Structural Biochemistry/Proteins/Purification/TEST

Determining the Composition of an Amino Acid
Edman sequencing is done best if the composition of the amino acid is known. To determine the composition of the amino acid, the peptide must be hydrolyzed. This can be done by denaturing the protein to its constituents, heating it to 110 ˚C and adding 6 M HCl for 24 hours. The amino acids can then be separated by ion exchange chromatography. Each amino acid in the protein can be identified by its elution level, the amount of buffer that was added in order remove the amino acid. . A ninjydrin or fluorescamine indicator dye is also added to the column, the amount of each amino acid in the protein can be identified by comparing the color intensity levels shown by each amino acid. This way, the composition but not the sequence can be determined.

Sequencing Amino Acids
A less useful way to sequence amino acids is to label the N-terminal. This can be done is to use Dabsyl chloride, which forms a covalent bond with the amine group, and also can be detected because of its fluorescent derivatives. A strong covalent bond is needed because it needs to remain stable when the protein is being hydrolyzed. After hydrolysis, the N-terminal can be determined; however, this method is not very practical because the protein is damaged by the hydrolysis conditions. This may lead to a lost in sequencing.

To solve the problem of damaging the protein by hydrolyzing conditions, Pehr Edman created a new way of labeling and cleaving the peptide. Edman thought of a way of sequentially removing only one residue at a time, which did not damage the overall sequencing. This was done by adding Phenyl isothiocyanate, which would react with the uncharged terminal group, and create a phenylthiocarbamoyl derivative with the N-terminal. The N-terminal is then cleaved under less harsh acidic conditions, creating a cyclic compound of phenylthiohydantoin PTH-amino acid. Chromatography can then be used to identify the amino acid in the cyclic compound. This does not damage the protein and leaves at least two constituents of the peptide. This method can be repeated for the rest of the residues.

Sequencing Larger Proteins
Larger proteins (ones that contain more than 50 residues) cannot be sequenced by the Edman sequencing because the peptides will no longer release a single amino acid at each step in the reaction. The process becomes less efficient as more amino acids are removed from the chain, they become increasingly impure. A strategy called divide and conquer successfully cleaves the larger protein into smaller, practical amino acids. This is done by using a certain chemical or enzyme (cyanogens bromide or proteolytic enzymes) which can cleave the protein at specific amino acid residues. This cleaving method breaks the amino acid residues at predicted areas of the chain, so it is easier to determine its overall sequence at the end -*The separated peptides can be isolated by chromatography. Then they can be sequenced using the Edman method, because of their smaller size.

In order to put together all the sequences of the different peptides, a method of overlapping peptides is used. The strategy of divide and conquer followed by Edman sequencing is used again a second time, but using a different enzyme or chemical to cleave it into different residues. This allows two different sets of amino acid sequences of the same protein to be separated, but at different points. By comparing these two sequences and examining for any overlap between the two, the sequence can be known for the original protein. Sometimes the original protein chain is actually several different protein chains and SDS-gel electrophoresis is used to count the N-terminal amino acids to determine the number of original chains. Afterwards, urea or guanidine hydrochloride is used to denature the several polypeptide chains held together and then their individual sequences determined through the use of the aforementioned identification procedures.