PROTEINS

Proteins

Polymer of amino acids linked by amide linkage.

 
Functions:
1. Nutritional: growth, digestion, metabolism (enzymes).
2. Physical structure: cheese, bread, foaming agent, gel-
forming.
 Amino Acids
O O
C H C OH
HO C H NH 2 C H
CH 2 OH CH 3

L -Glyceraldehyde L - Alanine

Zwitterion
O
O -
C OH C O
+
NH 2 C H NH 3 C H

CH 3 CH 3
Evidence of Zwitterion in Amino Acids at Neutral pH

O
+ -
1. NH 3 CH 2 C O At Neutral pH
O
-
CH 3 COOH CH 3 C O

+
CH 3 CH 2 NH 2 CH 3 CH 2 NH 3

2. Melting Point
CH 3 CH 2 COOH 22 C

CH3 CH CH 3 -101 C

NH 2

O
-
CH 3 CH C O 295 C
+
NH 3
 Acid and Base

+ CH3COO -
+
C H3C O O H H

+ +
RNH3 RNH2 + H

Acid = Proton donor

Base = Proton acceptor
Definition of Bronsted-Lowry
Amino Acid

H
+ -
NH 3 C COO
CH

CH 3 CH 3
L is naturally-occurring form for amino acids.

Ionizable groups
O
C OH NH 2
 
 
Zwitterion
O
pKa for C OH = 2.2
 
pKb for NH 2 = 9.5

What do 2.2 and 9.5 mean?

 Review

- log A/B = log B/A

log 1= 0
log 10 = 1
log 100 = 2
If log A/B= 2, The A is 100 times the B
-log [H+] = PH by the “Definition of Sorenson”
 Dissociation of Carboxyl Group (pKa)
H
+ -
[H] R C COO
+
NH 3
Ka =
H
R C COOH
+
NH 3

H H
- H
R C COOH R C COO + H+
+
R C COOH
+
NH 3 NH 3 NH 3
+
+
H = Ka
H
_
R C COO
+
NH 3

-log
H
-
R C COO
+
Henderson Hasselbalch Equation pH = pK a + Log
NH 3
H
What information do we get from the R C COOH
+
equation ? NH 3
 At pH=3.2

H H
+ - + -
NH 3 C COO H NH 3 C COO
CH 3 CH 3

A B
 Dissociation of Amino Group (pKb)
H
+ -
R C COO
[ H]
NH 2
Kb =
H
-
R C COO
+
NH 3

H
-
H R C COO
H +
- + + NH 3
R C COO - R C COO + H [ H] = K b
+ H
NH 3 NH 2 -
R C COO
NH 2
-log
-log
-log
-log H
-
-log H = ?
+
R C COO
NH 2
pH = pK b + Log
H
-
R C COO
+
NH 3
 At pH 10.5

H H
+ - + -
NH 3 C COO NH23 C COO
CH 3
CH 3

A B
Relationship between Dissociation of Amino
Acid and pH

[A-]
pH = pK + Log
[AH]
 
[COO-] [NH2]
pH - pK = Log or Log
[COOH] [NH3+]
 
Effects of pH on the Concentration of Different Groups

[COO -] * [NH2] **
Log Log
  [COOH] [NH3+]  
pH 1 -1.2 -8.5  

pH 2.2 0 -7.3

pH 3.5 1.3 -6  

pH 5.5 3.3 -3.3  

pH 7.5 5.3 -3  

pH 9.5 7.3 0  

pH 11.5 9.3 2

* pKa = 2.2 ** pKb = 9.5

 pK and pI of Amino Acids

  pK1 pK2 pKR pI

Glycine 2.34 9.78   6.06

Alanine 2.35 9.69   6.02
Isoleucine 2.36 9.69   6.02
Serine 2.21 9.15   5.68
Aspartic Acid 2.09 9.82 3.86 2.97
Asparagine 2.02 8.80   5.41
Glutamic Acid 2.19 9.67 4.25 3.22
Glutamine 2.17 9.13   5.65
Arginine 2.17 9.04 12.48 10.76
Lysine 2.18 8.95 10.53 9.74

What is pI?
 Groups of Amino Acids
1. Aliphatic amino acids
2. Hydroxy amino acids
3. Acidic amino acids
4. Amide amino acids
5. Basic amino acids
6. Sulfur-containing amino acids
7. Aromatic amino acids
8. Secondary amino acids
 Aliphatic Amino Acids

Glycine (GLY) Alanine (ALA)

H H
+ - + -
NH 3 C COO NH 3 C COO
H CH 3
 Aliphatic Amino Acids

Valine (VAL) Leucine (LEU)

