Chapter 4

Amino Acids
Instructor: Rashid Syed

Textbook: Biochemistry (4th Edition ) by Donald Voet Judith G. Voet

Amino Acids, Peptides and Proteins

Structure of 20 common amino acids

General structure of peptides and proteins
Ionization behavior of amino acids and peptides
Methods to characterize peptides and proteins

Amino Acids
Amino acids are building blocks for proteins
They have a central -carbon and -amino and carboxyl groups
20 different common amino acids
Same core structure, but different side group (R)
The -C is chiral (except glycine); natural proteins
contain only L-isoforms.
Amino acids are ampholytes, pKa of -COOH is ~2 and
of -NH2 is ~ 9
At physiological pH most aa occur as zwitterions.

Ampholytes
A molecule containing ionizing groups with both
acidic and basic pKa values is called an ampholyte.
The ionic form of each group in the compound is
dependent on the pH of the solution.
If the pH of solution is greater than the pKa, the
group is in the conjugate base form (deprotonated).
If the pH of solution is less than the pKa, the group
is in the conjugate acid form (protonated).

General Structure of Amino Acids

Two numbering systems (1,2 and ,,.)
COOH group is written at top, chiral carbon is in the center,
R-group is at the bottom
Absolute configuration:
L-aa : NH2 is on the left of the chiral C
D-aa : NH2 is on the right of the chiral C

Classification of Amino Acids

Aliphatic / non-polar R group: glycine, alanine,
valine, leucine, isoleucine, methionine, proline
Aromatic R group: phenylalanine, tyrosine,
tryptophan
Polar R group (net charge 0 at pH 7.0) uncharged:
serine, threonine, cysteine, asparagine, glutamine
Positively Charged R group (+ charge at pH 7.0):
lysine, arginine, histidine
Negatively Charged R group ( - charge at pH 7.0):
aspartate, glutamate

Amino Acid R-group Classification

Aliphatic: gly (G), ala (A) , val (V), leu (L), ile (I)
Aromatic: Trp (W), Phe (F), Tyr (Y),
Sulphur : Met (M), Cys (C)
Hydroxyl: Ser (S), Thr (T), Tyr (Y)
Cyclic: pro (P)
Carboxyl: asp (D), glu (E)
Amide: asn (N), gln (Q)
Amine: lys (K), arg (R)

These amino acid side chains absorb UV light at 270280 nm

These amino acids

side chains can form
hydrogen bonds.
Cysteine can form
disulfide bonds.

+H
at biological
pH

Ionization of Amino Acids

pKa values depend on chemical environment

Titration Curve
for Glycine
At pH > pI, net charge = At pH < pI, net charge = +

Calculation of pI for Glycine

Use the Henderson-Hasselbalch equation to calculate the pI.
At isoelectric point, pH = pI
pI = pKCOOH + log [H3N+CH2COO-]
[H3N+CH2COOH]
pI = pKNH3+ + log [H2NCH2COO-]
[H3N+CH2COO-]
Adding up: 2pI = pKCOOH + pKNH3+ + log [H2NCH2COO-]
[H3N+CH2COOH]
When pH=pI, [H2NCH2COO-]=[H3N+CH2COOH]
2pI = pKCOOH + pKNH3+ or pI = {pKCOOH + pKNH3+}/2

Titration
Curve for
Histidine
triphasic
pI = (pKR + pK2)/2

Chapter 6

Techniques of Protein Purification

Instructor: Rashid Syed

Textbook: Biochemistry (4th Edition ) by Donald Voet Judith G. Voet

Protein Biochemistry Methods

Methods can be analytical or preparative
Preparative: purification of protein of interest
Analytical: identification and
characterization
Methods based on solubility, size, charge,
binding affinity, activity

Protein Purification
Proteins can be purified from cell or tissue samples
Samples are homogenized and fractionated by
differential centrifugation to isolate the fraction
containing protein of interest
Protein purification is a multi-step procedure
Need to establish a specific method of identification.
Can be enzymatic, binding or activity assay
With each step, the purification level and specific
activity increases and the yield decreases

