2. Amino Acids
C H
HO C H
CH 2OH
O O
C OH
NH2 C H
CH 3
L -Glyceraldehyde L - Alanine
Zwitterion
O
C OH
NH 2 C H
CH 3
O
C O
-
NH 3
+
C H
CH 3
3. Evidence of Zwitterion in Amino Acids at Neutral pH
NH3
+
CH2 C O
-
O
CH3COOH
CH3CH2NH2
CH3 C O
-
CH3 CH2 NH3
+
O
At Neutral pH
1.
CH3CH2COOH
CH3 CH CH3
NH2
CH3 CH C O
-
NH3
+
O
Melting Point
-101 C
295 C
22 C
2.
4. 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?
C OH
O
NH2
C OH
O
= 2.2
NH 2 = 9.5
5. DISSOCIATION OF THE CARBOXYL GROUP (pKa)
R C COO
-
NH3
+
H
H
NH3
+
R C COOH + H+
H
+
K1 =
H
NH3
+
R C COO
-
R C COOH
NH3
+
H
H
+
= K1
H
NH3
+
R C COOH
R C COO
-
NH3
+
H
pH = pK1 Log
H
NH3
+
R C COO
-
R C COOH
NH3
+
H
[ ]
+
6. DISSOCIATION OF THE AMINO GROUP (pKb)
R C COO
-
NH2
H
H
+
H
NH3
+
R C COO- +
H
+
K2 =
H
NH2
R C COO
-
R C COO
-
NH3
+
H
H
+
= K2
H
NH2
R C COO
-
R C COO
-
NH3
+
H
pH = pK2 Log
H
NH2
R C COO
-
R C COO
-
NH3
+
H
[ ]
[ ]
+
7. [A-]
pH = pK + Log
[AH]
[COO-] [NH2]
pH - pK = Log or Log
[COOH] [NH3+]
RELATIONSHPI BETWEEN DISSOCIATION OF THE
AMINO GROUP (pKb) AND pH
8. 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
pKa = 2.2
Log
pKb = 9.5
Log
pH 11.5 9.3 2
[COOH]
[COO-]
[NH3+]
[NH2]
Effects of pH on the Concentration of Different Groups
9. pK' AND pI VALUES OF CERTAIN AMINO ACIDS
pK'1
a - COO-
pK'2
a - NH3+
pK'R
R Group
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
Histidine 1.82 9.17 6.00 7.58
Cysteine 1.71 10.78 8.33 5.02
Tyrosine 2.20 9.11 10.07 5.65
Arginine 2.17 9.04 12.48 10.76
Lysine 2.18 8.95 10.53 9.74
14. Amino Acids with Alcohol
Serine (SER) (a - amino b -
hydroxy propionate)
Threonine (THR) (a - amino b -
hydroxy butyrate)
NH3
+
C COO
-
H
CH2OH
NH3
+
C COO
-
H
CH3
CH OH
15. Amino Acids with additional Acidic Group
Aspartic Acid (ASP) (a -
amino succinate)
Glutamic Acid (GLU) (a - amino
glutarate)
NH3
+
C COO
-
H
CH2
COO
-
NH3
+
C COO
-
H
CH2
COO
-
CH2
16. Amino Acids with Amides of Acidic Amino Acids
Asparagine (ASN) (a -
amino b - succinamide)
Glutamine (GLN) (a -
amino g - glutaramide)
NH3
+
C COO
-
H
CH2
C NH2
O
NH3
+
C COO
-
H
CH2
CH2
C NH2
O
17. Basic Amino Acids
Lysine (LYS) (a, e - diamino
caproate)
Arginine (ARG) (a - amino d -
guanidine valerate)
NH3
+
C COO
-
H
CH2
CH2
CH2
CH2
NH3
+
NH3
+
C COO
-
H
CH2
CH2
CH2
NH C NH2
NH2
+
18. Histidine (HIS) (a - amino b -
imidazole propionate)
Basic Amino Acids
NH3
+
C COO
-
H
CH2
NH
HN
+
19. Sulfur-containing Amino Acids
Cysteine (CYS H) (a - amino
b - mercapto propionate)
Cystine (CYS-CYS) Di(a - amino b -
mercapto propionate)
NH3
+
C COO
-
H
CH2
SH
NH3
+
CH CH2 S S CH2 CH NH3
+
COO
-
COO
-
20. Methionine (MET) (a - amino
g - methyl mercapto butyrate)
Sulfur-containing Amino Acids
NH3
+
C COO
-
H
CH3
CH2
CH2
S
21. Aromatic Amino Acids
Phenylalanine (PHE) (a -
amino b - phenyl propionate)
Tyrosine (TYR) (a - amino b - [p -
hydroxyphenyl])
NH3
+
C COO
-
H
CH2
NH3
+
C COO
-
H
CH2
OH
22. Tryptophan (TRY) (a - amino b - indol
propionate)
Aromatic Amino Acids
H
NH3
+
C COO
-
H
CH2
N
24. AMIDE LINKAGE - Some double-bond character
H3N
+
C C O
-
R1 R2
O
H H
O
H3N
+
C C O
-
H O
R1
H3N
+
C C N C C O
-
H
H
R2 O
H2O
O
R2
H H
H3N
+
C C N
+
C C O
-
R1
O
-
H
-
+
Amide Linkage
25. Amide Linkage and Peptides
AMIDE LINKAGE - Some double-bond character
R1 C N C N C
COO
-
O
O
H
H
H R2
R3
C H CH
N
H
1 unit of amino acids
26. PROTEIN STRUCTURE
Primary Structure: due to covalent peptide bonds of amino acids.
