MadSci Network: Biochemistry
Query:

Re: separation of amino acids by paper chromatography

Date: Mon Feb 28 13:58:15 2000
Posted By: Chris Larson, Post-doc/Fellow Laboratory of Genetics
Area of science: Biochemistry
ID: 951026920.Bc
Message:

Amanda,

It sounds like someone else originally answered this question and you gave them more information, but I will just answer from the beginning and try to hit everything. You ask two questions, and I think the answers are related, so I will address them jointly.

From the procedure that you provided, one key thing for you to understand is that, at the end of Part A, you are not going to have individual amino acids but rather peptide fragments of albumin. Trypsin is a protease (protein- cleaving enzyme) that cuts polypeptide chains on the C-terminal side (where the carboxyl group of the amino acid in question links to the next amino acid in the chain) of either lysines or arginines only. It can't cut next to any other amino acids and thus you could not be generating individual amino acids, only peptides. Once the protein has been chopped up into peptides, the various peptides are separated by paper chromatography according to how their size and amino acid composition affect their ability to stick to the paper and resist being pulled up by the solvent butanol. Then they are visualized by looking at the colored products of the reaction of the amine group of the first amino acid in the peptide (reading the peptide N-end to C-end) with ninhydrin. The important thing to understand here is that proline is unique in that, while the main-chain amine group of all the other amino acids can, in effect, react twice with ninhydrin and form a purple cyclic compound, the main-chain amine group of proline can only react once and forms an acyclic, yellow compound. MO< So, a simple explanation for both of your observations (the Rf value of proline being wrong and the number of peptides being wrong) would be incomplete digestion by trypsin. If the trypsin didn't cut at every lysine and arginine, then several of the peptides that you are seeing should normally be cut into two peptides but aren't, giving you 9 peptides rather than 15. Also, that must include the one that contains the proline at the N-terminus, and the reason it runs at Rf=0.37 rather than Rf=0.43 is that it is bigger than it should be (since there must be a lysine or arginine in it where the trypsin would have cleaved it into two peptides but didn't) and thus runs more slowly relative to the solvent front (hence giving you the smaller Rf value, which is mobility of the spot over mobility of the solvent front).

An easy way for you to test this explanation would be to do the trypsin digestion for various times (for example, 6,12,24,48, and 72 hours, starting them all at the same time and stopping each one at the appropriate time by putting them in the freezer until the final one is done) and then doing the chromatography and ninhydrin test side-by-side and seeing that, with longer reaction, the number of peptides increases and that the mobility of the one yellow spot goes up.

Good luck, and feel free to email me directly with more specific questions.

Chris


Current Queue | Current Queue for Biochemistry | Biochemistry archives

Try the links in the MadSci Library for more information on Biochemistry.



MadSci Home | Information | Search | Random Knowledge Generator | MadSci Archives | Mad Library | MAD Labs | MAD FAQs | Ask a ? | Join Us! | Help Support MadSci


MadSci Network, webadmin@www.madsci.org
© 1995-2000. All rights reserved.