Amino Acid 3

Question 1

Terminal a-carboxyl group typical pKa

CARBOXYL

Answer 1

3.1

Question 2

Aspartic and Glutamic acid pKa

Answer 2

4.1

Question 3

Histidine pKa

Answer 3

6.0

Question 4

Terminal a-amino group kPa

Answer 4

8

Question 5

Cysteine kPa

Answer 5

8.3

Question 6

tyrosine kPa

Answer 6

10.9

Question 7

lysine kPa

Answer 7

10.8

Question 8

Arganine kPa

Answer 8

12.5

Question 9

pH, pKa and Charge On Amino Acid Side Chains

Answer 9

1. Hendersen - Hasselbalch equation

Question 10

Isoelectric Point

Answer 10

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

Question 11

For glycine:

Answer 11

which only has an amine and carboxylic acid group pKai

and pKaj represent pKa1 and

pKa2 in the titration curve.

Question 12

For aspartic and glutamic acids

Answer 12

s pKai and pKaj are pKa1 and pKaR

(pKaR represents the dissociation constant of the ionizable group
on the amino acid side chain).

Question 13

For arginine, histidine and lysine

Answer 13

e pKai and pKaj are pKaR and pKa2

Question 13

Buffers

Answer 13

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.

Question 14

Histidine as a buffer

Answer 14

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

Question 15

Buffering

Answer 15

• 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).

Question 16

Titration of a Buffer

Answer 16

The higher the concentration of a buffer the more OH- ( or H+ ) is required to change the pH in the range of the buffer.

Question 17

Why is Charge on Proteins Important?

Answer 17

1. Charges on proteins help bind to other proteins or molecules e.g.
2. Different charges on proteins can be used to separate and purify
   them by electrophoresis
3. Different charges on proteins can be used to separate and purify them by ion exchange chromatography

cation exchange chromatography

Question 18

1. Charges on proteins help bind to other proteins or molecules e.g.

Answer 18

• 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

Question 19

Electrophoresis Separation of Amino Acids

Answer 19

• 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.

Question 19

Different charges on proteins can be used to separate and purify
them by electrophoresis

Answer 19

• charged proteins move in an electric field
• positively charged protein move towards the
  cathode
• negatively charged proteins move towards the
  anode

Question 20

Why is Charge on Proteins Important?

Answer 20

3. Different charges on proteins can be used to separate and purify
   them by ion exchange chromatography
   cation exchange chromatography

Question 21

Proteins As Buffers

Answer 21

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