AMINO ACIDS

Contents
1. Introduction
2. Aminocidopathies
3. Amino Acid Analysis
Introduction
i. Building blocks of proteins
ii. Growth, repair and maintenance of cells

Peptide bond

Amino Acid
Introduction
i. Nutritionally essential amino acids is supplied by
diet (e.g. phenylalanine, lysine, etc.)
ii. Some amino are produced in the body
(e.g. tyrosine, glutamine, etc.)
Contents
1. Introduction
2. Aminocidopathies
3. Amino Acid Analysis
Aminoacidopathies
 Class of inherited errors of metabolism
 Enzymes defects that inhibits the body’s ability to
metabolize certain amino acids
i. PKU vi. Homocystinuria
ii. Tyrosinemia vii. Citrullinemia
iii. Alkaptonuria viii. Arginosuccinic Aciduria
iv. MSUD ix. Cystinuria
v. Isovaleric Acidemia
 Aminoacidopathies

Metabolism of
Phenylalanine and Tyrosine
Aminoacidopathies
i. Phenylketonuria
 Absence of phenylalanine hydroxylase (PAH)
 Musty odor of urine

PAH

 Laboratory Tests
1. Guthrie test
2. Microfluorometric assay
Aminoacidopathies
i. Phenylketonuria
 Laboratory Tests
1. Guthrie test
 Semi quantitative bacterial inhibition assay
 Uses phenylalanine to facilitate bacterial growth
(B. subtilis and β-2-thienylalanine).
 Aminoacidopathies

Metabolism of
Phenylalanine and Tyrosine
Aminoacidopathies
ii. Type II Tyrosinemia
 ↓ Tyrosine aminotransferase

iii. Type III Tyrosinemia

 ↓ 4-hydroxyphenylpyruvate dioxygenase
Aminoacidopathies
v. Alkaptonuria
 Lack of homogentisate oxidase
 ↑ homogentesic acid in urine

vi. Type I Tyrosinemia

 ↓ fumarylacetoacetate hydrolase
 Phenylalanine hydroxylase

Phenylketonuria

Tyrosine aminotransferase Tyrosinemia II

Tyrosinemia III

4-hydroxyphenylpyruvate dioxygenase

Alkaptonuria Homogentisate oxidase

Fumarylacetoacetate hydrolase Tyrosinemia I

Aminoacidopathies
vi. Maple Syrup Urine Disease
 ↓ branched-chain α-ketoacid decarboxylase
 ↑ Isoleucine
↑ Leucine
↑ Valine
 Burnt sugar
odor of urine
Aminoacidopathies
vi. Maple Syrup Urine Disease
 Laboratory Tests
1. Modified Guthrie test
 uses branched chain α-ketoacid to facilitate bacterial
growth (containing B. subtilis and 4-azaleucine).
Aminoacidopathies
vii. Isovaleric Acidemia
 Deficiency of isovaleryl-CoA dehydrogenase
 ↑ Isovaleric acid
 Sweaty feet odor
Aminoacidopathies
viii. Homocystinuria
 Lack of cystathionine β- synthetase
 ↑ Homocysteine
Methionine
Aminoacidopathies
viii. Homocystinuria
 Laboratory Tests
1. Modified Guthrie test
 Uses methionine to facilitate bacterial growth
(containing B. subtilis and L-methionine sulfoximime).
Aminoacidopathies
ix. Citrullinemia
x. Argininosuccinic aciduria
Aminoacidopathies
ix. Citrullinemia
i. Type I citrullinemia
 Lack of arginonosuccinic acid synthetase (ASAS)

Urea Cycle
Aminoacidopathies
ix. Citrullinemia
i. Type II citrullinemia
 Mutation of the gene that encodes for protein citrin

Urea Cycle
Aminoacidopathies
x. Argininosuccinic aciduria
 Lack of argininosuccinic acid lyase (ASL)