H H
+ - + -
NH 3 C COO
NH3 C COO
CH 2
CH
CH
CH3 CH3 CH 3 CH 3
Aliphatic Amino Acids

Isoleucine (ILE)

H
+ -
NH3 C COO
CH
CH CH2CH3
3
 Amino Acids with Alcohol

Serine (SER) Threonine (THR)

H
H
+
NH
+
3 C COO-
NH 3 C COO-

CH2 OH CHOH
CH 3
 Acidic Amino Acids

Aspartic Acid (ASP) Glutamic Acid (GLU)

H
H + -
+ - NH 3 C COO
NH3 C COO
CH2
CH2
CH2
-
COO COO
-
 Amino Acids with Amides
Asparagine (ASN) Glutamine (GLN)

H H
+ -
+ - NH 3 C COO
NH 3 C COO
CH 2
CH 2
CH 2
C NH2
C NH2
O O
 Basic Amino Acids
Lysine (LYS) Arginine (ARG)

H
+ - H
NH3 C COO + -
NH 3 C COO
CH2
CH 2
CH2
CH2 CH 2

CH2 CH 2 NH +
2
+
NH3
NH C NH 2
Basic Amino Acids

Histidine (HIS)

H
+ -
NH 3 C COO
CH2

HN NH
+
 Sulfuric Amino Acids

Cysteine (CYS H) Cystine (CYS-CYS)

H
+ -
NH 3 C COO COO
-
COO
-

+ +
NH 3 CH CH 2 S S CH 2 CH NH 3
CH 2
SH
Sulfuric Amino Acids

Methionine (MET)

H
+ -
NH3 C COO
CH2
CH2
S
CH3
 Aromatic Amino Acids

Phenylalanine (PHE) Tyrosine (TYR)

H H
+ - + -
NH 3 C COO NH 3 C COO

CH 2 CH 2

OH
Aromatic Amino Acids

Tryptophan (TRY)

H
+ -
NH 3 C COO
CH 2

N
H
 Secondary Amino Acids

Proline (PRO) Hydroxyproline (HYPRO)

-
- COO
COO +
+ H2N CH
H2 N CH
H2C CH 2
H2 C CH2
CH
CH2
OH
 pK and pI of Amino Acids

  pK1 pK2 pKR pI

Glycine 2.34 9.78   6.06

Alanine 2.35 9.69   6.02
Isoleucine 2.36 9.69   6.02
Serine 2.21 9.15   5.68
Aspartic Acid 2.09 9.82 3.86 2.97
Asparagine 2.02 8.80   5.41
Glutamic Acid 2.19 9.67 4.25 3.22
Glutamine 2.17 9.13   5.65
Arginine 2.17 9.04 12.48 10.76
Lysine 2.18 8.95 10.53 9.74

What is pI?
 Amide Linkage
R1 O R2 O
+ - + -
H3N C C O + H3N C C O
H H
- H2O

H O R2 O
+
H3N C C N C C O-
R1 H H
Amide Linkage

- R2 O
H O
+ + -
H3N C C N C C O
R1 H H
 Amide Linkage and Peptides

- O R3
COO
C H H CH
R1 LINKAGE
AMIDE C N C N double-bond
- Some C Ncharacter
H H R2 O H

1 unit of amino acid

 Protein Structure
Primary Structure: due to covalent peptide bonds of amino acids.

H O R O H O R O
+
HN3 C C (N C C )n N C C N C C O-
R H H H R H H

N-terminal amino acid C-terminal amino acid

Primary Structure of Protein
 Secondary Structure
Formation by hydrogen bonding between peptide bond
   
   
C O H N

Small negative charged oxygen atom = 

Small positive charged hydrogen atom = 

Kinds of Secondary Structure:

1.  - Helix
2. Pleated sheets structure
A. Parallel
B. Anti-parallel
Secondary Protein Structure
Secondary Structure of Protein
Secondary Structure of Protein
Tertiary Structure Aggregation of individual protein.

1. Hydrophobic attraction: the close association

attraction of hydrocarbon side-chains.
2. Ionic bond: between positively charged groups and
negatively charged groups.
3. Hydrogen bonds
4. Disulfide bonds

A protein has size and shape as well as unique arrangement through

hydrogen, ionic, hydrophobic and disulfide bonds.
i
i
i
i
i
Tertiary Structure of Protein
Tertiary Structure of Protein
Tertiary Structure of Protein
 Quaternary Structure

A protein has size and shape as well as unique

arrangement of its polypeptide chains. (Aggregation
of several peptide chains to form a definite molecule
by ionic bond, hydrogen bond, and/or hydrophobic
bond).
Quaternary Structure of Protein
Quaternary Structure of Protein
PROTEIN DETERMINATION
Primary Structure of Protein
Secondary Protein Structure
ii

i
 Protein Determination Methods

1. Kjeldahl Method.
2 Dye Binding Method.
3. Biuret Method.
4. Lowry Method.
5. Ultraviolet Method.
 Kjeldahl Method - Nitrogen Determination
(1) Digestion to conver nitrogen into an ammonium ion (NH4+).
+ conc. H2SO4
+ a catalyst (Copper sulfate)

(2) Neutralize NH4+ with NaOH to get NH3

(3) Steam distillation of NH3 and trap in boric acid.
(4) Titrate with hydrochloric acid.

