BIOCHEMISTRY

---A science explains life

at molecular level

Yiming Tao ( 陶毅明 ),

PhD, Prof.
 Introduction
Q1. What constitute humane body?

1% 5%
Carbo Minerals
-hydrate

65% O 10%
Lipid
18% C
10% H 20%
3% N Protein 64%
2% Ca Water

2% Others

made up of different types of atoms and molecules.

 Q2: What make living organisms distinguish
from nonliving material?

Growth and Metabolism

Reproduction and Transmission

of Genetic Information

The capacity for self-renewal, self-replication and self-

 Biochemistry describes the flowing contents:

Structure and function of biomolecules

(proteins, enzymes and nucleic acids in this course)

Metabolism of carbohydrates, fatty acids,

amino acids and nucleotides

Flow of genetic information-Molecular Biology

Biochemistry in Life

y ？
a r
e ss
e c
N
How wine is made?
 Biochemistry in Medicine

1. Biochemical basis of disease, e.g. Gout,

Sickle cell anemia

2. Diagnosis, e.g. Transferase and liver disease

. Drug and Treatment, e.g. antimetabolites,

antibiotics (e.g. sulfa)
References
 Contents in this Course

Protein and Enzyme: Unit 1: Chapter 1-3, 5

Flow of Genetic Information: Unit 6: Chapter 29-32

Metabolism: Unite 2-4: Chapter 6-16,

18-22
Chapter 1 Amino Acids
Outline

I. Overview

II. Structure

III. Acidic and Basic Properties

IV. Ultraviolet Absorption

V. Color Reaction
I. Overview

1) Proteins are the most abundant and functionally

diverse molecules in living systems.

E.g. enzymes, contractile proteins in muscle, collagen,

hemoglobin, immunoglobulins
20 α-amino acids (20 standard amino acids) constitute
the mammalian proteins.
COO-
+
H 3N Cα H

R Side chain

3) In protein, the carboxyl and amino groups are combined

through peptide bond.
+
H3N-CH-CO-NH-CH-COO-

R1 R2
II. Structure

1) Classification according to the polarities of R groups

A. Amino acids with nonpolar/hydryphobic side chains
1. Location of nonpolar amino acids in proteins

Location of nonpolar amino acids

in soluble and membrane protein.
B. Amino acids with uncharged polar side chains

Site of attachment for

phosphate, oligosaccharide
or formation of H bond

Site of formation of
disufide bond (–S-S-)
A disulfide bond is formed from the sulfhydryl group (-SH)
of 2 cysteines to produce a cysteine residue.
C. Amino acids with acidic side chains

D. Amino acids with basic side chains

E. Abbreviations and symbols for amino acids
 F. Optical properties of amino acids

With the exception of Gly, all amino acids found in proteins

are of L configuration.
 Exercises 1.1

( ) can participate in the formation of hydrogen bond(s)

using its/their hydroxyl group(s) in the side chain(s).

( ) can form disulfide bond(s) using its/their sulfhydryl group(s)

in the side chain(s).

At physiological pH, ( ) are negatively charged,

while ( ) are positively charged.

Most amino acids are ( ) forms.

A. L-alpha B. L-beta C. D-alpha D. D-beta
III. Acidic and Basic Properties of Amino Acids

Q: What will happen if an acid or a base is added

to the Gly solution?

COOH COO- COO-

+[H+] +
+
H 3N C H +
H 3N C H [ H 2N C H
O
H H H- H
positive net charge=0 ] negative

Isoelectric point (pI): the pH at which an amino acid

is electrically neutral.

pH=pI, neutral
pH<pI, positively charged;
pH>pI, negatively charged
 pIs of amino acids

Amino acids pI Amino acids pI Amino acids pI

Glycine 5.97 Alanine 6.00 Valine 5.96
Leucine 5.98 Isoleucine 6.01 Phenylalanine 5.49
Proline 6.30 Tryptophan 5.89 Threonine 6.53
Tyrosine 5.66 Cysteine 5.07 Methionine 5.74
Asparagine 5.41 Serine 5.68 Glutamine 5.65

Aspartic acid 2.97 Glutamic acid 3.22

Arginine 10.76 Histidine 7.59 Lysine 9.74

Q: What is the range of the most amino acids?

How about the pIs of acidic and basic amino acids?
IV. Ultraviolet Absorption of Trp and Tyr

Max. absorption
at 280 nm
V. Colored Chemical Reaction with Ninhydrin

purple or yellow (Pro) products

 Exercises 1.2
1. ( ) absorb the ultraviolet at 280nm.