Urea Cycle
Aminoacidopathies
ix. Citrullinemia
x. Argininosuccinic aciduria
Aminoacidopathies
xi. Cystinuria
 Defect in amino acid transport system
 Inadequate reabsorption of cystine in the kidneys
Aminoacidopathies
 Class of inherited errors of metabolism
 Enzymes defects that inhibits the body’s ability to
metabolize certain amino acids
i. PKU vi. Homocystinuria
ii. Tyrosinemia vii. Citrullinemia
iii. Alkaptonuria viii. Arginosuccinic Aciduria
iv. MSUD ix. Cystinuria
v. Isovaleric Acidemia
Disease Enzyme deficiency Amino acid increased
PKU Phenylalanine hyroxylase Phenylalanine
Tyrosinemia I Fumarylacetoacetate hydrolase Fumarylacetoacetate
Tyrosinemia II Tyrosine aminotranferase Tyrosine
Tyrosinemia III 4-Hydroxyphenylpyruvate p-Hydroxyphenylpyruvic
oxidase acid
Alkaptonuria Homogentisate oxidase Homogentisic acid (HGA)
MSUD Branched-chain Leucine, Isoleucine,
α-ketoacid decarboxylase Valine
Isovaleric acidemia Isovaleryl-CoA dehydrogenase Leucine, Isovaleric acid
Homocysteine,
Homocystinuria Cystathionine-β synthetase Methionine
Citrullinemia Arginosuccinic acid synthetase Citrulline, ammonia
Arginosuccinic Argininosuccinic acid lyase Argininosuccinic acid,
aciduria Citrulline, ammonia
Cystinuria Defective AA transport system Cystine ppt
Aminoacidopathies
 Diagnosis
i. 6-8 hours fasting
ii. Collected in heparin tube with the plasma
iii. Deproteinization performed within 30 minutes
iv. Screening by TLC stained with ninhydrin (blue)
v. Separated and quantitated by Ion exchange
chromatography and HPLC reversed-phase system
or capillary electrophoresis
End of Part I
PROTEIN
Contents
I. Introduction
II. Synthesis and Catabolism
III. Structure
IV. Classification
V. Plasma Proteins
VI. Other Plasma Proteins
VII. Total Protein Abnormalities
VIII. Method of Analysis
Introduction
i. Transport
ii. Structural proteins
iii. Receptors
iv. Enzymes
v. Transcription factors
Contents
I. Introduction
II. Synthesis and Catabolism
III. Structure
IV. Classification
V. Plasma Proteins
VI. Other Plasma Proteins
VII. Total Protein Abnormalities
VIII. Method of Analysis
II. Synthesis and Catabolism
II. Synthesis and Catabolism
II. Synthesis and Catabolism
i. Disintegration of protein to amino acids
1. Lysosomal pathway
 degrades extracellular proteins
2. Cytosolic pathway
 degrades intracellular proteins
Contents
I. Introduction
II. Synthesis and Catabolism
III. Structure
IV. Classification
V. Plasma Proteins
VI. Other Plasma Proteins
VII. Total Protein Abnormalities
VIII. Method of Analysis
Structure
 Levels of protein structure
i. Primary
ii. Secondary
iii. Tertiary
iv. Quarternary
Structure
 Levels of protein structure
i. Primary
 Amino acids in a specific sequence
ii. Secondary
 Regularly repeating structures stabilized by hydrogen
bonds between the amino acids within the protein
 α-helix and β pleated sheet
Structure
 Levels of protein structure
iii. Tertiary
 Overall conformation (fold) of the protein molecule
 Due to interaction of side chains (e.g. ionic)
iv. Quarternary
 Interaction of more than 1 protein molecule or subunits
Contents
I. Introduction
II. Synthesis and Catabolism
III. Structure
IV. Classification
V. Plasma Proteins
VI. Other Plasma Proteins
VII. Total Protein Abnormalities
VIII. Method of Analysis
Classification by Protein Functions
1. Enzymes
2. Hormones
3. Transport proteins
4. Immunoglobulins (antibodies)
5. Structural proteins
6. Storage proteins
7. Energy Source
8. Osmotic force
9. Homeostasis
10. Acid-Base Balance
 Classification by Protein Functions
Classification Function
Enzymes  Catalyze chemical reactions
Hormones  Chemical messengers that control
the actions of specific cells or organs
Transport proteins  Transport ions and macromolecules
across a biologic membrane
 Classification by Protein Functions
Classification Function
Immunoglobulins  Produced by B-cells that mediates
immune response
Structural proteins  Fibrous proteins that are the
structure of cells and tissues
Storage proteins  Serves as reserves of metal ions and
amino acids (e.g. ferritin)
 Classification by Protein Functions
Classification Function

