chemistry 501 handout 3 amino acids, peptides, and proteins chapter 3 dep. of chemistry &...
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Amino acids can be classified by R groupsTRANSCRIPT
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)