PROTEINS

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PROTEINS

• Polymer of amino acids linked by amide linkage.
•  
• Functions
• Nutritional growth, digestion, metabolism
  (enzymes).
• Physical structure cheese, bread, foaming agent,
  gel-forming.

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Amino Acids
L -Glyceraldehyde
L - Alanine
Zwitterion

O
O
-
C O
C OH

NH
C H
NH
C H
2
3
CH
CH
3
3
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Evidence of Zwitterion in Amino Acids at Neutral
pH
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L is naturally-occurring form for amino acids.
Ionizable groups
    Zwitterion pKa for   pKb
for What form do we get at pH 1? pH
3.5? pH 7.5? pH 9.5? and pH 11.5?
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DISSOCIATION OF THE CARBOXYL GROUP (pKa)
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DISSOCIATION OF THE AMINO GROUP (pKb)
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RELATIONSHPI BETWEEN DISSOCIATION OF THE AMINO
GROUP (pKb) AND pH

A- pH pK Log AH  
COO- NH2 pH - pK Log or Log
COOH NH3  
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Effects of pH on the Concentration of Different
Groups
pKa 2.2 Log  
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
 
 
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pK' AND pI VALUES OF CERTAIN AMINO ACIDS
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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
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Aliphatic Amino Acids
Glycine (GLY) (a - amino ethanoate)
Alanine (ALA) (a - amino propionate)
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Aliphatic Amino Acids
Valine (VAL) (a - amino isovalerate)
Leucine (LEU) (a - amino isocaproate)
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Aliphatic Amino Acids
Isoleucine (ILE) (a - amino b - methyl-valerate)
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Amino Acids with Alcohol
Serine (SER) (a - amino b - hydroxy propionate)
Threonine (THR) (a - amino b - hydroxy butyrate)
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Amino Acids with additional Acidic Group
Aspartic Acid (ASP) (a - amino succinate)
Glutamic Acid (GLU) (a - amino glutarate)
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Amino Acids with Amides of Acidic Amino Acids
Asparagine (ASN) (a - amino b - succinamide)
Glutamine (GLN) (a - amino g - glutaramide)
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Basic Amino Acids
Lysine (LYS) (a, e - diamino caproate)
Arginine (ARG) (a - amino d - guanidine valerate)
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Basic Amino Acids
Histidine (HIS) (a - amino b - imidazole
propionate)
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Sulfur-containing Amino Acids
Cysteine (CYS H) (a - amino b - mercapto
propionate)
Cystine (CYS-CYS) Di(a - amino b - mercapto
propionate)
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Sulfur-containing Amino Acids
Methionine (MET) (a - amino g - methyl mercapto
butyrate)
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Aromatic Amino Acids
Phenylalanine (PHE) (a - amino b - phenyl
propionate)
Tyrosine (TYR) (a - amino b - p -
hydroxyphenyl)
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Aromatic Amino Acids
Tryptophan (TRY) (a - amino b - indol propionate)
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Secondary Amino Acids
Proline (PRO) (2- carboxy pyrollidine)
Hydroxyproline (HYPRO) (2- carboxy-4-hydroxy-pyrol
lidine)
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AMIDE LINKAGE - Some double-bond character
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Amide Linkage and Peptides
AMIDE LINKAGE - Some double-bond character
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PROTEIN STRUCTURE
Primary Structure due to covalent peptide bonds
of amino acids.
Primary structure of a polypeptide chain showing
the N- and C- terminal amino acids.
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Secondary Structure of Protein
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Primary Structure of Protein
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Primary Structure of Protein
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Primary Structure of Protein
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Secondary Structure due to hydrogen bonding
between peptide bond.
Small negative charged oxygen atom d- Small
positive charged hydrogen atom d Kinds of
Secondary Structure 1. a - Helix 2. Pleated
sheets structure A. Parallel B. Anti-parallel
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a - Helix
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Secondary structure
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Secondary Structure of Protein
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Secondary Structure of Protein
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Secondary Structure of Protein
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Secondary Structure of Protein
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Secondary Structure
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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.
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Different Chemical Bonds in Tertiary Structure

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Tertiary Structure of Protein
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Secondary Structure of Protein
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Tertiary Structure of Protein
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Tertiary Structure of Protein
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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).
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Quaternary Structure of Protein
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Quaternary Structure of Protein
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Quaternary Structure of Protein
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PROTEIN DETERMINATION METHODS
1. Kjeldahl Method. 2 Dye binding
Method. 3. Biuret Method. 4. Lowry
Method. 5. Ultraviolet Method.
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Kjeldahl Method --- Nitrogen Determination
(1) Digestion conc. H2SO4 a
catalyst nitrogen converted into an ammonium
ion. (2) Neutralize to get NH3 with NaOH (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 standard
acid ? 0.014g/meq weight of sample  
ml of hydrochloric acid required to titrate
sample solution.
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Disadvantages not all N is protein. Purine Pyr
imidine DNA, RNA, etc. Urea Many plant tissues
have gt 50 non-protein N. N ? 6.25 Protein
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Conversion Factors from Nitrogen to Protein for
Foods
Corns
Milk
Whole wheat
Wheat flour
Nuts
Eggs
 