Energy source  Plasma proteins serve as a reserve

source of energy

 Plasma proteins function in the

Osmotic force distribution of water throughout the
compartments of the body.

Hemostasis  Participation in coagulation in blood

Acid base balance  Participation as buffers to

blood maintain pH
Classification by Protein Functions
1. Enzymes
2. Hormones
3. Transport proteins
4. Immunoglobulins
5. Structural proteins
6. Storage proteins
7. Energy Source
8. Osmotic force
9. Homeostasis
10. Acid-Base Balance
Classification by Protein Structure
1. Simple Proteins
2. Conjugated Proteins
Classification by Protein Structure
1. Simple Proteins
 Contain peptide chains composed of only amino acids.
 May be globular (hormone, enzymes, transport) or
fibrous (structural)
Classification by Protein Structure
2. Conjugated Proteins
 Consist of a protein and a nonprotein prosthetic group
a. Metalloprotein
 Metal ions attached – Ferritin, Ceruloplasmin
 Complex metal – Hemoglobin, Flavoproteins

b. Lipoproteins
 Lipids attached
 HDL, LDL, VLDL
Classification by Protein Structure
2. Conjugated Proteins
 Consist of a protein and a nonprotein prosthetic group
c. Mucoproteins or proteoglycans
 With higher carbohydrate
 Mucin

d. Glycoproteins
 10%-40% carbohydrate
 Haptoglobin and α1-antitrypsin

e. Nucleoproteins
 Nucleic acids attached
 Chromatin
Classification by Protein Structure
1. Simple Proteins
2. Conjugated Proteins
a. Metalloprotein
b. Lipoprotein
c. Mucoprotein and glycoprotein
d. Nucleoprotein
Contents
I. Introduction
II. Synthesis and Catabolism
III. Structure
IV. Classification
V. Plasma Proteins
VI. Other Plasma Proteins
VII. Total Protein Abnormalities
VIII. Method of Analysis
IX. Protein in Other Body Fluids
Plasma Proteins
A. Albumin
B. Globulin
1. α1-Globulins
2. α2- Globulins
3. β-Globulins
4. γ-Globulins
α1 β1 γ

Alb α2 β 2
Plasma Proteins

Function
• Indicator of nutrition
Prealbumin • Binds thyroid hormones (T3, T4)
• Binds retinol-binding protein
Albumin • Binds bilirubin, steroids, fatty acids
• Major contributor to oncotic pressure
 Plasma Proteins

α1-Globulins Function
α1 - Antitrypsin • Acute phase reactant
• Protease inhibitor
α1 - Fetoprotein • Principal fetal protein
• ↑ spina bifida, ↓ - Down syndrome
α1 - Acid glycoprotein • Acute phase reactant
 Plasma Proteins

α1-Globulins Function
α1 – Lipoprotein • Transport lipids (HDL)
α1 - Antichymotrypsin • Inhibits serine proteinases
Inter-α-trypsin inh. • Inhibits serine proteinases
Gc-globulin • Transports Vit. D and binds actin
Plasma Proteins

α1-Globulins
α1 - Antitrypsin α1 – Lipoprotein
α1 - Fetoprotein α1 - Antichymotrypsin
α1 - Acid glycoprotein Inter-α-trypsin inhibitor
Gc-globulin
 Plasma Proteins

