Protein Structure and Function! Lecture 4: ph, pka and pi!

Definition of ph and pk a! ph is a measure of the concentration of H +.! + ph = log 10[H ] For a weak acid,! HA #!!"! H + + A!, K a = [H + ][A! ] [HA] pk a = log K a The pk a of an acid is the ph at which it is half-dissociated.!

Henderson-Hasselbalch equation! 1 1 [A ] = + [H ] K a [HA] + [A ] log[h ] = log K a + log [HA] [A ] ph = pk a + log [HA]

Amino acid pk a! Amino acid! Ionizable group! s.c.! b.b. H 3 +! b.b. CH! Arginine! -HC(H 2 ) 2 +! 12.5! 9.0! 1.8! Aspartic Acid! -CH! 3.9! 10.0! 2.0! Cysteine! -SH! 8.3! 10.8! 1.8! Glutamic acid! -CH! 4.3! 9.7! 2.2! Histidine! ImH +! 6.0! 9.2! 1.8! Lysine! -H3 +! 10.5! 9.2! 2.2! Tyrosine! -C 6 H 4 -H! 10.1! 9.1! 2.2! In a polypeptide, the pk a s may shift about +/- 1 ph unit.! In a folded protein, specific interactions may shift them even more.!

pka and environment 4.3 H 3.9 H + H 3 + H 3 + H 3 10.0 H 2.0 9.7 2.2 9.2 H 10.5 H 3 + H 2.2 Inductive effect Through bond interaction Carbonyl C is electron-withdrawing H 3 + is electron-withdrawing Charge effect Through space interaction H 3 + stabilizes C CH 3 CH ClCH 2 CH Cl 3 CH 4.8 2.9 0.6

Glycine versus Diglycine + H 3 9.6 H 2.3 H H + H 3 8.2 H 3.1 10.6 + H 3 Inductive effect Through bond interaction Carbonyl C is electron-withdrawing H 3 + is electron-withdrawing Charge effect Through space interaction H 3 + stabilizes C C stabilizes H 3 +

pka and resonance forms H 2 H 2 + H + H 2 H 2 H H 2 H 2 H + Arg side chain, pka = 12.5 H - Tyr side chain, pka = 10.1

Amides not protonated! Amide has delocalized electrons and partial double bond character! H 2 - H 2 + Same for internal amide bonds in a polypeptide! + H 3 H + H 3 H + H 3 R1 H R3 H R5 R2 H R4 H

Amino acid pk a! Amino acid! b.b. H +! 3 b.b. CH! Alanine! 9.9! 2.3! Asparagine! 8.8! 2.0! Glutamine! 9.1! 2.2! Glycine! 9.8! 2.4! Isoleucine! 9.7! 2.4! Leucine! 9.6! 2.4! Methionine! 9.1! 2.3! Phenylalanine! 9.1! 1.8! Proline! 10.6! 2.0! Serine! 9.2! 2.1! Threonine! 10.4! 2.6! Tryptophan! 9.4! 2.4! Valine! 9.6! 2.3! In a polypeptide, amine pka ~ 8, CH pka ~ 3!

Cyclic peptides! + H 3 R1 H R3 H R5 R2 H R4 H R1 H R3 H R5 H R2 H R4 H o free backbone carboxylate or ammonium

Isoelectric point! pi = ph at which the net charge = 0. pi = pk 1 + pk 2 2

Isoelectric point! ph at which the molecule has no net charge! From Biochemical Calculations, Irwin Segel

Ionization state! Group! Start! pk 1! 1 st eq! pk 2! 2 nd eq! pk 3! 3 rd eq! C-term CH! 0! -0.5! -1! -1! -1! -1! -1! Asp s.c. CH! 0! 0! 0! -0.5! -1! -1! -1! -term H3+! +1! +1! +1! +1! +1! 0.5! 0! et charge! +1! +0.5! 0! -0.5! -1! -1.5! -2!

Lysine isoelectric point!

Lys, isoelectric point! Group! Start! pk 1! 1 st eq! Ionization state! pk 2! 2 nd eq! pk 3! 3 rd eq! C-term CH! 0! -0.5! -1! -1! -1! -1! -1! -term H3 +! +1! +1! +1! 0.5! 0! 0! 0! Lys s.c. H3 +! +1! +1! +1! +1! +1! 0.5! 0! et charge! +2! +1.5! 1! 0.5! 0! -0.5! -1! pk 1 = 2.2 pk 2 = 9.0 pk 3 = 10.5! pi = 0.5 * (pk 2 + pk 3 ) = 0.5 * (9.0+10.5) = 9.75!

