3 | Amino Acids,

Peptides, and Proteins

© 2017 W. H. Freeman and Company

 CHAPTER 3
Amino Acids, Peptides,
and Proteins

Learning goals:
• Structure and naming of amino acids
• Structure and properties of peptides
• Ionization behavior of amino acids and peptides
• Methods to characterize peptides and proteins
 Amino Acids:
Building Blocks of Protein
• Proteins are linear heteropolymers of -amino acids.

• Amino acids have properties that are well suited to carry

out a variety of biological functions:
– capacity to polymerize
– useful acid-base properties
– varied physical properties
– varied chemical functionality
Amino Acids Share Many Features,
Differing Only at the R Substituent
Amino Acids Have Three Common Functional
Groups Attached to the α Carbon
• The α carbon always has four substituents and is
tetrahedral.
• All (except proline) have:
– an acidic carboxyl group connected to the α carbon
– a basic amino group connected to the α carbon
– an α hydrogen connected to the α carbon
• The fourth substituent (R) is unique in glycine, the
simplest amino acid. The fourth substituent is also
hydrogen.
All Amino Acids Are Chiral (Except Glycine)
Proteins only contain L amino acids
 Amino Acids: Classification

Common amino acids can be placed in five basic

groups depending on their R substituents:
• nonpolar, aliphatic (7)
• aromatic (3)
• polar, uncharged (5)
• positively charged (3)
• negatively charged (2)
These amino acid side chains absorb UV light at 270–280 nm
These amino acids side chains can form hydrogen bonds.
Cysteine can form disulfide bonds.
 Ionization of Amino Acids
• Amino acids contain at least two ionizable protons,
each with its own pKa.
• The carboxylic acid has an acidic pKa and will be
protonated at an acidic (low) pH:
−COOH ↔ COO− + H+
• The amino group has a basic pKa and will be
protonated until basic pH (high) is achieved:
−NH4+ ↔ NH3 + H+
 Ionization of Amino Acids
• At low pH, the amino acid exists in a positively
charged form (cation).
• At high pH, the amion acid exists in a negatively
charged form (anion).
• Between the pKa for each group, the amino acid
exists in a zwitterion form, in which a single molecule
has both a positive and negative charge.
Cation  Zwitterion  Anion
 Amino Acids Can Act as Buffers

Amino acids with uncharged side chains, such as glycine,

have two pKa values:
• The pKa of the -carboxyl group is 2.34.
• The pKa of the -amino group is 9.6.

As buffers prevent change in pH close to the pKa, glycine

can act as a buffer in two pH ranges.
Buffer
Regions
Ionization of water, weak acids and bases

Ammonium Ion

Phosphoric Acid

Acetic Acid

Buffering capacity

Fig 2.18
Buffering against pH change in biological
systems

• Buffer
– A solution that resists pH change when acids
(H+) or bases (OH-) are added

• Henderson-Hasselbach equation
– For HA <===> H+ + A-
– pH = pKa + log [A-] / [HA]
– pH = pKa + log [proton acceptor] / [proton donor]
– When [A-] = [HA] , [A-] / [HA] =1, log 1= 0, pH =pKa
• Midpoint of titration curves
 Buffering against pH change in biological
systems

– Proteins & NA serve to buffer

– Phosphate buffer system
– Bicarbonate buffer system
 Buffering against pH changes in biological systems
Enzymes are highly pH dependent

pH must be maintained in cells & fluids

Therefore buffer systems
Fig 2.21
 Buffering against pH changes in biological systems

• Reactions in biological systems are pH dependent

• Organisms require constant pH
• Cytoplasm of cells has high [proteins]
• AA contribute buffering capacity
– Example: Histidine is a weak acid
– pKa of protonated side chain R, is 6.0

Fig 2.20
 Buffering against pH changes in biological systems

In Vivo Buffering

• Examples
1. Proteins
• functional groups that are weak acids or weak bases
2. Nucleotides
• Ionizable groups contributing to cytoplasmic buffering
3. Phosphate buffer system
• H2PO4- <===> H+ + HPO42-
• pKa = 6.86, therefore buffers at approximately neutral pH
• Cytoplasm of all cells
4. Bicarbonate buffer system
• Keeps blood plasma at neutral pH
• Controlled by rate of breathing
 Buffering against pH changes in biological systems

Bicarbonate buffer system

Bicarbonate
Exercise ---> Lactic acid --->---->---->--->---->

Carbonic acid

Box 2-4 Fig. 1

 Peptide Ends Are Not the Same
Numbering (and naming) starts from the amino terminus (N-terminal).
AA1 AA2 AA3 AA4 AA5
 Naming Peptides:
Start at the N-terminal

• Using full amino acid names:

– serylglycyltyrosylalanylleucine
• Using the three-letter code abbreviation:
– Ser-Gly-Tyr-Ala-Leu
• For longer peptides (like proteins) the one-
letter code can be used:
– SGYAL
Chapter 3.2 Peptides & Proteins

Peptides
Can have very important biological activities
Examples of small “active” peptides
1. Oxytocin (9 AA) - stimulates uterine contractions
2. Bradykinin (9 AA) - Inhibits tissue inflamation
3. Many antibiotics are small peptides
4. Nutrasweet (Aspartame) - Aspartate-Phenylalanine
5. Insulin - 2 polypeptides of < 30 AA each
6. Glucagon - 29 AA (opposite effects to insulin)
Chapter 3.2 Peptides & Proteins
Number of amino acids in Proteins

• Average MW of AA is 138

• Actually 128 (smaller AA predominate)

