Chapter 3 Amino Acids Peptides and Proteins Amino

Chapter 3 Amino Acids, Peptides, and Proteins

Amino Acids

Amino Acids n 20 Residues n Numbering of carbons § 1, 2, 3. . . from C of COO§ a , b, g… from C bonded to NH 3+ and COO- n Absolute configuration of amino acids § L amino acids in biological system 1 2 a

Classification of Amino Acids Nonpolar UV absorption at 280 nm

Classification of Amino Acids • Nonpolar • Structural role

Classification of Amino Acids

Uncommon Amino Acids n Collagen n Myosin n Cell wall n Collagen n Prothrombin n Ca 2+ binding proteins n Elastin n A few proteins n Incorporation during translation

Amino Acids as Acids and Bases n Zwitterion § Acts as either an acid or a base § Ampholytes § Amphoteric substances § Substances with dual nature n Amino acid is a diprotic acid

Titration of Amino Acids n Two p. Ka and two buffering regions n p. I: ioselectric point or isoelectric p. H § The point with zero electric charge § Above p. I : negative charge § Below p. I : positive charge n p. I = (p. K 1 + p. K 2)/2 = 5. 92

Effect of Chemical Environment on p. Ka

Amino Acids with Ionizable R Group n p. I = (p. K 1 + p. KR)/2 = 3. 22 n p. I = (p. KR+ p. K 2)/2 = 7. 59

Peptides and Proteins

Peptides and Proteins n Peptide bond § Dehydration reaction n Polypeptide vs. protein § Polypeptide: Mr<10, 000 n Amino-terminal (N-terminal) n Carboxyl-terminal (C-terminal)

Ionization of Peptide n Ionization of peptide § One free a-amino group § One free a-carboxyl group § Inonizable R groups n p. Ka of R groups in peptide § Different from p. Ka of free amino acid

Biologically Active Peptides and Polypeptides n Size § Small peptide § Vertebrate hormones § § Oxytocin (9), thyrotropin-releasing factor (3), insulin (30 + 21) Antibiotics § Large proteins § Most of the proteins § Titin: vertebrate muscle protein (27, 000 a. a. ) § < 2, 000 a. a. n Oligomeric status § Single polypeptide chain § Multisubunit proteins § Oligomeric : at least two subunits are identical § Protomers : identical units n Calculation of the number of amino acid residues § Mr / 110 § Average Mr of 20 a. a. : 138 § Average Mr of protein a. a : 128 § Removal of water during peptide bond formation : 128 -18 =110

Conjugated Proteins n Conjugated proteins § Proteins with permanently associated chemical components § Prosthetic group § Non-amino acid part of a conjugated protein

Working with Proteins

Protein Purification n Cell lysis § Crude extract n Fractionation § Use differences in protein solubility § Depending on p. H, temperature, § salt concentration etc. Salting out § Addition of ammonium sulfate ((NH 4)2 SO 4) for differential precipitation of proteins n Dialysis § Exchange of salts and buffer using semipermeable membrane n Column chromatography § Separation of proteins based on charge, size, binding affinity etc. § Stationary phase and mobile phase containing protein

Ion-exchange chromatography n Cation-exchange chromatography § Solid matrix : negatively charged group § Positive charged proteins migrate slowly n Anion-exchange chromatography § Solid matrix : positively charged group

Size-Exclusion Chromatography n Solid matrix : Beads with engineered pores of cavities n Small proteins enter the pores § Slow migration

Affinity Chromatography n Beads with covalently attached chemical group § Binding of proteins with affinity for the chemical group

Protein Purification n HPLC (high-performance liquid chromatography) § Use high pressure pump that speed the movement of the protein molecules § Limited diffusion High resolution n Determining the methods for protein purification § Mostly empirical

Separation of Protein by Electrophoresis n Electrophoresis § Separation of charged proteins in an electric field § Electrophoretic mobility of proteins § Depending on size and shape of proteins n SDS gel electrophoresis (SDS PAGE) § SDS (sodium dodecyl sulfate) binds to proteins roughly proportional to the molecular weight of the protein § Binding of 1 SDS/ 2 amino acids § § § Similar chare to mass ratio for all the proteins § Similar shape for all the proteins Separation of proteins depending on the mass § Useful to determine molecular weight Visualization of the bands by staining (e. g. Goomassie blue)

Determining Molecular Weight of a Protein SDS PAGE (polyacrylamide gel electrophoresis)

Isoelectric Focusing n Procedure to determine the p. I of a protein § Establishment of p. H gradient § Gel containing a mixture of low molecular weight organic acids and bases (ampholytes) § Application of electric field § Each protein migrates until it reaches the p. H corresponding to its p. I

Two-Dimensional Electrophoresis § 1 o : Isoelectric focusing § 2 o : SDS-PAGE

Quantification of Proteins During the Purification n Enzyme assay § 1 unit of enzyme activity § The amount of enzymeo causing transformation of 1. 0 mmol of substrate/min at 25 C under optimal conditions § Activity § Total units of enzyme in solution § Decrease during purification § Specific activity § The number of enzyme units/ mg of total proteins § Measure of enzyme purity § Increase during purification n Other assays § Biological activities § DNA binding, alteration of cell growth § Detection of the presence of the target protein § Western blotting