Chapter 2.

Protein
 Amino acids
General formular L- amino acids

“CORN“

pK1  2.2
 Aminoacid
pK2  9.4

• Vary by R side chain: greatly vary in

properties
• L- amino acids: Protein building blocks
 20 L- axit amin forming protein

• Only 20 (22) L- axit amin forming protein

- dipeptid (2 amino acids): AA1 - AA2 20 x 20 = 400 peptids

- Tripeptid (3 amino acids): AA1 AA2 AA3 20 x 20 x 20 = 8000 peptids

- Protein from 100aa: 20100 = 1.27 x10130

-
 D- amino acids

- Join constructing antibiotics

(gramidicine-S,
Actinomycine-D…)
- D-glutamic acid and D-
alanine found in bacterial
membrane
- D-Serine; D- aspartate in
brain tissue
- …..
 Classification

1. Based on essentiality:

Non-essential amino acids Essential amino acids

Can not be synthesized in the
body
Must be provided by
nutrition
Depending species, age

Histidin
 2. Based on the nature of R groups Polar
Non-polar Non-ionizable:

-Non- or less dissolves in water Ionizable:

-Forming Val Der Wall interaction. Acidic:
-Folding inside protein
-Forming S-S linkage (covalent bond)
stabilizes protein conformation Basic:
-Possible forming hydrogen bonds, stabilizes
protein conformation dissolves in water

Linkage between Rs -anion/cation forming salting 7

bridge, stabilizes protein conformation
Selenocysteine (Sec, U): 21st amino acid Pyrrolysin (Pyl, O): 22nd amino acid
• Rare, cysteine but replacing S = Se
• Found in several eukaryotic and prokaryotic, • Lysin replacing NH2 by NH-pyrrole
in 1970s • Coding by stop codon: UAG
• Found in active sites of some enzymes
• Found 2002 in some archaea
(formate dehydrogenase of E. coli
; glutathione peroxidase of some mammal)
• Coding by stop codon UGA
• Strong nucleophile

Join in some protein: antioxidant

property
 Function of amino acids
❑ Biological:
✓ Energy
✓ Construction of protein and Enzyme: composition and conformation of
proteins
✓ Active Peptides: antibiotics, antioxydants, anticancer, ….
❑ Food industry
✓ Energy
✓ Food additives and functional food (L-Theanine)
✓ Food structure
✓ Food sensorial properties
✓ Enzymes
❑ Pharmaceuticals
❑ Agriculture
✓ Animal and aquaculture feeds
✓ …………….
 Physical Properties
Solubility
• Depending on
– Nature of R groups
– pH : at pI, less soluble

– Solvent nature:
» Polarity of R
» Polarity of solvent
Configuration and optical activity: L- and D-

-At least one chiral center C* (except

glycine): optical activity
-Only L-amino acids forming protein
(except L-cysteine)
- D-amino acid found in cell wall…
“CORN“

(S)- amino acid (R)- amino acid

UV absorption

• Amino acids absorb UV light

Aromatic: Phenylalanine, tyrosine, tryptophane
max at 250-290 nm
200-230 nm
Histidine, cysteine, methionine
max at 200-210 nm

• Law Beer Lambert : A= .L.C

A

C mg/ml
A = εLC
 Beer Lambert Law

I0 I

Monochromatic
light L

Transmittance, T = I / I0
%T = 100 T
A=L.C
Absorbance, A
(Optical density- OD)

: molar extension (mol-1.cm-1) A = log10 I0 /I

A = log10 1 / T
The Beer-Lambert law: linear relationship between A = log10 100 / %T
the absorbance and the concentration, molar absorption A = 2 - log10 %T
coefficient and optical coefficient of a solution.
Dissociation
- Zwitterion: dipolar ions both a formal positive and negative charge (Neutral)

pKa ~ 5 pKa ~ 9

- Isoelectric point (pI): The pH at which the amino acid neutral, zwitterionic
form (influenced by the nature R):
- Pr/E loss function
- Viscosity maximal
- Minimal solubility- precipitation
 Chemical reactivity

• Reactivity of group –COOH

• Reactivity of group–NH2
• Reaction of group -R
• Reaction of both –COOH and –NH2
– Peptide bond
– Ninhydrin reaction
 PEPTIDE BOND