Calculation:
Gram nitrogen/ gram of sample =
*(ml of sample - ml of blank)  N (normality) of standard acid 
0.014g/meq
weight of sample

 * ml of hydrochloric acid required to titrate sample solution.

Disadvantages: not all Nitrogen is protein.
Purine
Pyrimidine DNA, RNA, etc.
Urea
Many plant tissues have > 50% non-protein
Nitrogen.
% Nitrogen  6.25 = % Protein
Primary Structure of Protein
 Aliphatic Amino Acids
Valine (VAL) Leucine (LEU)

H H
+ - + -
NH 3 C COO
NH3 C COO
CH 2
CH
CH
CH3 CH3 CH 3 CH 3
 Basic Amino Acids

Lysine (LYS) Arginine (ARG)

H
+ -
H
NH3 C COO + -
NH 3 C COO
CH2
CH 2
CH2
CH 2
CH2
CH2 CH 2 NH +
2
+
NH 3 NH C NH 2
Conversion Factors from Nitrogen to Protein
for Foods

6.25 6.38 5.83 5.70 5.30

Corns Milk Whole wheat Wheat flour Nuts

Eggs   Barley    

Peas   Oats    

Meat   Rye    

Beans   Millet    
Conversion of Nitrogen Content to Protein Content
Amino Acid Nitrogen % Conversion Factor from
R=NH2CHCOOH Nitrogen to Protein

R
(56 / 174) x 100 = 32.2 3.11
(CH ) 3
2

NH C NH 2
NH

Arginine
(14 / 75) x 100 = 18.7 5.36
R

H
Glycine
R

CHOH
(14 / 119) x 100 = 11.76 8.50
CH 3

Threonine
R

(14 / 131) x 100 = 10.69 9.36

CH
CH 3 CH 2 CH 3

Isoleucine
R

CHOH
(14 / 119) x 100 = 11.76 8.50
CH 3
Threonine

(14 / 131) x 100 = 10.69 9.36

CH
CH 3 CH 2 CH 3
Isoleucine
 Dye Binding Method
Principle:At low pH, basic groups of protein are (+) charged. These
will quantitatively bind a (-) charged dye.
What are these basic amino acids with positive charge at low pH?
+
NH 3
CH 2 H
+
CH 2 CH 2 N C NH 2
Lysine
CH 2 CH 2 NH2
CH2 O CH 2 Arginine
H
CH N C CH
N C C N
H H
O CH 2
C NH+

HC CH Histidine
N
H
 Dye Binding Method
Acid Orange 12:

-
SO3
HO
N=N

Procedure:
1. Mix protein, dye, buffer pH = 2.
2. Filter or centrifuge.
3. Measure absorbance of filtrate.
 Dye Binding Method
Absorbance of dye bound by protein = A initial (free) die concentration - A.
filtrate die concentration

2
Absorbance at 470 nm

2
x
x
Skim milk
11 x

x
6 8 10 12 14 16

% Protein (Kjeldahl)
 Dye Binding Method

Factors Influencing Dye Binding determination:

1. Temperature
2. Non-proteins.
3. Buffers systems.
4. Protein quality.
 Biuret Method

Principles: Cu++ in alkaline solution form complexity with

peptide bonds - give pinkish-purple color.

Measure the intensity of color at 540 nm.

A at 540 nm

% Protein (Kjeldalh)
 Lowry Method
• Cu++ in alkaline solution to form complexity with protein.
• Cu++ catalyses oxidation of phenol group of tyrosine with
phosphomolybdic-phosphotungstic acid.
Absorbance at 750 nm

 g of protein (Kjeldahl)
 Ultra-violet Absorption (UV) at 280 nm

1. Chromophoric side chains of aromatic amino acids

(Tyrosine, Tryptophan).
2. Absorption at 280 nm. “Non-destructive means to
determine protein”.
3. Calculation of protein concentration based upon
absorption
 Fluorescence Method

Tyrosine and Tryptophaneare fluorescent compounds.

