The first step is to expose the purified sample of protein with HCl 6m for 16 to 72 hours. This will break down the peptide bonds into separate amino acids. Then you expose the amino acids to PITC or Edman’s reagent. This will form a bond with the separate amino acids PTC-aa. Then the PTC-aa residues are subjected to HPLC (high pressure liquid chromatography), where residues are subjected to an organic solvent and then forced in column chromatography under high pressure. The
eluant is then measured for absorbance at 254 nm. The absorbance is plotted as a function of time. Each peak corresponds to a specific amino acid, while the amount corresponds to the integral function of the area underneath the peak.
In order to perform Edman degration and n-terminal sequencing… you follow the same procedures as above except that you don’t treat the sample with HCl because that will cleave the peptide chain. Treat the purified peptides with PITC… Once this occurs, you get PTC-peptide. You then treat the PTC-peptide with f3ccooh or (tfc). This will separate the PTC-peptide into anilothiazolinine and a peptide at n-1. Repeat the procedure for the n-1 peptide now. You then treat the anilinothiazolinine compound with an aqueous acid, which forms phenylthiahydantoin. This phenylthiahydantoin is then treated similarly to by putting it through high performanc eliquid chromatography. In hplc, you put the compound of interest into an organic solvent and then force the mixture into column chromatorpaghy under super high pressure… anyways, you take the eluant and test its absorbance under 254 nm and plot it as a function of time.
Edman’s degradation or n-terminal sequencing is effective but to only 30 aa residues maximum. That’s why you got you use prteins like cyanogen bromide and hydroxaline as chemical bond breakers or trypsin, thrombin, chymotryprsin, and carboxypeptidase c to make the peptide that was purified into smaller pieces. Recall that cynogen bromide cleaves carboxyl met, hydroxylamine cleaves bonds between asn(n) and glycine(g)… trypsin cleaves carboxyl ends of lysine and arginine, thrombin: arginine, chymotrypsin (w, f, y, l, m) and carboxypeptidase, the carboxyl end.
Also a note, iodoacetate is used in Edman's reagent for n-terminal sequencing.
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