Question

1) Estimate the isoelectric point (pI) for the dipeptide Ser-Ile.

Round the answer to one decimal place.

2) Estimate the isoelectric point (pI) for the tripeptide Gly-Lys-Val.

Round the answer to one decimal place.

3)A mixture of Cys, Glu, Tyr, Lys and Ser was applied to a cation exchange column at pH 6.

Which of these amino acids eluted from the column first?   

Which of these amino acids eluted from the column last?   

Tyr Glu Lys Ser Cys

Answer 1

2

Lets calculate the charge on the compound at pH = 0

Step 1:

If pH < pKa, H+ is on. It means that the group is protonated

If it happens to be for -NH2 group, it will have +1 charge.

For -COOH group in this case, there will be no charge

If pH > pKa, H+ is off. It means that the group is de protonated

If it happens to be for -COOH group, it will have -1 charge.

For -NH2 group in this case, there will be no charge

Step 2: find individual amino acids

Gly-Lys-Val is composed of Gly, Lys, Val amino acids

Step 3: find pka's of all group in each amino acids from internet or textbook

For Glycine:

pKa of acidic part, pKa1 = 2.34

pKa of basic part, pKa2 = 9.6

It has neutral side chain

For Lysine:

pKa of acidic part, pKa1 = 2.18

pKa of basic part, pKa2 = 8.95

pKa of side chain, pKa3 = 10.53

It has basic side chain

For Valine:

pKa of acidic part, pKa1 = 2.32

pKa of basic part, pKa2 = 9.62

It has neutral side chain

Step 4: find charge on individual amino acid

Lets calculate charge on Gly

acidic part is involved in forming bond with next amine group

pH is less than pKa2. So, basic part will have H+ on it

So, positive charge is present because of it

Hence, net charge on the Gly is +1

Lets calculate charge on Lys

acidic part is involved in forming bond with next amine group

basic part is involved in forming bond with previous amine group

pH is less than pKa3 and since it is basic, it will have H+ on it

So, positive charge is present because of it

Hence, net charge on the Lys is +1

Lets calculate charge on Val

pH is less than pKa1. So, acidic part will have its H+ on

So, no charge because of it

basic part is involved in forming bond with previous amine group

Hence, net charge on the Val is 0

Step 5: find charge on molecule

Hence, net charge on the molecule is +2

This is the charge on the compound at pH = 0

pKa values part of compound and that have not formed bonds are to be arranged in ascending order

2.32 of Val

9.6 of Gly

10.53 of Lys

With increasing pka, charge on the molecule will decrease by 1

We need to find the 2 pKa values between which charge is 0

After pH > 2.32 charge on molecule becomes 1

After pH > 9.6 charge on molecule becomes 0

After pH > 10.53 charge on molecule becomes -1

Charge on molecule is 0 between pH = 9.6 and pH = 10.53

So, pI = average of this 2 pH values

So, pI = (9.6 + 10.53)/ 2

pI = 10.1