2. At pH 8.5, which electrode will Ala, Glu and Arg migrate towards?
 Key Points in Chapter 1

1. Amino acids with –OH, -SH groups

2. 2 acidic and 3 basic amino acids

3. Amino acids absorb ultraviolet

4. pI
Chapter 2 Structure of Proteins
Outline

I. Overview

II. Primary Structure

III. Secondary Structure

IV. Tertiary Structure

V. Protein Misfolding

VI. Quaternary Structure

 I. Overview

. 20 amino acids are joined together by peptide bond

to form proteins.

2. The protein structure is analyzed

at 4 organizational levels: primary, secondary,
tertiary and quaternary.

3. Some diseases are related to the incorrect

structure of proteins.
II. Primary Structure of Proteins
1. Peptide

amino acid residues

(C-N)
Oligopeptide: <10 amino acids

Polypeptide: > 10 amino acids

Protein: > 50 amino acids

 Tyr Ala Leu
Ser Gly

Seryl glycyl tyrosyl alanyl leucine, SGTAL (from N to C terminals)

2. Biologically Active Peptides

① Glutathione (GSH): reductant

② Neuropeptides: enkephalin, endorphine,

dynorphine

③ Hormones: glucagon, thyrotropin-releasing

hormone, somatostatin, corticotrophin
3. Characteristics of Peptide Bond

α α

① Resistant to conditions that denature protein,

such as heating and urea

② Partial double-bond character (C-N)

③ Cα-C and N-Cαare freely rotated.

④ Coplanar with other 4 atoms (Cα-CONH-Cα)
--Peptide Unit

⑤ Polar groups(-CO, -NH) are involved

in the formation hydrogen bond.
4. Primary Structure of Proteins
*The primary structure of protein is the sequence of amino acid.

Insulin consists of 2 polypeptide chains joined by disulfide bonds.

Frederick Sanger first worked out the complete amino acid sequence
of bovine insulin.
III. Secondary Structure of Proteins

1. The secondary structure of protein is the spatial

conformation of the main chain
(polypeptide backbone),
regardless of the distributions of side chains.

2. Major elements of secondary structure:

α -helix, β-sheet,
β-turn and coil
A. α-helix
① Coiled polypeptide backbone
② Right-handed spiral
③ Side chains (R groups) extending outward
 O H
-C-[-NH--CaH--CO-]3-N-

R
The H bonds are formed between
the –CO- and the –NH- ahead.

④ Stabilized by hydrogen bonds (dash line)

which are parallel to spiral
⑤ 3.6 residues/turn
⑥ Proline, large numbers of charged amino acids
disrupt an α-helix.
 B. β-pleated sheet

① Pleated, almost fully extended

② Composed of two or more peptide chains

or segments of polypeptide chains

③ Stabilized by hydrogen bonds

that are perpendicular to polypeptide backbone
* Parallel and antiparallel sheets

A. Antiparallel
B. Parallel
C. β-bends/turns

①Reverse the direction of a polypeptide chain

②Pro,Gly frequently found

③Stabilized by hydrogen and ionic bonds

D. Nonrepetitive secondary structure-random coil
E. Supersecondary structures (motif)
IV. Tertiary Structure of Proteins

1. The tertiary structure

describes the overall 3D
arrangement of all atoms
in a protein.

2. It is determined by its
primary structure.

Tertiary structure of myoglobin

A. Domain: fundamental functional
and three-dimensional structural units

The E.coli protein Malonyl-CoA:Acyl Carrier Protein

transacylase has two domains:
the catalytic domain (blue and green)
and the ACP-binding domain (red and yellow).
B. Interactions stabilizing tertiary structure
C. Protein Folding and Role of Chaperones in Folding
Video

①Folding information contained in the primary structure.

② Nonrandom, ordered pathways

③ The fully folded native proteins are in low-energy state.

④Many proteins require chaperones or chaperonins

and ATP to complete the folding.
D. Denaturation of Protein

①Unfolding and disorganization of a protein tertiary

structures without hydrolysis of peptide bond

②Denaturing agents include organic solvents,

strong acids or bases, detergents, ions of heavy metal,
and heat.

③Denatured proteins are often insoluble.

V. Protein Misfolding
A. Prion Diseases

In bovine spongiform encephalopathy, the α-helixes in prion

protein (PrP) are replaced by β-sheets, forming the infectious
prion protein scrapie (PrPsc) which is highly resistant to
proteolytic degradation and tends to form insoluble aggregates
of fibrils.
B. Amyloid diseases

Abnormal cleavage of protein leads to the formation of

fibrils consisting of β-sheets.

Alzheimer disease: Amyloid-β peptide (Aβ) is derived from

an amyloid precursor protein (Fig. A).
Aβ peptides aggregate to form
insoluble β-sheets (amyloid)
which are neurotoxic and
lead to cognitive impairment (Fig. B).
VI. Quaternary Structure of Proteins

β2 β1

α2 α1
Structure of hemoglobin

*For proteins that consist of 2 or more polypeptides,

the arrangement of these subunits is called
the quaternary structure.