• 128 - 18 (MW H2O) = 110

• Therefore the #AA in a protein = MW protein / 110

• Polypeptide of 10,000/ 110 = 90 AA

• So proteins are approximately > 100 AA

 Polypeptide Size and Number
Varies Greatly in Proteins
TABLE 3-2 Molecular Data on Some Proteins
Molecular Number of Number of polypeptide
Protein
weight residues chains

Cytochrome c (human) 12,400 104 1

Ribonuclease A (bovine pancreas) 13,700 124 1

Lysozyme (chicken egg white) 14,300 129 1

Myglobin (equine heart) 16,700 153 1

Chymotrypsin (bovine pancreas) 25,700 245 1

Chymotrypsinogen (bovine) 25,700 245 1

Hemoglobin (human) 64,500 574 4

Serum albumin (human) 66,000 609 1

Hexokinase (yeast) 107,900 972 2

RNA polymerase (E. coli) 450,000 4,158 5

Apolipoprotein B (human) 513,000 4,536 1

Glutamine synthetase (E. coli) 619,000 5,628 12

Titin (human) 2,993,000 26,926 1

Chapter 3.2 Peptides & Proteins

Disulfide bonds
Flexibility
Buffering at neutral pH
(ATG) 1st AA
Rigidity
Frequently phosphorylated
 Proteins are comprised of:
• Polypeptides (covalently linked -amino acids) + possibly:
• cofactors
 functional non-amino acid component
 metal ions or organic molecules
• coenzymes
 organic cofactors
 NAD+ in lactate dehydrogenase
• prosthetic groups
 covalently attached cofactors
 heme in myoglobin
• other modifications (posttranslational modifications)
 Conjugated Proteins Are Covalently
Bound to a Nonprotein Entity

TABLE 3-4 Conjugated Proteins

Class Prosthetic group Example

Lipoproteins Lipids β1-Lipoprotein of blood

Glycoproteins Carbohydrates Immunoglobulin G

Phosphoproteins Phosphate groups Casein of milk
Hemoproteins Heme (iron porphyrin) Hemoglobin
Flavoproteins Flavin nucleotides Succinate dehydrogenase
Metalloproteins Iron Ferritin
Zinc Alcohol dehydrogenase
Calcium Calmodulin
Molybdenum Dinitrogenase
Copper Plastocyanin
Common Questions About Peptides
and Proteins
What is its sequence and composition?
What is its three-dimensional structure?
How does it find its native fold?
How does it achieve its biochemical role?
How is its function regulated?
How does it interact with other macromolecules?
How is it related to other proteins?
Where is it localized within the cell?
What are its physico-chemical properties?
Studying Proteins and Peptides Sometimes
Requires Purification from a Mixtureprotei
1. Cells/ Tissues

2. Process of Cell fractionation

a) Crude extracts (homogenate)
b) Differential Centrifugation
Studying Proteins and Peptides Sometimes
Requires Purification from a Mixtureprotei

Perform Purification Techniques

a) Dialysis
b) Salting out
A Mixture of Proteins Can Be Separated
• Separation relies on differences in physical and
chemical properties:
– charge
– size
– affinity for a ligand
– solubility
– hydrophobicity
• Chromatography is commonly used for
preparative separation in which the protein is
often able to remain fully folded.
 Working with proteins

Column Chromatography of Proteins

1. Exchange Resins
» Cation (binds +ve)
» Anion (binds -ve)
2. Gel Filtration
» Size exclusion
3. Affinity
» Binding to ligand
Column Chromatography

• Column chromatography
allows separation of a
mixture of proteins over a
solid phase (porous matrix)
using a liquid phase to
mobilize the proteins.
• Proteins with a lower
affinity for the solid phase
will wash off first; proteins
with higher affinity will
retain on the column longer
and wash off later.
Separation by Charge: Ion Exchange
Separation by Size: Size Exclusion
Separation by Binding: Affinity
 Electrophoresis for Protein Analysis

Separation in analytical scale is commonly done

by electrophoresis.
– The electric field pulls proteins according to their
charge.
– The gel matrix hinders mobility of proteins
according to their size and shape.
– The gel is commonly polyacrylamide, so separation
of proteins via electrophoresis is often called
polyacrylamide gel electrophoresis, or PAGE.
 SDS PAGE Separates Proteins by
Molecular Weight
• SDS – sodium dodecyl sulfate – a detergent

• SDS micelles bind to proteins and facilitate unfolding.

– SDS gives all proteins a uniformly negative charge.
– The native shape of proteins does not matter.
– The rate of movement will only depend on size: small
proteins will move faster.
Chapter 3.3 Working with proteins

Crude extract Final purification

Fig. 3-19b
SDS-PAGE Can Be Used to Calculate the
Molecular Weight of a Protein
Isoelectric Focusing Can Be Used to
Determine the pI of a Protein
Isoelectric Focusing and SDS-PAGE Are
Combined in 2D Electrophoresis
Aromatic Amino Acids Absorb Light in a
Concentration-Dependent Manner
• The aromatic amino acids absorb light in the UV region.
• Proteins typically have UV absorbance maxima around
275–280 nm.
• Tryptophan and tyrosine are the strongest
chromophores.
• For proteins and peptides with known extinction
coefficients (or sequences), concentration can be
determined by UV-visible spectrophotometry using the
Lambert-Beer law: A = cl
 Protein Sequences as Clues to
Evolutionary Relationships
• Sequences of proteins with identical functions from
a wide range of species can be aligned and analyzed
for differences.
• Differences indicate evolutionary divergences.
• Analysis of multiple protein families can indicate
evolutionary relationships between organisms,
ultimately the history of life on Earth.
 Chapter 3: Summary

In this chapter, we learned about the:

• many biological functions of peptides and proteins

• structures and names of amino acids found in
proteins
• ionization properties of amino acids and peptides
• methods for separation and analysis of proteins