Polypeptide isoelectric point! Example: Ala-Asp-Gly-Arg-Leu! Internal amino acids have no ionizable backbone amino or carboxy group.! Ionizable groups! -terminal H3! Asp side chain CH! Arg side chain guanidino group! C-terminal CH! Approximate the pk a s of each ionizable group based on the values for an isolated amino acid.!

Ala-Asp-Gly-Arg-Leu! Ionization state Group Start pk 1 1st pk 2 2nd pk 3 3rd pk 4 end -CH 0-0.5-1 -1-1 -1-1 -1-1 Asp CH 0 0 0-0.5-1 -1-1 -1-1 -H3 +1 +1 +1 +1 +1 0.5 0 0 0 Arg guanid. +1 +1 +1 +1 +1 +1 +1 0.5 0 et charge +2 +1.5 +1 +0.5 0-0.5-1 -1.5-2 pi = 0.5 * (pk 2 + pk 3 ) = 0.5 * ( 3.9 + 9.9) = 6.9!

Applications of the isoelectric point! Basis for purification techniques such as! isoelectric focusing! ion-exchange chromatography!

Isoelectric focusing! Stable ph gradient 4 5 6 7 8 9 10 + V + + + + + + At high ph, the highly negatively charged molecule is repelled by the negative potential.! As it moves towards the +V, the surrounding ph drops and the molecule acquires protons.! At ph = pi, net charge = 0. Migration stops.! Diffusion away from the pi location is prevented by the electric potential.! V

Isoelectric focusing! Stable ph gradient 4 5 6 7 8 9 + V + + + + + + + + + + + + At low ph, the highly positively charged molecule is repelled by the positive potential.! As it moves towards the -V, the surrounding ph rises and the molecule loses protons.! At ph = pi, net charge = 0. Migration stops.! Diffusion away from the pi location is prevented by the electric potential.! 10 V

Establishing a ph gradient! 4 5 9 + V 6 Mixture of amphoteric buffers with different pi values.! Large buffering capacity at pi.! Stable ph gradient 6 7 8 9 7 6 5 8 9 7 Gel starts out at a uniform ph.! Application of electric field causes migration of the buffer electrolytes.! A stable ph gradient is created.! 10 8 5 V

2D gel electrophoresis! basic Stable ph gradient acidic SDS migration (MW) From Biochemistry, L. Stryer

Ion-exchange chromatography! The resin carries ionic charges that retard molecules of opposite charge.! Positively charged: Diethylaminoethylcellulose (DEAE-cellulose)! egatively charged: Carboxymethylcellulose (CM-cellulose), phosphocellulose, sulfonated polystyrene (Dowex-50)! The degree of retardation depends on both the ph and ionic strength of the solution.! The combination of electrostatic and nonpolar interactions determines the elution time.! Example: protein X and protein Z have pi values of 5 and 9, respectively.! In a sample containing both proteins X and Z at a ph of 7, which protein is expected to elute first from a DEAE-cellulose column?! Ans: Protein X will have a net negative charge while protein Z will have a net positive charge. Hence, we expect Z to elute first.!!

Charged moieties in resin! Positively charged: Diethylaminoethylcellulose (DEAE-cellulose)! +! +! +! +! +! +! Resin! +! +! +! C 2 H 4 C 2 H 5 H+ C 2 H 5 egatively charged: Carboxymethylcellulose (CM-cellulose), phosphocellulose, sulfonated polystyrene (Dowex-50)! Resin CH 2 CH 2 CH CH 2 CH - - S 3 S 3 n

Determinants of elution time! Interplay of electrostatic and nonpolar interactions! Dependence on ph and ionic strength! δ δ+ C 2 H 4 C 2 H 5 H+ C 2 H 5 δ δ δ δ+ δ+ δ+ Glu δ+ Resin CH 2 δ+ δ+ δ+ δ δ δ + H 3 More info on ion-exchange chromatography: http://ntri.tamuk.edu/fplc/ion.html! δ Lys

Sample problems! Calculate the isoelectric point of the following peptide: Ala-Glu-Gly-Tyr-Arg.! You have a mixture of two polypeptides: Ala-Glu- Ala and Ala-Lys-Ala. What would be a suitable ph to use to separate the mixture on a Dowex-50 (negatively charged) ion-exchange column?!