Marmara Üniversitesi Fen-Edebiyat Fakültesi Kimya Bölümü / Biyokimya Anabilim Dalı
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1 EXPERIMENT IX Marmara Üniversitesi DETERMINATION OF N-TERMINAL AMINO ACID RESIDUE OF PROTEINS BY THIN LAYER CHROMATOGRAPHY Functions of the proteins depend upon its amino acid sequence. Because amino acid sequence determine chemical properties and three dimensional structures of proteins. Therefore determination of amino acid sequence of a peptide is important. A sample analysis by Edman degradation are given below. Biyokimya Laboratuvarı I Güz 0
2 General steps of a sequence determination analysis could be given as: 1- Separation of the subunits of the proteins and purification. 2- Reducing of inter- and intra- disulfide bonds with 2-mercaptoethanol and dithiyotreithol (DTT) and acetylation of SH bonds with iodoacetamide. 3- Protein hydrolyzed with 6N HCl at boiling temperature and total amino acid composition determined. 4- Carboxyl and amino terminal amino acids determined. 5- Peptide is degraded into smaller pieces using certain protease enzymes. 6- Sequence of each peptide fragment analyzed using Edman degradation. 7- All gathered information is used to determine polypeptide sequence. Biyokimya Laboratuvarı I Güz 1
3 Proteases are enzymes that break down peptide bonds before or after specific amino acids. Protease or Chemical Agent * Trypsin Chymotrypsin Pepsin Thermolysin Submaxillarus protease Endoproteinase Lys C CyanogenBromide * Carboxypeptidase A Peptide Bond Breakage Lysine, Arginine (C) Phenylalanine, Tryptophan, Tyrosine (C) Phenylalanine, Tryptophan, Tyrosine (N) Phenylalanine, Tryptophan, Tyrosine (C) Arginine (C) Lysine (C) Methionine (C) C-Terminal amino acid (N) (C): Break from C-terminal. (N): Break from N-terminal. Biyokimya Laboratuvarı I Güz 2
4 Protein N-terminal analysis is done by Sanger method. However after the process peptide completely turns its amino acids therefore it is not usable in sequence analysis. The Edman degradation procedure labels and removes only the amino-terminal residue from a peptide, leaving all other peptide bonds intact. The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions, which converts the aminoterminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to the PTC adduct is then cleaved in a step carried out in anhydrous trifluoroacetic acid, with removal of the amino-terminal amino acid as an anilinothiazolinone derivative. The derivatized amino acid is extracted with organic solvents, converted to the more stable phenylthiohydantoin derivative by treatment with aqueous acid, and then identified. Biyokimya Laboratuvarı I Güz 3
5 Novadays, proteins that purified by electrophoresis can be sequenced using MALDI-TOF and protein databases. But in order to analyse proteins by this methodology, interested protein should be in the database. 3-dimensional structure of a purified and sequenced polypeptide is done by x-ray crystallography and NMR analysis. CHEMICALS Aspartic acid, leucin, valin, arginine, proline, cysteine, cystin, glycine, Na 2 CO 3, 2,4- dinitrofluorobenzene, HCl, chloroform, benzyl alcohol, and glaciel acetic acid. Method 20 mg of amino acid dissolved in10 ml 5% Na 2 CO 3 and 0,5 ml of 2,4- dinitroflurobenzene (DNFB) is added to this solution. Since DNFB is an allergen gloves should be used at all times. Mixture incubated at 40 C for 30 minutes under constant shaking. Temperature should not be higher than 40 C. Unreacted DNFB is removed using a Pasteur pipette and 1.5 ml of concentrated HCl added to the solution. DNFB derivatives of the amino acids precipitate at this stage. If precipitation does not occur, solution cooled in an ice bath. Precipitate is taken in a test tube and solved in a small amount of acetone. Mobile phase: (Chloroform: Benzyl alcohol: Glaciel acetic acid) ; (45:20:1) Using a pencil, a line is drawn on the TLC (thin layer chromatography) sheet at 3 cm from the lower edge. DBFB derivatives are applied on the line parallel to the lower edge of the plate with an interval of 2cm. After TLC sheet completely dried it is placed in the chromatography chamber. After TLC sheet developed, it is removed from the chamber, solvent front is marked, dried infume hood and Rf values determined for each amino acid and itsdnfb derivatives. Question 1- How three dimensional strucures of proteins determined? Biyokimya Laboratuvarı I Güz 4
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