Chemistry 501 Handout 3

Amino Acids, Peptides, and ProteinsChapter 3

Dep. of Chemistry & BiochemistryProf. Indig

Lehninger. Principles of Biochemistry.by Nelson and Cox, 5th Edition; W.H. Freeman and Company

Amino Acids

Steric relationship of the stereoisomers of Alanine to the absolute configuration of L- and D-glyceraldehyde

The amino acid residues in proteins are the L isomers

Amino acids can be classified by R groups

Reversible formation of disulfide bond by the oxidation of two molecules of cysteine

e.g. two polypeptide chains of insuline

indole ring

Absorption of ultraviolet light by aromatic amino acids

Lambert-Beer Law

log (Io/I) = C L

guanidino

imidazole

Uncommon amino acids also have important functions

Residues created by modification of common residues already incorporated into a polypeptide

12

34

5

plant cell wall,collagen

collagen

myosin

prothrombim, a # of Ca+ binding proteins

elastin

Lysine residues

~ 300 additional amino acidshave been found in cells

rare, introduced during proteinsynthesis rather than created

through a postsynthetic modification

Reversible amino acid modifications involved in regulation of protein activity

Titration of glycine

Amino acids can act as acids and bases

Nonionic and zwitterionic forms of amino acids

amphoteric

(ampholytes - amphoteric electrolytes)

Titration curves predict the electric charge of amino acids

Isoelectric point (or isoelectric pH)

pI = ½ (pk1 + pk2) = ½ (2.34 + 9.60) = 5.97

Effect of chemical environment on pKa

Amino acids differ in their acid-base properties

three stages (three ionization steps three pka values)

Amino acids with ionizable R groups

Amino acids with R groups that do not ionize

pka of the –COOH group: 1.8 – 2.4

pka of the –NH3+ group: 8.8 – 11.0

e.g.

Peptides are chains of amino acids

Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond,

to yield a dipeptide

Serylglyciltyrosylalanylleucineor

Ser-Gly-Tyr-Ala-Leuor

SGYAL

Pentapeptide

Peptides are named beginning with the amino-terminalresidue, which by convention is placed at the left.

condensationhydrolysis

just a few residues oligopeptidemany residues polypeptide

The 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 sizes

Multisubunit proteins: have two or more polypeptide chains associated noncovalently

If 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 compositions

The non-amino acid part of the conjugated protein is usually called its prosthetic group.

Protein Separation and Purification

ColumnChromatography

Crude extract --> --> --> fractionation

Ion-Exchange Chromatography

Example: Cation-exchange chromatography

Exclusion Chromatography

(gel filtration)

Affinity Chromatography

1.0 unit of enzyme activity = amount of enzyme causing the transformation of 1.0 mol of 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 the

enzyme is pure.

Electrophoresis

Cross-linked polymerpolyacrylamide

acts as a molecular sieve, slowing the migrationof proteins approximately in proportion to theircharge-to-mass ratio.

SDS-polyacrylamide gel

SDSCH3(CH2)11SO4

-Na+

Purification of RNA polymerize from E. coli

gel stained with a protein-specific dye (e.g. coomasie blue)

Isoelectric focusing Two-dimensional electrophoresis

There are several levels of protein structure

Includes disulfide bonds

Particularly stable arrangements of amino acid residues giving rise to

recurring structural paterns

All aspects of the 3-D folding of a polypeptide

Multisubunit proteinsArrangement is space of

polypeptide subunits

Determination of amino acid sequence

Amino acid sequence of bovine insulin

(10 years of work by Sanger)

The amino acid sequences of millions of proteins have been

determined

Identical in human, pig, rabbit and sperm whale

Identical in cow, dog, goat and horse

Short polypeptides are sequenced using automated procedures

Sanger’s method for identifying the amino-terminal

residue

The Edman degradation procedure (carried out

on a sequenator)

reveals the entire sequenceof a peptide

Large proteins must be sequenced in smaller fragments

Breaking disulfide bonds Cleaving the polypeptide chain

Some proteases cleave only the peptide bondadjacent to particular amino acid residues

Ordering the peptide

fragments

Met (C)

Amino acid sequences can also be deduced by other methods

Correspondence of DNA and amino acid sequences

codon

Investigating proteins with mass spectrometry

Small peptides and proteins can

be chemically synthesized

(9-fuorenylmethoxycarbonyl)