BIOC2000 Lecture Notes - Lecture 25: Conjugate Acid, Acid Dissociation Constant, Amine
Lecture 25
*Be familiar with ways in which amino acids are ionized
-COOH group (uncharged) ionizes to form COO-(–vely charged) –
therefore carboxyl group can exist in an uncharged state or –vely charged
state
- Lysine’s primary amino group can exist in pronotated form (+vely
charged) and deprotonated form (uncharged)
- Arginine’s amino group can exist in pronotated form (+vely charged) and
deprotonated form (uncharged)
General equations for weak acids and bases:
HA + H2O ⇌ A- + H3O+
acid + base ⇌ conj. base + conj. acid
B(aq)+H2O(l)⇌BH+(aq)+OH−(aq)
B + H2O ⇌ BH+ + OH-
base + acid ⇌ conj. acid + conj. base
Conjugate acid is always protonated form, conjugate base is always the
deprotonated form
Red – for Henderson-Hasselbalch equation
For each amino acid, there is a conj. acid (protonated form) and a conj.
base (deprotonated form)
HA to A- class: Cys, Tyr, Ser, Thr
Neutral in protonated form (SH, OH) and negatively charged in the
deprotonated form
Typically an acid has a pKa of 3-4, Cys, Tyr, Ser and Thr are very weak
acids (high pKa values)
The ionization state of acidic and basic groups on proteins changes with pH
% Ionisation = % in charged state
pKa is defined as the pH were amino acids exist as 50% ionized and 50%
unionized
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Henderson-Hasselbalch equation
pH = pKa + log [conjugate base]/[conjugate acid]
Basic amino acids = Arg, Lys, His
Acidic amino acids = Glu, Asp, and polar amino acids = CysH, Tyr, Ser and
Thr)
Example: Acidic side chain Glu, HA⇌A- +H+
Rearranging Henderson-Hasselbalch: pH - pKa = log [conjugate
base]/[conjugate acid]. Now at any given pH, the average charge of Glu
residue can be deduced.
Conjugate base is charged form, conjugate acid is uncharged form
e.g. if pH is one unit above the pKa for an acid
1= log10[A-]/[HA]
10 = [A-]/[HA] i.e. for each one HA there are 10A- (as expected, when
pH>pKa, -vely charged A- is dominant); there are 10 charged side chains, 1
uncharged side chain
We want to know proportion of charged side chains = 10/11
10/(10+1) = 0.909 of side chains are in the charged A- form of a Glu acid in
pH 1 unit above pKa of Glu
So the average charge on that side chain is -0.909. Average is that 1/11 will
be neutral and 10/11 will be –vely charged.
HA + H2O ⇌ A- + H3O+
- pH<pKa = high H+ present, these will react with A-, equilibrium pushed to
LHS, HA predominant Overall protein charge: +ve
- pH=pKa = at equilibrium, 50% ionized(A-), 50% unionized (HA)
Overall protein charge: neutral
- pH>pKA = low H+ present, equilibrium pushed to LHS, A-predominant
Overall protein charge: -ve
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Isoelectric point pI: the
pH at which the amino
acid is neutral, i.e. the
zwitterion form is
dominant.
Example: Basic side chain Histidine, BH+ ⇌ B + H+
pH - pKa = log [conjugate base]/[conjugate acid]
Conjugate base is now uncharged form, conjugate acid is charged form
We want to work out what proportion is in conjugate acid form
e.g. if pH is one unit above the pKa for a base
1= log [conjugate base]/[conjugate acid]
[B]/[BH+] = 10 i.e. for every 1 BH+ there are 10 B molecules
1/(10+1) = 0.091 of side chains are in the charged BH+ form
So the average charge on that side chain is = +0.091
B + H2O (= acid ”H+”) ⇌ BH+ +
OH-
- pH<pKa = high H+ present,
these will react with B,
equilibrium pushed to RHS, BH+
predominant Overall protein
charge: +ve
- pH=pKa = at equilibrium, 50%
ionized(BH+), 50% unionized (B)
Overall protein charge: neutral
- pH>pKA = low H+ present,
equilibrium pushed to LHS, B
predominant
Overall protein charge: neutral
The net charge on a protein is the sum of all individual charges at a given
pH
At a pH below its pI a protein will be positively charged
At a pH above its pI it will be negatively charged
More information on acidic and basic amino acids:
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Document Summary
*be familiar with ways in which amino acids are ionized. Cooh group (uncharged) ionizes to form coo-( vely charged) therefore carboxyl group can exist in an uncharged state or vely charged state. Lysine"s primary amino group can exist in pronotated form (+vely charged) and deprotonated form (uncharged) Arginine"s amino group can exist in pronotated form (+vely charged) and deprotonated form (uncharged) Conjugate acid is always protonated form, conjugate base is always the deprotonated form. For each amino acid, there is a conj. acid (protonated form) and a conj. base (deprotonated form) Ha to a- class: cys, tyr, ser, thr. Neutral in protonated form (sh, oh) and negatively charged in the deprotonated form. Ha + h2o a- + h3o+ acid + base conj. base + conj. acid. B + h2o bh+ + oh- base + acid conj. acid + conj. base. Typically an acid has a pka of 3-4, cys, tyr, ser and thr are very weak acids (high pka values)