Specific Activity (activity/total protein)

can be Used to Assess Protein Purity

Solubility
Salting Out: For most proteins, solubility
decreases as salt concentration increases.
Greater the polarity, greater the solubility.
Proteins can be fractionated by sequential salt
precipitation
Isoelectric Precipitation: Proteins are least
soluble when pH = pI. (because at pI, net
charge is 0)

Size
Dialysis: Diffusion through a semi-permeable
cellulose membrane. Different pore sizes allow
removal of molecules smaller than specific MW
Gel-Filtration Chromatography: Also called sizeexclusion chromatography. Column packed with
porous beads made of a cross-linked polymer. Can
get different pore sizes. Smaller molecules get
trapped within the porous beads and their flow down
the column is retarded. Larger molecules are
excluded from the beads and move down between
loosely packed bead. Smaller the molecule longer
the elution time.

Dialysis

Size-Exclusion Chromatography

Charge
Ion Exchange
Chromatography: Columns
are made of charged
cellulose particles.
Carboxymethyl (CM)
cellulose: - charge, cation
exchange column
Diethylaminoethyl (DEAE)
cellulose: + charge, anion
exhange column
Proteins are eluted using a
pH gradient

Binding Affinity
Affinity
Chromatography: The
column matrix is modified
by covalent linkage to a
compound with high
specific binding affinity to
protein of interest
Eg: Lectins, antibodies,
ligands, substrates
3 steps: Specific binding of
protein, washing unbound
proteins, elution of bound
protein by specific
displacement, high salt or
low pH

HPLC
High Pressure Liquid Chromatography
Enhanced version of all column chromatography
techniques
Column material are very fine and closely packed
for better resolution
High pressure has to be applied to maintain flow
Clean, rapid separation of very small samples

Electrophoresis
Primary analytical technique
Electrophoresis is the movement of charged proteins in
an electric field
Movement is from the electrode (cathode) to +
electrode (anode). Migration is related to charge: mass
ratio. Generally, smaller proteins migrate further
Separation on slabs of polyacrylamide cross-linked with
methylenebisacrylamide: inert, porous and readily
formed
Visualization by staining (coomassie blue, silver)

Gel Electrophoresis

Determination of M. Wt. of Protein

Types of Electrophoresis
Denaturing gels: SDS disrupts all non-covalent
interactions. Associates with all proteins and imparts high
charge making native charge insignificant. Reducing agent
opens disulfide linkages
Native gels: No SDS, native charge contributes to
migration, usually protein activity maintained
Isoelectric focusing: Polyampholytes are used to form a
pH gradient in the gel. Proteins focus where pH = pI,
because at this point they have 0 net charge
2D electrophoresis: IEF + SDS-PAGE. Proteins separated
by pI horizontally, then by size vertically for complete
resolution of complex samples

Isoelectric Focusing can be Used to

Determine the pI of a Protein

Isoelectric Focusing and SDS-PAGE are

Combined in 2D Electrophoresis

Peptide Synthesis
The N-terminal aas NH2 group is blocked using 9-fluorenylmethoxycarbonyl(Fmoc) group

The C-terminal aa is covalently attached to a solid support via its

carboxlate group

The Fmoc group is removed (aa is deprotected)

The n-1 aa is protected with Fmoc.
The COOH group of n-1 aa is activated by
dicyclohexylcarbodiimide (DCC)
The two are reacted to form a peptide bond
The 2nd cycle starts with deprotection of n-1 NH2
After multiple cycles, the synthetic peptide is released from the
resin by HF hydrolysis

Chapter 7

Covalent Structures of Proteins

Instructor: Rashid Syed

Textbook: Biochemistry (4th Edition ) by Donald Voet Judith G. Voet

Proteins
Linear polymers of aa via amide linkages form peptides
(1-10), polypeptides (11-100) and proteins (>100)
Eg: Aspartame (2), glutathione (3), vasopressin (9),
insulin (51)
Proteins have an amino-end and carboxyl-end
In the lab, proteins can be hydrolyzed (to aa) by strong
acid treatment
Physiologic hydrolysis by peptidases and proteases

Protein Sequence
Amino acid sequence determines primary structure
Unique for each protein; innumerable possibilities
Gene sequence determines aa sequence
Each aa is called a residue; numbering (& synthesis)
always from NH2 end toward COOH end
Amino acids covalently attached to each other by an
amide linkage called as a peptide bond.