*Subunits are held together by noncovalent bonds

(hydrogen, ionic bonds and hydrophobic interactions)
Summary Video
 Key Points in Chapter 2

1. Characteristics of peptide bond

2. Primary/secondary/tertiary/quaternary structure of
protein

3. Interactions stabilizing tertiary structure

4. Protein misfolding, Prion diseases, amyloid diseases

 Supplementary Information
S1. Protein Physicochemical Properties & Purification

S1. 1 Protein Physicochemical Properties

S1. 1. 1 Amphoteric dissociation of proteins

*Dissociable groups: N-terminal amino group,

C-terminal carboxyl group,
dissociable groups in side chains

*Isoelectric point (pI): the pH at which the protein is

electrically neutral.
pH=pI, no charges;
pH<pI, possitively charged;
pH>pI, negatively charged

The range of pI for most human proteins is 5-7.

S1. 1. 2 Colloidal properties of proteins

* Molecular weights > 10 000, diameter 1-100 nm

* Coated with a water layer as
a hydration shell and electric charges
hydration
shell

+ － －
+ +
acid base －
－
+ +
－ －
+ + －－
PI
Positively Negatively
charged charged
 +
+ +
+ +
+ +

+
+ +
+ +
+ +

Two major factors for stabilizing proteins in solutions,

the electrostatic repulsion and hydration shell.
S1.1.3 Denaturation and precipitation

Denaturation: Some physicochemical factors can disrupt

the weak forces within the proteins to disturb
their native spatial structures
and dissipate their biological activities.

* Factors: strong acids or bases, organic solvent,

ionic detergents,
chaotropic agents (urea, guanidine),
heavy metals, and heat

* Spatial conformation disrupted,

but primary structure not affected
Precipitation: separating out from the aqueous solution

* 2 factors for stabilizing protein

in solution are destroyed.

* Denatured protein precipitates or not

depends on the pH.
If the pH is far away from its pI,
the electric charges may keep
the denatured protein in solution.
If near by its pI,
the protein often precipitates.
* Protein can be precipitated without denaturation
by removing the hydration shell (adding cold ethanol or acetone)
or neutralizing electric charges (adding high concentration of
ammonium sulfate—salting out).
* Protein can be precipitated without denaturation
by removing the hydration shell
(adding cold ethanol or acetone)

or neutralizing electric charges

(adding high concentration of
ammonium sulfate—salting out).
S1.1.4 Properties of spectral absorbance
and coloring reactions

* Ultraviolet absorption at 280 nm

* Biuret reaction
* Reaction with phenol reagent (Lowry method)
* Reaction with Coomassie brilliant blue G-250
(Bradford assay)
S1.2 Protein purification

1) Salting-out
2) Dialysis and Centrifuge
3) Chromatography
4) Electrophoresis
Dialysis and Centrifuge

Centrifuge
Dialysis
Chromatography used in protein purification

1. Ion exchange column-separate proteins by charges

2. Gel filtration/molecule exclusion chr.-by MW
3. Hydrophobic column-by hydrophobic property
4. Affinity column-especially for antigen and antibody
Gel Filtration Chromatography

Beads/Resins: Sephadex or Sephacryl

Proteins are separated

according to their molecular weights.

Large proteins could not

enter the beads,
they are first eluted.

e.g. Using Sephacryl S-200

to separate protein
A(110 kDa),
B(40 kDa)
and C(63 kDa).
 Electrophoresis

tank
filled with buffer(pH 8.8)

mould

gel-making
mould

Polyacrylamide gel electrophoresis

(PAGE)
 - -
① pI of most proteins are 5-7,
pH>pI,
proteins are negatively charged.
② Proteins are separated
according to their
charge quantities
and molecular weights.
③Protein staining to see the bands.
(AgNO3 or Coomassie Brilliant Blue)

④PAGE can be used to identify

+ +
the purity of proteins.

Sliver staining Coomassie Brilliant

Blue R250 staining
 Measurement of Molecular Weight by SDS-PAGE
(MW of Subunits)

marker samples ①SDS, sodium dodecylsulfate, is

an anionic detergent, causing
multimeric proteins to dissociate
into their subunits.

②SDS coats proteins with an even

distribution of
negative charge per unit mass,
eliminating the effects of different
charge quantities of the proteins themselves

③Coated polypeptide chains/subunits

can then separated by molecular mass.
(method to determine MW)
 Key Points in Supplementary Materials

2 major factors for stabilizing proteins in solutions

Methods to precipitate proteins without denaturation

Electrophoresis of proteins