Dr. Ziegler, © Spring 2008 Peptide ionization practice problems, p. 3 ...
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This note was uploaded on 01/30/2010 for the course BIOC 460 taught by Professor Ziegler during the Spring '07 term at University of Arizona- Tucson.

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