• Covalent bond:–NH2 at C and -

COOH at C of 2 aa.
• CONH on the same plan
 Ninhydrin reaction
a.a 1
Qualitative/quantit
ative analysis (-
NH2)
Spetrophotometry
a.a 2 (by Law Lamber
Beer)

570 nm
 Peptide
Peptide: amino acids linked via peptide bonds

– Dipeptide: two amino acid linked

Aspartame, sweetener : Aspartic-phenylalanine- metyl-este
– Oligopeptide: chain of < 10 amino acids linked via peptide Aspartame
bonds
Glutathion-GHS -Gamma Glutamylcysteinglycine: glutamic-
Cysteine-Glycine, an antioxydant
– Polypeptide: < 50 amino acid residues
Insulin, hormone
Structure of GSH
 Protein
Protein: Composititon
Consists of one or several • 20 (22) L- amino acids
polypeptide chains, forming • Atoms:
conformation – C, H, O, N
– N: 16%
Mw> 10,000 Da (>50 amino – Other:
acid residues)
•S
• P
(1Da = 1/12 mass of 12C)
• Other
•
 Protein function
Biological function

• Catalysis – enzymes
• Structural – keratin
• Transport – hemoglobin
• Trans-membrane transport – Na+/K+
ATPases
• Toxins – rattle snake venom, ricin
• Contractile function – actin, myosin
• Hormones – insulin
• Storage Proteins – seeds and eggs
• Defensive proteins – antibodies
 Protein function
• Technological function (Food industry)

• Energy
• Product structure – foaming, emulsion,
• Catalysis – enzymes
• Sensorial
 Remind main interaction types in bio-moleculars
Bản chất liên kết Năng lượng liên kết/mol

Lực hút tĩnh điện giữa nguyên tử H liên kết với

12-29 KJ
nguyên tử âm điện (O, N..)

Liên kết tĩnh điện giữa các nhóm tích điện ~ 20 kJ

Sự sắp xếp các phân tử có cực quanh phân tử

không cực (hoặc ngược lại), 4-30 KJ

Lkết giữa hai nguyên tử không tiếp xúc do 4-8 KJ

tương tác các lớp điện tử ngoài,

Sự góp chung cặp điện tử của các nguyên tử

Covalent bond
350-400 KJ
Four Levels of Protein Structure
Primary sequence of
protein

• Characterized by:
• Type, sequence and number of amino
acids in polypeptide(s)
• Stabilized by peptide bond: strong
covalent bond

Polypeptide Is Made Up of a Series of Planes

Linked at α Carbons
 SECONDARY STRUCTURE
 helix  pleated sheet  turn

• Popular secondary structure

• Myoglobin, hair, …. Characteristics :
• Chiral C*→ peptide turns
Fibroin Flavodoxine
Cacbonic anhydrase
• Stability: hydrogen bonds, supplemented
by van der Walls interaction
- Thermostability • Weak hydrogen bonds
Tertiary structure

-Sheet

Xoắn 

Characteristics
• Minimal in energy
• Consists of helices, sheets, turns and loops-
form domain (section of protein active surface)
• Folding with hydrophobic R inside, hydrophilic R
group at the surface: solubility of protein
• Weak bonds stabilize protein conformation
 Quaternary structures
Homodimer protein Homoteramer protein

Heteroteramer protein

Hemoglobin
• 2  globin subunits
• 2  globin subunits

-Protein consists of two or more polypeptide chains (one polypeptide chain: one sub-unit)
- Stabilized by: weak bonds + covalent bonds (S-S)
Summary protein structure
Change
Amino acid sequence

Protein structure Change

Functionality
Ex. Diseases caused by changes
in protein structure

• Spongiform
encephalopathy (BSE or
mad cow disease) seen in  sheet

cattle and livestock and

Creutzfeldt-Jakob disease
(CJD) seen in humans. Normal Prion Unnormal Prion
(trên bề mặt tế bào thần kinh)

Mal folding protein Prion (proteinaceous infection particles, Nobel 1997)

causes nerve cell death in mad cow disease , Alzheimer)
Sickle Cell Anemia – single amino acid change in
hemoglobin related to disease
 Protein shapes

