Amino Acid 3 Flashcards
Terminal a-carboxyl group typical pKa
CARBOXYL
3.1
Aspartic and Glutamic acid pKa
4.1
Histidine pKa
6.0
Terminal a-amino group kPa
8
Cysteine kPa
8.3
tyrosine kPa
10.9
lysine kPa
10.8
Arganine kPa
12.5
pH, pKa and Charge On Amino Acid Side Chains
- Hendersen - Hasselbalch equation
Isoelectric Point
The pI - isoelectric point is the pH at which the molecule
carries NO NET ELECTRIC CHARGE.
For amino acids the pI is:
pI = 1/2(pKai + pKaj)
Isoelectric Point
* Kai and Kaj are the pKas of the two ionizations
For glycine:
which only has an amine and carboxylic acid group pKai
and pKaj represent pKa1 and
pKa2 in the titration curve.
For aspartic and glutamic acids
s pKai and pKaj are pKa1 and pKaR
(pKaR represents the dissociation constant of the ionizable group
on the amino acid side chain).
For arginine, histidine and lysine
e pKai and pKaj are pKaR and pKa2
Buffers
T he presence of a buffer (a weak acid/base) in a solution results in a smaller change in
pH near the pKa of the buffer on the addition of a strong acid or a strong base compared to when the acid or base is added to water.
A buffer acts to reduce pH changes (in the pH range of its pK) due to increasing
or decreasing [H+] ([H3O+]), hence around the pKas of histidine in the above titration curve, the rate of increase in pH as OH- is added is less than at other points in the titration.
Histidine as a buffer
Free His has three weak
acid/base groups:
α-carboxyl (pKa= 1.82);
side chain (pKa = 6.0)
α-amino (pKa = 9.17)
Hence a solution of
histidine will buffer at 3
pH ranges corresponding
to the ranges around the
pKas
Buffering
- If OH- were added in the absence of histidine it would directly react with
protons in the solution (H3O+, H+) to form H2O, thus directly reducing the [H+] ([H3O+]) in proportion to the added [OH-
] and hence increasing pH (pH = -log10[H+]) - When OH- is added in the presence of His over the pH range of about 5.1-7.1,
we can consider that most of the OH- reacts with protons from the protonated
imidazole group of the side chains of His (ImH+). This results in no change in
[H+] ([H3O+]) but consumes most of the OH-
, leaving less to react with H+ (H3O+)
and thus reducing the change in [H+] ([H3O+]) and thus the increase in pH. - Once all of the ImH+ side chains of His have been consumed, it no longer acts as
a buffer. (Note from titration it takes about 1 equivalent of OH- to cover the
buffering range of the ionisable groups).
Titration of a Buffer
The higher the concentration of a buffer the more OH- ( or H+ ) is required to change the pH in the range of the buffer.
Why is Charge on Proteins Important?
- Charges on proteins help bind to other proteins or molecules e.g.
- Different charges on proteins can be used to separate and purify
them by electrophoresis - Different charges on proteins can be used to separate and purify them by ion exchange chromatography
cation exchange chromatography
- Charges on proteins help bind to other proteins or molecules e.g.
- Blue areas on surface of DNA-binding protein
indicate areas of positive charge due to basic
amino acids. - DNA is a negatively charged molecule due to
many phosphate groups. - Positive charged areas on protein helps bind
DNA
Electrophoresis Separation of Amino Acids
- The differences in the acid base properties of amino acids allows them to be separated on the basis of their pI values.
- Apply a mixture of amino acids to a pieces of filter paper or to a gel.
- Place the gel or paper in a buffered solution between two electrodes and apply and electric field.
- The amino acids with pI greater than the pH of the solution will have an overall positive
charge and will migrate to the cathode (the negative electrode). - The further the amino acid’s pI is from the pH of the buffered solution, the more positive the amino acid will be and the farther it will migrate towards the cathode.
- The amino acids with pI less than the pH
of the solution will have an overall negative charge and will migrate to the
anode (the positive electrode). - If two molecules have the same charge,
the larger one will move more slowly
during electrophoresis because the same
charge has to move a greater mass.
Different charges on proteins can be used to separate and purify
them by electrophoresis
- charged proteins move in an electric field
- positively charged protein move towards the
cathode - negatively charged proteins move towards the
anode
Why is Charge on Proteins Important?
- Different charges on proteins can be used to separate and purify
them by ion exchange chromatography
cation exchange chromatography
Proteins As Buffers
Example: In skeletal muscle, anaerobic exercise leads to a build up of lactic acid and acidification (lowering of pH) of the muscle which reduces its ability to contract.
This is offset by histidine side chains in the muscle proteins which buffers this this build up of acid and allows the muscle to
operate longer in anaerobic exercise
