Amino acid sequencing in proteins from the N terminal

The determination of amino acid sequence in proteins is known as protein sequencing. First of all, the protein should be extracted from the tissue & should separate the protein. before analyzing the amino acid sequence, should determine whether the protein contains more than one polypeptide chain. As the number of polypeptide chains is equal to the number of N terminal a.a. residues, we determine the no. of N terminal a.a. residues per molecule of protein. If the polypeptide chains have no covalent cross linkages, they can be separated by altering PH, 8M Urea/6M Guanidium HCl, high salt concentration. But if the chains are covalently cross linked by disulfide bonds, commonly they are cleaved by reducing the disulfide bond to sulfhydryl groups with mercaptoethanol. Then check the purity by using a method like polyacrylamide gel electrophoresis.

Now complete hydrolysis is done either by acid hydrolysis or by alkaline hydrolysis. In the acid hydrolysis, excess 6N HCl is added in a sealed evacuated tube at 110-120 C for 24-72h. In the alkaline hydrolysis, add 5N Ba(OH)2 under reflux for 18-24h.

Identification of N terminal amino acids -
The first useful method for this was described by Sanger. He used Fluorodinitrobenzene for this. Free unprotonated alpha amino group of peptides react with this & forms a yellow coloured derivative. Now subject to acid hydrolysis with 6N HCl. Then all the peptide bonds hydrolyses, but the bond between DNFB & alpha amino group of N terminal amino acid is stable. Then this labelled residue is separated from other amino acids by chromatography.

Another common method is Edman's method. Here, Phenyl isothiocyanate is used to cleave N terminal a.a. from peptide without disrupting other amino acids. It binds with the free amino group of a peptide to yeild the corresponding phenylthiocarbamoyl peptide. Then treat with anhydrous acid. Then the N terminal a.a. splits off as a phenylthiocarbamoyl a.a. Then it is crystallized separated & identified usually by gas liquid chromatography. The great advantage of this method is that it does not disturb the rest of the peptide chain after the removal of the N terminal a.a.