Excite the amino acids at 280 nm.
Measure emission at 348 nm.
 
Advantage: more sensitive than UV absorption.
 Fluorescence Method

What is fluorescence and how to measure it?

Excited State
Emits radiation (fluorescence)
Decay yields fluorescence
Energy At longer wavelength

Ground State

By using specific  (wavelength) to excite and measure output at

a specific . It is rather specific.
 Fluorescence Method

Fluorescence at 348 nm

mg of protein/ml of solution
Determination for Amino Acid Compositions of Proteins

A. Hydrolysis
1. Overnight in 6 M HCl at 100 C.
2. Enzymes.
 
B. Separation by ion exchange chromatography.
Mechanism of Ion-Exchange Chromatography of
Amino Acids
Na
+ pH 2
+
H3N
-
SO3
COOH
+
Na
OH
- +
So3 H3N
COOH
Exchange Resin

-
SO3 H3N+
pH3.5
COOH

-
OH
So3 +
H3N
+ - + -
Na COO H OH = H2O

+
Na

-
SO3 H3N
+
- + -
COO H OH = H2O
- + pH4.5
So3 Na
Chromatogram of Amino Acids

Moles/Liter VAL

ALA

HIS LEU

ASP
LYS GLU

pH 2.25 pH 3.25 pH4.25

 Protein Denaturation
Denaturation

o o o
Changes in 2 , 3 , or 4 structure by
• Heat.
• Heavy metals (Hg is most common).
• pH (trichloroacetic acid or phosphotungstic acid)
• Salt (NaCl or ammonium sulfate [NH4]2 SO4)

Reasons for Precipitating Proteins

1. Purify, concentrate protein.
2. Remove protein which cause turbidity or emulsion troublesome.
Secondary Protein Structure
Secondary Protein Structure
Tertiary Protein Structure
 Protein Denaturation
Effects of Denaturation
1. Decreased solubility.
2. Alteration of size and shape
3. greater reactivity
4. Decreased biological activity (enzyme +
immune proteins)
5. Increased sensitivity to electrolytes.
6. Nutritive value.
Protein Quality
Proteins
 What is Essential Amino Acids?

Amino acids which the body cannot make (or make

enough of) for protein synthesis due to lack of enzymes.

Essential Amino Acids:

Histidine, Isoleucine, Leucine, Lysine,
Methionine, Phenylalanine, Threonine, Valine
Proteins
Limiting Amino Acid
The essential amino acid which is lacking in the protein to have a
balanced protein.

Product Limiting Amino Acid

Corn Lysine
Oats Lysine
Rice Lysine
Wheat Lysine
Cow’s Milk Methionine
Potato Methionine
Green Pea Methionine
Cotton Seed Isoleucine
Beef Valine
Chicken Tryptophan
Protein Quality Determination

1. Protein Efficiency Ratio.

2. Biological Value.
3. Net Protein Utilization.
 Protein Efficiency Ratio

For labeling purposes

1. If PER = casein (2.5), the RDA = 45 g/day.

2. If 0.5 < PER < 2.5, then RDA = 65 g/day.
3. If PER < 0.5 (20% of casein), then “not a
significant source of protein”.
 Protein Efficiency Ratio Determination

1. Male lab rats ≥ 21 days,  28 days of age, at least 10 rats/group.

2. Feed a standardized diet containing salt mix, vitamins, cotton seed oil,
cellulose, starch or sucrose + water for 28 days.
3. Measure weight gain and food intake at regular intervals, not > 7 days.
4. PER = Weight Gain/Gram of Protein in Diet.
5. Usually normalized for casein = 2.5.
6. Determine protein quality of sample as ratio of sample PER to reference
casein PER.

Protein Efficiency Ratio = Gain in weight per gram protein taken.

 Protein Efficiency Ratio for Foods

Product Product PER

Rice 100% 2.30

Rice 70% + Black Beans 30% 2.70
50% + Black Beans 50% 2.60
20% + Black Beans 80% 1.30
Black Beans 100% NIL

Corn 100% < 2.0

+ 0.4% Lysine + 0.07% Tryptophan 2.14
Corn 50% + Black Beans 50% 2.05
.
 Protein Efficiency Ratio for Foods

Product PER
Soybean 2.32
Cotton Seed 2.25
Egg 3.90
Chick Peas 1.68
Peanuts 1.65
Kidney Beans 0.88
 Biological Value & Net Protein Utilization

BV = Retained Nitrogen (nitrogen intake - fecal &

urinary nitrogen) /Absorbed Nitrogen (nitrogen intake -
fecal nitrogen)
 
NPU = Retained Nitrogen / Intake Nitrogen = BV 
Digestibility