2 The sequence of a peptide is as follows GSEKEALYRWNKRTTGNN

2. The sequence of a peptide is as follows: GSEKEALYRWNKRTTGNNDNCGGVHV

a.

Find the pI of this peptide. Use as many pH intervals as you need to take into account the pKa values of the different ionizable groups. Transfer the table with the pH intervals and charges to the answer sheet. Based on your calculations, what is the pI for this peptide.

b. Based on the pI of the peptide, what type of ion exchange column (IEC) can be used to purify this peptide? What is the pH of the buffer of your choice? (Chose an ideal buffer based on the pKa of the buffer, a list of ideal buffers for biomolecules and their pKas are given in lecture notes). How many buffers are needed to purify the peptide over an IEC? Give the ideal composition of the buffer(s) that is/are needed for purifying this peptide using an IEC.

SHOW WORK TO HELP ME LEARN PLEASE!

Solution

GSEKEALYRWNKRTTGNNDNCGGVHV=26 amino acids.

3 acidic amino acid (EE and D).

5 basic amino acid (KRKR and H)

these amino acids contribute to the net charge on the protein at acidic and basic condition.

now your protein has net charge = 5-3 = +2 charge because it has more basic amino acids.

since it has + charge means you have to provide OH^- charge so that the net charge will be zero. means isoelectric point is in basic range.

now take the average of the pKa of two basic amino acid. that will be your pI. (10.5 of Lys +6.1 of His)/2= 8.3.

(we did not take pKa of Arg (12.5) because it will neutralize faster as compare to Lys.

b) at physiological pH (7 or 7.4) your peptide is positively charged. you can choose tris chloride buffer or phosphate buffer of pH 7 or 7.4. for IEC you choose those type of matrix which will interact with the positive charge molecules (means they have negative charge on themselves). so sulfo propyl (SP) or carboxy methyl (CM) would be a good choice.