α2-Globulins Function
Haptoglobins • Acute Phase reactant, Binds Hgb.
Ceruloplasmin • Contains copper
• ↓ - Wilson’s disease. Menkes synd.
α2 - Macroglobulin • Inhibits protease
 Plasma Proteins
β-Globulins Function
Pre-β-lipoprotein • Transports lipids (VLDL triglyceride)
β-Lipoprotein • Transports lipids (LDL cholesterol)
Trasferrin • Transport Iron,↑-IDA,↓ Hemochromatosis
Hemopexin • Acute phase reactant, Binds heme
β2-Microglobulin • Component of HLA molecules
 Plasma Proteins
β-Globulins Function
C4, C3, C1q complement • Immune response (Opsonins)
Fibrinogen • Precursor of fibrin clot
C-reactive protein • Acute phase reactants
• Promotes phagocytosis
 Plasma Proteins

γ-Gamma-Globulins Function
Immunoglobulin G • Antibodies
Immunoglobulin A • Antibodies in secretions
Immunoglobulin M • Antibodies in early response
Immunoglobulin E • Antibodies (reagen, allergy)
Immunoglobulin E • Surface antibody
Plasma Proteins
A. Albumin
B. Globulin
1. α1-Globulins
2. α2- Globulins
3. β-Globulins
4. γ-Globulins
α1 β1 γ

Alb α2 β 2
Contents
I. Introduction
II. Synthesis and Catabolism
III. Structure
IV. Classification
V. Plasma Proteins
VI. Other Plasma Proteins
VII. Total Protein Abnormalities
VIII. Method of Analysis
IX. Protein in Other Body Fluids
 Other Proteins

Function
•Oxygen carrier in muscles
Myoglobin •Cardiac marker (AMI)
•↑ 2-3 hrs of onset, peak at 8-12 hrs
Troponin (cTn) •Cardiac marker for acute coronary
syndrome
Fibronectin • Cellular interaction
• Placental adherence to the uterus
Fetal fibronectin (fFN) • ↑ - Preterm labor and delivery
 Other Proteins

Function
Cross-Linked • Proteolytic fragment of collagen I
C-Telopeptides • Marker of bone resorption
• Syn: Prostaglandin D synthase
β-Trace Protein • Marker for CSF leakage
Cystatin C • Cysteine proteinase inhibitor
• Marker for kidney function (GFR)
• Fibrous protein aggregates formed
Amyloid from alteration of β pleated sheats
• ↑ Amyloidoses
Contents
I. Introduction
II. Synthesis and Catabolism
III. Structure
IV. Classification
V. Plasma Proteins
VI. Other Plasma Proteins
VII. Total Protein Abnormalities
VIII. Method of Analysis
IX. Protein in Other Body Fluids
Hypoproteinemia

TP Albumin Globulin Disease

N, Hepatic Damage
↓ ↑ • Cirrhosis β-γ bridging
↓ • Hepatitis ↑ γ-globulins
Infections
• Acute - α1 , α2 globulins
• Chronic - ↑ α1, α2 γ,
globulins
 Hypoproteinemia

TP Albumin Globulin Disease

↓ ↓ N Inadequate diet
Nephrotic syndrome
↑α2,β-globulins;↓γ-
globulins
↓ N ↓ Immunodeficiency syndrome
Hypoproteinemia

TP Albumin Globulin Disease

↑ ↑ ↑ Dehydration
Multiple myeloma
↑ N ↑ Monoclonal &
Polyclonal gammopathies
Contents
I. Introduction
II. Synthesis and Catabolism
III. Structure
IV. Classification
V. Plasma Proteins
VI. Other Plasma Proteins
VII. Total Protein Abnormalities
VIII. Method of Analysis
IX. Protein in Other Body Fluids
 Method of Analysis

Total Protein Albumin and Globulin

Kjeldahl Salt Precipitation
Refractometry Dye Binding (Methyl orange, HABA, BCG, BCP)
Biuret Electrophoresis (Coomassie blue)
Dye Binding
 Total Protein

