Amino Acids as Acids, Bases and Buffers: = Amino acids are weak acids 5 \ : ai -SH - Allhave at least 2 titratable protons ptsine Coy, ¢) ba -S (shown below as fully protonated species) — “Tyrgsin CY, Typ) hee 04 and therefore have 2 pKa’s iia a es © a-carboxyl (COOH) e ine (Lae y kK) kee = bY © a-amino (-NH;*) W tae 1 \ ;e = ini hao = NH; - Some amino acids have a third titratable Olaginive Oey, ®) Nee sisi excised Mee erealis a dhsh ding Cs, 4) es Sa Pa © © Showing all protonated: = ee eee ees : COOH (pk) ~ Clulemate (lu, &) bus — coor (pk) H3N—C—H XH (pK) coo" yt $00" S,N, C—O7 wonky > wont § Nut q H* NH} Base Acid Histidine side chain ionization Iidazolium ion (an acid) coo- + H3NCH + H* CH; B goo" coo" nskcH HANCH + Ht CH; cH; B CcTable 3.2 ino acids: Table of pXvalues for the 20 aminoacids found in proteins. pX; values are pKa Table for amino acids: assigned to the c-carboxyl group, pK values to the c-amino group, and pK einen tolonizable groups in the R side chain (see structure). Name Pe PK PK Second column (pKa:) = NH" iy 24 98 Alanine 23 97 * Third column (pKaq)=R group H* Valine eS A Iso 24 97 Methionine 23 92 Phonvlalanine 1s a Proline 20 106 Sexine 21 92 Threonine 26 to Cysteine 18 ios 83 Asparagine 20 88 Giitamine 22 31 ‘Tyrosine 22 sa 109 ‘Tryptophan 24 94 Aspartate 20 10 39 Gitutamate 22 97 a3 Histidine Ls 92 60 Ly 2 92 108 is 90 225 eodishnay aon AMINO ACIDS AS WEAK ACIDS: = Properties of amino acids in proteins and peptides are determined by the R group but also by the charges of the titratable group. Will ultimately affect protein structure - Important to know which groups on peptides and proteins will be protonated at a certain pH. PHA. Net charge +1 PHT Net charge 0 pH 19. Not charge —1 + + oor goo G00" nsdn = vagiad— a_i hn On 1 I i R R R Cationic form Zwitterion (neutral) Anionic form The degree of dissociation depends on the PH of the solution. NH,CH,COO- +H" ['NH,CH,COO][H'] INH,CH,COO]H*] PNH,CH,COOH] Keo ['NH,CH,COO] How do we do this? Example ~ Alanine +1 net charge 1 Draw the fully protonated structure 4 Q: ‘Which protons come:cff when?’ Hoe -4 — COOH A: Look at pKa table for amino acids NH" ‘Alanine has 2 pKas: a-COOH (pKa = 2.3) comes off first (has lower pKa) a-NH' (pKa = 9.9) Others come off SEQUENTIALLY in ascending order of pKa 2. Write out structures for sequential deprotonation and place pKa values over the equilibrium arrows. i H\N—C—COOH pK,=9.7 CHy pk, =23 Alanine Fann EEnEnnnnannEnECT anno oo 23 60 99 iv oh) it i i * a HN—C—COOH —HN—C—COOT HNC —COO™ cH; Hs cHy Seiden Soe Fully protonated —_1“ proton removed. 24 proton removed Net charge = +1 Net charge 0 Net charge = -1So, from looking at the net charges, at different pH’s, amino acids can have different charges! ‘Very important for protein structure!! Remember that the pKa = pH when ¥ of an available amount of an ionizable group is ionized. = Let's take a look at the titration curve for Alanine ~ Looks very much like what we saw for acetic acid last time except that it has 2 midpoints (pKa’s) — one for each proton -COOH and a-NHs” y-60 = Atbeginning, all protonated facumcoonsy = Need one equivalent of base for each fo men Proton i = At each HALF equivalent = pKa Giscrncoony ©. 50% protonated/50% Aeon" depofonatad oo w vo 7 31 = At end all deprotonated ~ Flat parts of curve are BUFFERING REGIONS: Acts as buffer in TWO pH ranges. © #/-1 pH unit from pKa - For our purposes, to determine whether the proton is ON or OFF at a certain pH use the following RULES © pH=pKa Equal amounts of protonated and deprotonated species exist PER pie if pH is LESS than the pKa of a particular group, that Hé on, substance protonated group will be predominantly protonated pH > pk, ifpH is GREATER than the pKa of a particular ionizable Hi olf, substance deprotonated group, that group will be predominantly deprotonated For example: Alanine at different pHs (see pKa table) 4 AtpH 15: — pHis less than the pKa of both the a-COOH_ te ¢—coon and the a-NHs", therefore, both protons are ON Nn 4 AtpH7: pH is greater than the pKa of the e-COOH > H’ OFF a pH is less than the pKa of the a-NH3* > H" ON has IsAtpH 10.5 pHis greater than the pKa of the -COOH > H’ OFF pH is greater than the pKa of the c-COOH - H” OFF Apply same rules if there are 3 titratable protons: 1. Determine what the pKa’s of the titratable protons are by looking at the pKa table 2. Draw the structures and the equilibria representing the complete deprotonation of the amino acid a. Start with fully protonated and then remove in order of pKa values b. Put pKa values above equilibrium 3. Determine at the pH of interest whether the proton is ON or OFF using the above rules For example, Aspartate (D, Asp): Asp has 3 titratable protons Net charge coo" HaN-C=H1 on boo- Starthere: COOH coo™ coo- Ha-cH PK Hac H PKR HCH cH, cH, Gt, oon oon boo- Net charge = +1 Net charge = 0 Net charge = etipns pate Corinna ie Sse sae 1, _pKa’s for the three groups (look at Table3.2) 2. Draw the structures from fully protonated to fully deprotonated‘Note that all amino acids are at one point, electrically neutral at some pH value, This pH = isoelectric point (pI) How do you calculate pI? Draw out the complete ionization of amino acid = Determine net charge on each ionized form Find the structure that has no net charge Take the average of the pKa’s that are around the struct pl=pKa,+ pKa: 2 Note do NOT just take the average of all pKa’s, ‘What about Asp?? pKas: 2, 3.9, 10 (from Table 3.2) pl=(2+3.99/2=2.95 Amino acids can be separated on the basis of their charges at a certain pH