I need help with these questions!!

Question

This particular sequence of DNA encodes the peptide N’-PRANCED-C’:



1.). If the pKa values of this fragment are 12.48, 4.07, 3.90, 2.20, 10.70 (N-

terminus), 8.37 calculate the pH where you are most likely to find a +1 charged form.

2.) Calculate the fraction of ONLY the Cysteine sidechain that is protonated at pH=7.8

3.) Devise a system to separate the N’-PRANCED-C’ peptide from N’-HARK-C’ (pKas 6.04, 10.54, 12.48, 2.1, 9.8) at pH 7. You must use at least two different methods to isolate them from one another. Please clearly state what kind of separations you will be using in each step and the fate of each molecule after each step.

Answer 1

Hi Bay, Happy to help with this.

This particular sequence of DNA encodes the peptide N’-PRANCED-C’:



1.). If the pKa values of this fragment are 12.48, 4.07, 3.90, 2.20, 10.70 (N-

terminus), 8.37 calculate the pH where you are most likely to find a +1 charged form.

So for this, we need to try and assign the pKa's to various parts of the peptide. We need to identify all of the polar charged parts of the peptide.

P = Proline this doesn't have a side chain with a pKa

R = Arginine (a positively charged amino acid) has a side chain with a pKa of 12.48

A= Alanine doesn't have a side chain with a pKa

N= Asparagine doesn't have a side chain with a pKa

C= Cysteine has a side chain with a pKa of 8.37

E= Glutamic Acid (a negatively charged amino acid) has a side chain with a pKa of 4.07

D= Aspartic Acid (the other negatively charged amino acid) has a side chain with a pKa of 3.90

The 4 amino acids with pKas match 4 of the numbers given in the problem. The other two, one is labeled as N-terminus so we know that is the N-terminus, and then the other is 2.20 which at such a low pKa that is the C terminus where the carboxylic acid is.

Now we are going to do a modified version of finding the pI. Normally you calculate pI by protonating everything, calculating the net charge, writing the pKas from low to high, then skipping over the number of pKas based on charge and then taking the average of the 2 pKas you land in the middle of. But we know we are looking for a charge of +1 so we will skip step 1, and just start with writing the pKas in order from low to high.

2.20, 3.90, 4.07, 8.37, 10.70, 12.48

now we are looking for +1 so we will move from left to right the charge. so its 1 we move from the left of 2.20 to between 2.20 and 3.90. and it is only 1 so we stop. then take the average of those 2 numbers. (2.20+3.90)/2 = 3.05

So the pH where you are most likely to find a +1 charge is pH 3.05.

2.) Calculate the fraction of ONLY the Cysteine sidechain that is protonated at pH=7.8

Ok so we need to find the fraction of protonation and we can do this with the [HA]/[HA]+[A-] equation simplified to 1/(1+10^(pH-pKa) )

so 1/(1+10^(7.8-8.37))= 1/(1+10^(-0.57))=1/(1+0.27)=0.79 so about 79% of your cysteine is protonated at pH 7.8.

3.) Devise a system to separate the N’-PRANCED-C’ peptide from N’-HARK-C’ (pKas 6.04, 10.54, 12.48, 2.1, 9.8) at pH 7. You must use at least two different methods to isolate them from one another. Please clearly state what kind of separations you will be using in each step and the fate of each molecule after each step.

Ok for this you need two different methods and there are honestly several ways you could do this, but two easy ones that I can come up with are:

1. They are different sizes. PRANCED is 7 peptides long and HARK is only 4, this should be a big enough difference to be able to separate them based on size exclusion chromatography. For this, the smaller peptides go inside the pores of the column and get slowed down so the large peptide will elute of the column first and you can collect it. Usually, it is linked to a computer or a graph machine so you can collect the fraction until the peak drops, and then when the second peak starts you collect that peak and that will be your smaller peptide HARK.
2. The second way is to separate them based on charge. At pH 7 PRANCED has a charge of -1 and HARK has a charge of +2 So you could do ion exchange either anion or cation. If you do cation exchange then your positive peptide (cation) will bind to the negative column, and the negative peptide will flow through, so you can collect the flow through and then elute the HARK off with a buffer with a high pH to release the HARK from the column. Alternatively, you can do anion exchange (a positive column) where the PRANCED will bind and HARK will flow through and then you will need to use a buffer of low pH (like 3.05 from above) to elute off the PRANCED from the column.

I hope this helps, I am happy to assist with a more in-depth tutoring session if you find yourself struggling with this further, feel free to reach out!

Happy Studying!