Chemistry 501 Handout 3Amino Acids, Peptides, and ProteinsChapter 3Dep. of Chemistry & BiochemistryProf. IndigLehninger. Principles of Biochemistry.by Nelson and Cox, 5thEdition; W.H. Freeman and CompanyAmino AcidsSteric relationship of the stereoisomersof Alanine to the absolute configuration of L-and D-glyceraldehydeThe amino acid residues in proteins are the L isomersAmino acids can be classified by R groupsReversible formation of disulfide bond by the oxidation of two molecules of cysteinee.g. two polypeptide chains of insulineindoleringAbsorption of ultraviolet light by aromatic amino acidsLambert-Beer Lawlog (Io/I) = ε C LguanidinoimidazoleUncommon amino acids also have important functionsResidues created by modification of common residues already incorporated into a polypeptide12345αβγplant cell wall,collagencollagenmyosinprothrombim, a # of Ca+binding proteinselastinLysine residues~ 300 additional amino acidshave been found in cellsrare, introduced during proteinsynthesis rather than createdthrough a postsynthetic modificationReversible amino acid modifications involved in regulation of protein activityTitration of glycineAmino acids can act as acids and basesNonionic and zwitterionic forms of amino acidsamphoteric(ampholytes - amphoteric electrolytes)Titration curves predict the electric charge of amino acidsIsoelectric point (or isoelectric pH)pI = ½ (pk1+ pk2) = ½ (2.34 + 9.60) = 5.97Effect of chemical environment on pKaAmino acids differ in their acid-base propertiesthree stages (three ionization steps → three pkavalues)Amino acids with ionizable R groupsAmino acids with R groups that do not ionizepkaof the –COOH group: 1.8 – 2.4pkaof the –NH3+group: 8.8 – 11.0e.g.Peptides are chains of amino acidsTwo amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptideSerylglyciltyrosylalanylleucineorSer-Gly-Tyr-Ala-LeuorSGYALPentapeptidePeptides are named beginning with the amino-terminalresidue, which by convention is placed at the left.condensationhydrolysisjust a few residues → oligopeptidemany residues → polypeptideThe vast majority of naturally occurring proteins contain fewer than 2,000 amino acid residues.Biologically active peptides and polypeptides occur in a vast range of sizesMultisubunit proteins: have two or more polypeptide chains associated noncovalentlyIf at least two chains are identical → the protein is said to be oligomeric, and the identical units (consisting of one or more chains) are referred to as protomers.Some proteins contain chemical groups other than amino acids (conjugated proteins).Polypeptides have characteristic amino acid compositionsThe non-amino acid part of the conjugated protein is usually called its prosthetic group.Protein Separation and PurificationColumnChromatographyCrude extract --> --> --> fractionationIon-Exchange ChromatographyExample:Cation-exchange chromatographyExclusion Chromatography(gel filtration)AffinityChromatography1.0 unit of enzyme activity = amount of enzyme causing the transformation of 1.0 μmolof substrate per minute at 25oC under optimal conditions of measurement.Refers to the total number of units of enzyme in a solution.Number of enzyme units per milligram of total protein.A measure of enzyme purity: it increases during purificationof an enzyme and becomes maximal and constant when theenzyme is pure.ElectrophoresisCross-linked polymerpolyacrylamideacts as a molecular sieve, slowing the migrationof proteins approximately in proportion to theircharge-to-mass ratio.SDS-polyacrylamide gelSDSCH3(CH2)11SO4-Na+Purification of RNA polymerize from E. coligel stained with a protein-specific dye (e.g. coomasie blue)Isoelectric focusing Two-dimensional electrophoresisThere are several levels of protein structureIncludes disulfide bondsParticularly stable arrangements of amino acid residues giving rise to recurring structural paternsAll aspects of the 3-D folding of a polypeptideMultisubunit proteinsArrangement is space of polypeptide subunitsDetermination of amino acid sequenceAmino acid sequence of bovine insulin(10 years of work by Sanger)The amino acid sequences of millions of proteins have been determinedIdentical in human, pig, rabbit and sperm whaleIdentical in cow, dog, goat and horseShort polypeptides are sequenced using automated proceduresSanger’s method for identifying the amino-terminal residueThe Edman degradation procedure (carried out on a sequenator)reveals the entire sequenceof a peptideLarge proteins must be sequenced in smaller fragmentsBreaking disulfide bonds Cleaving the polypeptide chainSome proteases cleave only the peptide bondadjacent to particular amino acid residuesOrdering the peptide fragmentsMet (C)Amino acid sequences can also be deduced by other methodsCorrespondence of DNA and amino acid sequencescodonInvestigating proteins with mass spectrometrySmall peptides and proteins can be chemically