The Peptide Bond

A Pentapeptide
Numbering (and naming) starts from the amino terminus
AA1

AA2

AA3

AA4

Ser-Gly-Tyr-Ala-Leu
SGYAL
serylglycyltyrosylalanylleucine

AA5

Ionization of Peptides & Net charge

Each ionizable group

ionizes according to its pKa
and solution pH.
Net charge is sum total of
all charges on molecule.

4 Levels of Protein Structure

Protein structure is stabilized by non-covalent interactions and disulfide linkages

Disulfide Linkages

The Proteome
Proteome is the protein equivalent of the genome
The proteome consist of all of the proteins expressed
by a cell under specific conditions
The proteome of a cell depends on the cell type, its
developmental stage, environment/stimuli,
nutritional and metabolic status etc
The genome of a cell is fixed, the proteome is
dynamic
The proteome is much larger than the genome. Each
gene can translate into multiple isoforms of proteins

Protein Mass and Concentration

Protein mass is measured in Daltons (Da) or kDa
One Dalton is 1/12 the mass of a 12C atom
On average, the MW of each aa is 110 Da
Most proteins range from 30 to 80 kDa
Trp and Tyr have a high ability to absorb light
with maximum absorption at 280 nm. Since most
proteins contain these aa, protein concentration
can be estimated spectrophotometrically.

Characterization of Protein Primary Structure

Amino Acid Composition

Complete hydrolysis for 24 hr at 110 oC in 6 M HCl
Separation of amino acids by ion exchange
chromatography on sulfonated polystyrene resin
Elution using a pH gradient
Detection of amino acids by reaction with ninhydrin
or fluorescamine (spectrophotometry)
Identification of amino acids by position of peak on
chromatogram
Determination of amino acid ratios by size (height) of
each peak

Amino Acid Analysis

Identification of amino-terminal residue

Identification of N-terminal amino acid

The N-terminal aa can be identified by Sangers
method. This method involves modification of the
N-terminal residue by flurodinitrobenzene followed
by complete hydrolysis of the peptide.
More recently, fluorescent compounds such as
dansyl chloride or dabsyl chloride are used because
of their higher sensitivity.
The N-terminal aa is the only modified aa and it is
identified by chromatography.
The peptide is completely hydrolyzed and cannot be
reused

Protein Sequencing
It is essential to further biochemical analysis that we know
the sequence of the protein we are studying
Actual sequence generally determined from DNA sequence
Edman Degradation (Classical method)
Successive rounds of N-terminal modification, cleavage, and identification
Can be used to identify protein with known sequence

Mass Spectrometry (Modern method)

MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus
the amino acid sequence
Can be used to determine post-translational modifications

Edmans Degradation

The next cycle releases residue 2. It is possible to

identify ~50 aa from each sample by this method

MS Procedures for Sequence IDs

MS Procedures for Sequence IDs

Proteins with
disulfide linkages
Disulfides are reduced
using DTT
-SH groups are blocked by
treatment with iodoacetate
to form carboxymethylated
derivatives

Specific Cleavage of Polypeptides

Proteins larger than 50 aa are first hydrolyzed into
shorter peptides
Chemical or enzymatic methods hydrolyze
proteins at specific sites
Peptides are separated by chromatography
Peptides generated by 2 or more cleavage methods
are each sequenced separately.
Sequences of individual peptides are overlapped
together to deduce the entire protein sequence

Protein Sequencing Example

Method 1 (Trypsin):
ser-glu-phe-his-lys
ala-ile-cys-asp-tyr-thr-ala
gly-leu-pro-arg
Method 2 (staphylococcal protease):
gly-leu-pro-arg-ser-glu
phe-his-lys-ala-ile-cys-asp
tyr-thr-ala
Overall protein sequence:
Gly-leu-pro-arg-ser-glu-phe-his-lys-ala-ile-cys-asp-tyr-thr-ala

Protein Synthesis by Bruce Merrifield Method