N C C N C C N C C N C C
H
H H
H
H
H
O
O O
O
R
R
R
R
H
H3
+
N-terminal
amino acid
C-terminal
amino acid
( )n
Primary structure of a polypeptide chain showing the N- and
C- terminal amino acids.
31. Secondary Structure: due to hydrogen bonding between
peptide bond.
C O H N
d
-
d
d
d
-
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
40. 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.
41. SH HS S S + H2O
Dehydrogenation
Different Chemical Bonds in Tertiary Structure
47. 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).
52. 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.
53. Disadvantages: not all N is protein.
Purine
Pyrimidine DNA, RNA, etc.
Urea
Many plant tissues have > 50% non-protein N.
% N 6.25 = % Protein
54. Conversion Factors from Nitrogen to
Protein for Foods
Corns Milk Whole wheat Wheat flour Nuts
Eggs Barley
Peas Oats
Meat Rye
6.25 6.38 5.83 5.70 5.30
Beans Millet
55. 2. Dye Binding Method
Principle:At low pH, basic groups of protein are (+) charged. These
will quantitatively bind a (-) charged dye.
What are these basic groups?
NH3
+
CH
CH2
CH2
CH2
CH2
N
N
H
H
C
O
C
CH2
C NH
+
CH
CH
N
HC
H
C
O
N
H
CH2
CH2
CH2
N
H
C
NH2
NH2
+
Lysine
Arginine
Histidine
56. Acid Orange 12:
N = N
HO
SO3
-
Procedure:
1. Mix protein, dye, buffer pH = 2.
2. Filter or centrifuge.
3. Measure optical density (O.D.) of filterate.
Dye Binding Method
57. Abosrbance of dye bound by protein A dye initial - A. filterate
% Protein (Kjeldahl)
6 8 10 12 14 16
Skim milk
Dye Binding Method
58. Factors Influencing Dye Binding determination:
1. Temperature
2. Non-proteins.
3. Buffers systems.
4. Protein quality.
Dye Binding Method
59. 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.
% Protein (Kjeldalh)
60. 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
61. 5. Ultra-violet Absorption (UV) at 280 nm
1. Chromophoric side chains of aromatic amino acids
(Trosine, Tryptophan).
2. Absorption at 280 nm. “Non-destructive means to
determine protein”.
3. Calculation protein conc. based upon absorption
62. 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.
64. Fluorescence Method
What is fluorescence and how to measure it?
Ground State
Excited State Emits radiation
(fluorescence)
Decay yields
fluorescence at
longer wavelength
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.
65. Amino Acid Determination
A. Hydrolysis
1. Overnight in 6 M HCl at 100 C.
2. Enzymes.
B. Separation by ion exchange.
66. MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS
Na
+
Na
+
H
+
OH
-
= H2O
= H2O
OH
-
H
+
Na
+
Na
+
COO
-
H3N
+
Na
+
OH
COO
-
N
+
H3
H3N+
COOH
OH
COOH
COOH
H3N
+
So3
-
SO3
-
SO3
-
So3
-
So3
-
SO3
-
H3N
+
Exchange Resin
pH 2
pH3.5
pH4.5
68. 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 [NH4]2 SO4)
Reasons for Precipitating Proteins
1. Purify, concentrate protein.
2. Remove protein which cause:
turbidity/emulsion/troublesome.
69. 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.
70. 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
71. 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
Cow’s Milk Methionine
Potato Methionine
Chick Pea Methionine
Green Pea Methionine
Cotton Seed Isoleucine
Beef Valine
Product Limiting Amino Acid
Chicken Tryptophan
73. 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 < PER < 2.5, then RDA = 65 g/day.
3. If PER < 0.5 (20% of casein), then “not a
significant source of protein”.
74. How does one determine PER?
1. Male lab rats e 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.
75. Protein Efficiency Ratio for Different Foods
Product PER
Rice 100% 2.30
Rice 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
.
76. 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
77. 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
OTHER PROTEIN QUALITY DETERMINATION METHOD