Barley
 
 
Peas
 
Oats
 
 
Meat
 
Rye
 
 
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2. Dye Binding Method
PrincipleAt low pH, basic groups of protein are
() charged. These will quantitatively bind a
(-) charged dye.  What are these basic groups?
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Dye Binding Method
Acid Orange 12
Procedure 1. Mix protein, dye, buffer pH
2. 2. Filter or centrifuge. 3. Measure optical
density (O.D.) of filterate.
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Dye Binding Method
Abosrbance of dye bound by protein A dye initial
- A. filterate
A. at 470 nm
Skim milk
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6
8
10
12
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Protein (Kjeldahl)
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Dye Binding Method
Factors Influencing Dye Binding
determination 1. Temperature 2. Non-proteins. 3.
Buffers systems. 4. Protein quality.
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3. 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)
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4. Lowry Method (one of most sensitive methods)

• Cu in alkaline solution to form complexity with
  protein.
• Cu catalyses oxidation of phenol group of
  tyrosine with phosphomolybdic-phosphotungstic
  acid.

A at 750 nm
g of protein (Kjeldahl)
m
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5. Ultra-violet Absorption (UV) at 280 nm

• Chromophoric side chains of aromatic amino acids
  (Trosine, Tryptophan).
• Absorption at 280 nm. Non-destructive means to
  determine protein.
• Calculation protein conc. based upon absorption

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6. Fluorescence Method
Tyrosine is a fluorescent compound. Tryptophane
is a fluorescent compound. Excite the amino
acids at 280 nm. Measure emission at 348
nm.   Advantage more sensitive than UV
absorption.
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Fluorescence Method
at 348 nm
Emitted Fluorescence
mg of protein/ml of solution
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Fluorescence Method
What is fluorescence and how to measure it?
Excited State
Emits radiation
(fluorescence)
Decay yields
fluorescence at
longer wavelength
Ground State
By using specific l (wavelength) to excite and
measure output at a specific l. It is rather
specific. Problems Turbidity/Quenching (self or
others)/Expensive/ Quantitation is difficult.
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Amino Acid Determination
A. Hydrolysis 1. Overnight in 6 M HCl at 100
C. 2. Enzymes.   B. Separation by ion exchange.
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MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO
ACIDS
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CHROMATOGRAM OF AMINO ACIDS
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Some Important Reactions of Proteins

• Denaturation
• Changes in 2o, 3o, 4o structure.
• By heat.
• Heavy metals (Hg is most common).
• pH (trichloroacetic acid, phosphotungstic acid)
• Salt (NaCl or ammonium sulfate NH42 SO4)
• Reasons for Precipitating Proteins
• Purify, concentrate protein.
• Remove protein which cause turbidity/emulsio
  n/troublesome.

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Protein Denaturation
Manifestation 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.
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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 Thr
eonine, Valine
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Limiting amino acid is the essential amino acid
which is lacking in the protein to have a
balanced protein.
Corn
Lysine
Oats
Lysine
Rice
Lysine
Wheat
Lysine
Sesame Seed
Lysine
Cows Milk
Methionine
Potato
Methionine
Chick Pea
Methionine
Green Pea
Methionine
Cotton Seed
Isoleucine
Beef
Valine
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PROTEIN QUALITY DETERMINATION
1. Protein Efficiency Ratio. 2. Biological
Value. 3. Net Protein Utilization.
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What are the measurements of protein quality?
For labeling purposes, one needs to know the
protein efficiency ratio. 1. If PER casein
(2.5), the RDA 45 g/day. 2. If 0.5 lt PER lt 2.5,
then RDA 65 g/day. 3. If PER lt 0.5 (20 of
casein), then not a significant source of
protein.
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• How does one determine PER?
• Male lab rats 21 days, 28 days of age, at
  least 10 rats/group.
• Feed a standardized diet containing salt mix,
  vitamins, cotton seed oil, cellulose, starch or
  sucrose water for 28 days.
• Measure weight gain and food intake at regular
  intervals, not gt 7 days.
• PER Weight Gain/Gram of Protein in Diet.
• Usually normalized for casein 2.5.
• Determine protein quality of sample as ratio of
  sample PER to reference casein PER.
• Protein Efficiency Ratio Gain in weight per
  gram protein taken.

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Protein Efficiency Ratio for Different Foods
Product PER Rice 100 2.30 R
ice 70 Black Beans 30 2.70 50 50 2.60
20 80 1.30 100 NIL Corn 0.4
Lysine 0.07 Tryptophan 2.14 Corn
(50) Black Beans (50) 2.05 .
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Protein Efficiency Ratio for Foods
Product PER   Soybean 2.32 Cotton Seed
Meal 2.25 Egg 3.90 Chick
Peas 1.68 Peanuts (ground nuts) 1.65 Kidney
Beans 0.88
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OTHER PROTEIN QUALITY DETERMINATION METHOD
Biological Value (BV) Net Protein Utilization
(NPU)   BV Retained Nitrogen (nitrogen intake -
fecal urinary nitrogen)/Absorbed Nitrogen
(nitrogen intake - fecal nitrogen)   NPU
Retained Nitrogen/Intake Nitrogen BV ?
Digestibility