Peptides - Biochemistry - Lecture Slides, Slides of Biochemistry

Download Peptides - Biochemistry - Lecture Slides and more Slides Biochemistry in PDF only on Docsity! Peptides 1. Little Proteins A. Dipeptides, tripeptides, …polypeptides 2. Basic Structure O C N H3N C C C O H H H O + C-terminal N-terminal Glycylalanine H CH3 rotation rotation Peptide bond Docsity.com O N C H C H3N H + H C C O H O CH3 O N C H C H3N H + H C C O H O CH3 y f Primary Structure Docsity.com Rotation Around the Bonds in a Polypeptide Chain Amide plane Amide plane Side group - Docsity.com Conformation Around the Bonds at d=0 yw=0 Interaction \ / Conformation is NOT Allowable Docsity.com Steric Interference in Peptide Groups van der Waals distance Docsity.com H3N- -COO - S-S K R M c c R S-S K SO3 - SO3 - Trypsin Performic acid K M Homoserine lactone CNBr 1 2 3 4 Preparing Peptides for Sequence Analysis Docsity.com A. Absorption of light at 280 nm. If the peptide in question absorbs uv light, this is an indication of aromatic amino acids. Look for residues of phenylalanine, tyrosine, or tryptophan. Failure to absorb uv light would indicate the absence of these residues. B. Cleavage by Trypsin. Sensitivity to trypsin indicates the present of a basic amino acid (lysine or arginine) at the cleavage site. Trypsin cleaves the bond and leaves the basic amino acid as the C-terminus of the split product. C. Cleavage by Chymotrypsin. The same as trypsin, only with an aromatic amino acid residue is at the cleavage site. D. Cleavage by Carboxypeptidase. The residue released by carboxypeptidase is the C- terminal residue of the peptide. This exopeptidase cleaves from the C-terminus one amino acid residue at a time. The enzyme is used to identify residues at or near the C-terminus of the peptide. E. Cleavage by Aminopeptidase. The residue released by aminopeptidase is the N- terminal residue of the peptide. Like carboxypeptidase, only working from the other end of the peptide. F. Cleavage by CNBr. CNBr (cyanogen bromide) cleaves at methionine residues. CNBr is one of the few chemical reagents that cleave at a specific location in the peptide. See p 42-43 in Strategies Docsity.com A. Cleavage by Sulfhydryl Reagents. This indicates that the peptide contains a disulfide bond. Reagents such as -mercaptoethanol, dithiothreitol, and glutathione split the bond by reducing action. A disulfide bond is created by an interaction between two cysteine –SH (sulfhydryl) groups. A disulfide bond may not be broken by hydrolysis. Reagents such as performic acid can, however, oxidize it. B. Residue split does not rotate light. This indicates that the glycine (the only amino acid lacking a chiral center) was released in the procedure. C. Residue split is an acidic amino acid. The residue is either aspartic acid or glutamic acid. D. Residue split is a basic amino acid. The residue is either lysine or arginine. E. Residue split has a group with a pK = 6. The residue is histidine F. Peptide releases NH3 when hydrolyzed by 6 N HCl. The amino acids asparagine or glutamine are in the structure. Both contain an amide group that releases NH3 in response to strong acid hydrolysis. G. Peptide requires more than 2 equivalents of OH- to titrate. One or more residues with charged R groups are present. If no charged R groups were present, only two equivalent of base (one for the NH3 and one for –COOH) would be required. H. Peptide does not react with N-terminal reagents. Peptide is either cyclic or has a blocked N-terminus. Docsity.com A peptide fragment with a Mwt of about 300, upon treatment with 6 N HCl gave 2 amino acids. One did not rotate light. The N- and C- terminal residue had a pKa of 6. What is the structure? CLUE 1: 300 Mwt means 3 residues CLUE 2 pKa 6 means Histidine CLUE 3 Not able to rotate light means Glycine ANSWER: His-Gly-His Docsity.com A tripeptide did not react with chymotrypsin. It did react with trypsin, liberating a dipeptide. The peptide required 4 equivalents of base to titrate all protons It absorbed light a 280 nm. One residue was valine, and one had a pKa around 12. 1 Chymotrypsin cleaves on -COO side of aromatic groups 2 Trypsin cleaves on -COO side of lysine and arginine 3 4 equivalents means 2 charged amino acids 4 280 nm absorption means aromatic amino acid pKa of 12 identifies arginine ANSWER: Arg-Val-Tyr 5 Docsity.com