Method Principle
Reference method. Assume average
1. Kjeldahl
nitrogen content of 16%
2. Refractometry Measurement of refractive index due to
solutes in serum
3. Biuret Formation of violet-colored chelate between
Cu2+ ions and peptide bonds
4. Dye Binding Protein binds to dye and causes a spectral
shift in the absorbance maximum
1. Kjeldahl
 Acid precipitation (TCA or tungstic acid) of
protein with measurement of total nitrogen
a. Kjeldahlization – conversion of nitrogen to ammonia
Nitrogen H2SO4 NH3
b. Ammonia measurement
1. Nessler’s reaction (HgI2/KI)

Ammonia + Nessler’s rgt Gum ghatti 

Yellow solution (NH2Hg2I)
Berthelot reaction
2.
Ammonia + alkaline hypochlorite Na nitroprusside
Indophenol blue
2. Refractometry
 Measurement of refractive index (velocity of
light in air and water) due to solutes in serum.
3. Biuret
 Formation of violet-colored chelate between
Cu2+ ions and peptide bonds (540 nm)
 Composition
1. Cupric ions – breaks the peptide bonds
2. Tartrate salt – keeps copper in solution
3. Potassium iodide – stabilizes cupric ions
3. Dye binding
 Protein binds to dye and causes a spectral
shift in the absorbance maximum of dye.
1. Bromphenol blue
2. Ponceau S
3. Amido black 10B
4. Lissamine green
5. Coomassie brilliant blue
 Total Protein

Method Principle
Reference method. Assume average
1. Kjeldahl
nitrogen content of 16%
2. Refractometry Measurement of refractive index due to
solutes in serum
3. Biuret Formation of violet-colored chelate between
Cu2+ ions and peptide bonds
4. Dye Binding Protein binds to dye and causes a spectral
shift in the absorbance maximum
 Method of Analysis

Total Protein Albumin and Globulin

Kjeldahl Salt Precipitation
Refractometry Dye Binding (Methyl orange, HABA, BCG, BCP)
Biuret Electrophoresis (Coomassie blue)
Dye Binding
 Albumin

Method Principle

Globulins are precipitated

Salt Precipitation Albumin in supernatant is quantitated by
biuret reaction

Dye Binding Principle

Methyl orange Nonspecific for Albumin
HABA Many Interferences (salicylates, bilirubin)
BCG (Bromcresol green) Sensitive, Most commonly used dye
BCP (Bromcresol purple) Specific, Sensitive and Precise
 Method of Analysis

Total Protein Albumin and Globulin

Kjeldahl Salt Precipitation
Refractometry Dye Binding (Methyl orange, HABA, BCG, BCP)
Biuret Electrophoresis (Coomassie blue)
Dye Binding
 Albumin and Globulin

Method Principle
Proteins separated based on electric
Electrophoresis charge densities
Electrophoresis

 Cellulose acetate/agarose gel (support media)

 After separation, protein fractions are immersed in
acid solution then stained by dyes (Coomassie blue)
Electrophoresis

 The medium is placed in scanning densitometer

which compute the area under the absorbance
 Electrophoresis

 Reference values
 Albumin, 53-65% (3.5-5 g/dL)
 α1–Globulin, 2.5-5% (0.1-0.3 g/dL)
 α2–Globulin, 7-13% (0.6-1.0 g/dL)
 β–Globulin, 8-14% (0.7-1.1 g/dL)
 γ–Globulin, 12-22% (0.8-1.6 g/dL)
Electrophoresis
Electrophoresis (High Resolution)
 Uses higher voltage couple with a cooling system
and more concentrated buffer
 Method of Analysis

Total Protein Albumin and Globulin

Kjeldahl Salt Precipitation
Refractometry Dye Binding (Methyl orange, HABA, BCG, BCP)
Biuret Electrophoresis (Coomassie blue)
Dye Binding
Contents
I. Introduction
II. Synthesis and Catabolism
III. Structure
IV. Classification
V. Plasma Proteins
VI. Other Plasma Proteins
VII. Total Protein Abnormalities
VIII. Method of Analysis
Thank You!