Globular /oval
Fibrous /elongated - Hemoglobin,
- Keratin: hair, nails, horns - Prolamines: gliadin (wheat), zein (maize)
- Collagen: skin, - Albumin
- Elastin: tendon, - Glutelins (wheat, oryzenin in rice)
Insoluble, resistant to digestion Soluble and digestive
 Properties

• Molecular weight: depends on number of aa (40-4000 aa; Mw:

4-440 Kda)
• Dissociatiton: anion/cation and pI
• Solubility
• UV absorption
• Denaturation
• Catalytic (Ch. Enzyme)
• Biure reaction
• Hydrolysis
• Functional
• Sensorial
Molecular weight
Peptide: < 10 000 Da (10 Kda) (1Da = 1/12 12C)
Protein: > 10,000 Da- 10 KDa (polymer)
Dissociation
- Depends on compositional amino acids, dissociation of R groups of aa
- Depends on pH
- pI: Zwitter ions- and dipolar ions→
- Electrically neutral
- Minimum solubility
- Maximum precipitability
- Less buffering capacity
- pH  pI: charged→ pI viết tắt là độ pH=0

- pH>pI: anion (-) x`

- pH<pI: cation (+)

- solubility increased sự hòa tan tăng
Solubility
- Depends on R (amino acids, folding...)
- Depends on pH, (pI or other than pI)
- Solvent: polar or less
- Salt concentration
- Low concentration: soluble: salting in
- High concentration: precipitation: salting out
kết tủa

coagulate: đông lại

Proteins coagulation at low pHs

Yogurt: lactic fermented, Tofu: lower pH : protein coagulated

lower pH : protein
coagulated
UV absorption hấp thụ tia UV
dùng để phát hiện protein

– Law Lambert beer Lowry method

– Maximum absorption Bradford method
• Amino acids: 200-270 nm
• Peptit/Protein: 280 nm - Detection pf protein
- Protein analysis (!)
Biure reaction

NaOH

• Specific for peptide bonds (>2 peptide bonds): protein, peptide

• Ion Cu2+/NaOH protein denaturation : protein biến tính

• A750nm = f (C), Law Lamber Beer

• Hydrolysis : cleaves peptide bonds,
forms amino acids or peptide: damage
polypeptide/protein
• Agents:
– Enzyme: proteases
• Products: L amino acids
• Depends on specificity of enzymes
– Acids
• Damage S- containing amino
acids
– Alkaline:
• Isomeration, forming racemic
mixture (L/D: 1/1)
• Apply?
Protein hydrolysis

• Examples?
• Agents
• Condition
• Problems?
Maillard reaction: non enzyme browning reaction
phản ứng tạo mùi

-NH2 + -C=O → Melanoidin x

(Chapter 4. Glucide)
Functional properties
 Functional properties

1. Water binding and holding

ability:
– Hydrogen bond to H2O on the
Protein surface (to hydrophilic
R)
• Juiciness
• Weight
Depends on:
- Protein type/structure,
concentration
- Thermo-treatment severity
- Salt concentration:
hexametaphosphat
- pH  pI
 Irreversible gels

2. Gelation:
- Interaction protein-protein-
starch- water-lipide (sausage,
cheese, yogurt …) forming 3D
network
- Denaturation agents:
- Temperature/cooling
- pH
- Pressure
- Mechanical
- Salt
 3. Emulsification
nhũ tương

emusipier : chất tạo nhũ

- Emulsion: A suspension of
small globules of two liquids
not mixed

- Protein contains both

hydrophobic and hydrophilic
groups, can be good
emulsifiers

Charateristics:
- Emulsification capacity
- Emulsification stability
4. Foaming
- Dispersion system of air in water phase
- Influencing factors:
- Mixing
- Partially denatured protein: hydrophobic groups on
the surface of protein
- Globular Protein > fibrous protein
- pH  pI
- Salting in/out
- Lipide inhibits foaming, thinning the protein film
(except cream)
- Viscosity: adding starch, sugar..)
- Processing time/force
Charateristics:
- Foaming capacity
- Foaming stability
 Protein denaturation
protein biến tính

Alteration of native conformation of protein caused by disruption of secondary

bonds (unchanged amino acid sequence), destroys properties, functionality
tất cả cấu trúc bậc cao sẽ bị thay đổi vì lk lm bền cấu trúc bậc cao thay đổi ko thay đổi polipeptit

Disruption of secondary bonds :

- Hydrogen
- Hydrophobic interaction Weak
- Van der Waal bonds
- Ionic bonds
- Disulfide bridge-S-S

- May be Reversible or Irreversible

1 chiều

Reversible
thuận nghịch
 Denaturation agents
Physical: alters
• Temperature >50oC: chaotic, hydrogen bonds
Irreversible
• UV:
• Pressure Weak bonds

• Mechanical force:
Chemical : affects on
• pH/acid/alkaline: ionic interaction, salt bridge
• Solvent: hydrophobic, Van der Waal intereaction Reversible
• Urea, hydrocloride guanidine: alters S-S
• Salting out: competes forming hydrogen bonds with water
• Metalic ions: ionic interaction
 Denatured protein properties

– Altered native structure leading to alter functionality (enzyme,

antibody, vaccine, hormone..)
– Surface properties: change in hydrophobic/hydrophilic (R) groups
presented on the surface of protein
• Interactivity (internal or external molecular)
• Functional /technological properties
• Sensorial properties
 Properties of denatured protein
• Dysfunctionality (enzyme, antibody, vaccine,
hormone..)
• Change in surface properties: change in
Altered native Interactivity (internal or external molecular)
structure leading of hydrophobic/hydrophilic (R) groups
to alter presented on the surface of protein
functionality • Reduce toxicity
• Digest ability (due to hydrolysis)khả năng tiêu hóa

• Change in functional properties

• Change in sensorial features
• Reversible or irreversible
Nutritional characteristics
• Balanced amino acids
– Essentials
– Non-essentials
• Chidrend : 10 axit amin(Leu, Ile, Val,
Thr, Lys, Arg, Met, Phe, Trp, His)
• Aldults : 8 (Leu, Ile, Val, Thr, Lys, Met,
Phe,Trp

• Protein content
• Digest ability
• SENSORIAL properties
Essential Amino Acids – Veg.vs. Meat
 Protein classification
• Protein structure Homodimer protein Homoteramer protein

o Number of polypeptide chains:

– One polypeptide chain - monomeric
protein
– More than one: multimeric protein (-
subunits)
o Protein composition
– Hom(l)omultimer – all one kind of chain
– Heteromultimer - two or more different
chains
By compositional

Hom(l)oproteins
(proteins đơn giản) • consists of amino acids only

Classification of conjugated proteins

class prosthetic group example

immunoglobulin (antibody)
glycoprotein saccharide
interferon (antiviral agent)

hemoglobin (O2 carrier in blood)

hemoprotein heme
myoglobin (O2 storage in muscle)
Heteroproteins
conjugated (protein LDL - low-density lipoprotein (lipid carriers)
lipoprotein lipid
phức hợp) HDL - high-density lipoprotein (lipid carriers)

Ca2+ in calmodulin (muscle contraction)

Fe2+ in hemoglobin/myoglobin
metalloprotein metal ion Fe2+ in ferritin (Fe2+ storage)
Zn2+ in carboxypeptidase (digestive
enzyme)

nucleoprotein nucleic acid RNA-bound protein

• Protein source

– Recombinant
(genetically
modified
organism-GMO)

Plant Animal Microorganism

 Analysis
Kjeldahl’s: total nitrogen
 Lowry method: protein measurement

Stage 1: Biure
reaction +
reduce Cu+2 in Axit amin
thơm Tyr,
alkaline medium Tryp

Stage 2: aromatic
amino acids in Cu+1
Màu vàng
complex reducing
Folin agent into
Molybdennum blue

- Law Lamber Beer

- Protein standard curve (BSA) (0-2mg/mL)
Stage 2: reducing Folin agent
 Bradford method: Protein determination
Agent: Comassie Blue G-250

- Simple ,
- Fast (1 step, 5 min.)
- Sensibility: 1-20 µg
- Specificity
- Influenced by polyphenol and detergent
Learning objectives

• Understand amino acid system, functions. Relationship between

structure and properties
• Know the definitions of primary, secondary, tertiary, and quaternary
structures or protein
• Understand the differences between globular and fibrous proteins
• Know the forces that stabilize these structures
• Understand the type of bonds that stabilize these structures
• Understand that certain changes in protein structure can change the
structure and function of protein
• Have some examples illustrated for products/process.