PROTEIN

Contents
I.

II.
III. IV. V. VI. VII. VIII.

Introduction Synthesis and Catabolism Structure Classification Plasma Proteins Other Plasma Proteins Total Protein Abnormalities Method of Analysis

Introduction
i.

ii.
iii. iv. v.

Transport Structural proteins Receptors Enzymes Transcription factors

Contents
I.

II.
III. IV. V. VI. VII. VIII.

Introduction Synthesis and Catabolism Structure Classification Plasma Proteins Other Plasma Proteins Total Protein Abnormalities Method of Analysis

II. Synthesis and Catabolism

Steps in Protein Synthesis:

Activation Step. Bonding of the correctamino acid are bonded to the correct transfer RNA (tRNA) (catalyzed by the enzyme aminoacyltRNAsynthetase) Step 1: Initiation Step. AUG codon (amino acid methionine or Met) on mRNA signals both the interaction of the ribosome with mRNA and tRNA with the anticodons UAC. The interaction is on a particular region near the 5 end of the mRNA. The large ribosomal subunit has two binding sites: P and A site. The P site of the ribosome is where the initiator tRNA (containing the AUG codon) binds. The A site of the ribosome functions as the acceptor for the growing protein.

Step 2: Chain Elongation. When the next tRNA binds to the ribosome. The ribosomes move along them RNA and the protein is assembled as new amino acids are added. The preceding amino acid (Met at the start of translation) is covalently linked to the incoming amino acid with a peptide bond. Then initiator tRNA is released from

Step 3: Chain Termination. Termination of the polypeptide happens when the A site of the ribosome encounter a stop codon (UAA, UAG, or UGA).

Protein Synthesis

Catabolism Digestion of Proteins

Dietary Protein
1.Mouth. Saliva no effect on digestion
Blood Stream 2. Stomach : HCl denatures proteins

Pepsin: Hydrolyze peptide bonds Large Polypeptides

Amino acids

Intestinal lining

Active Transpor t

3. Small Intestine: Hydrolyze Peptide bonds: Trypsin, Chymotrypsin, Carboxypeptidase, and Aminopeptidase

II. Synthesis and Catabolism

i.

Disintegration of protein to amino acids

1.

Lysosomal pathway
degrades extracellular proteins

2.

Cytosolic pathway
degrades intracellular proteins

Structure

Levels of protein structure

i.
ii. iii.

iv.

Primary Secondary Tertiary 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 -helix and pleated sheet

Structure

Levels of protein structure

iii.

Tertiary
Overall conformation (fold) of the protein molecule Due to interaction of side chains

iv.

Quarternary
Interaction of more than 1 protein molecule or subunits

Classification by Protein Functions

1.

2.
3. 4.

5.
6. 7.

8.
9. 10.

Enzymes Hormones Transport proteins Immunoglobulins (antibodies) Structural proteins Storage proteins Energy Source Osmotic force Homeostasis Acid-Base Balance

Classification by Protein Functions

Classification Enzymes Hormones Transport proteins Function Catalyze chemical reactions Chemical messengers that control the actions of specific cells or organs Transport ions and macromolecules across a biologic membrane

Classification by Protein Functions

Classification Immunoglobulins Function Produced by B-cells that mediates immune response Fibrous proteins that are the structure of cells and tissues Serves as reserves of metal ions and amino acids (e.g. ferritin)

Structural proteins
Storage proteins

Classification by Protein Functions

Classification Function

Energy source

Plasma proteins serve as a reserve source of energy

Plasma proteins function in the distribution of water throughout the compartments of the body. Participation in coagulation in blood Participation as buffers to blood maintain pH

Osmotic force
Hemostasis Acid base balance

Classification by Protein Functions

1.

2.
3. 4.

5.
6. 7.

8.
9. 10.

Enzymes Hormones Transport proteins Immunoglobulins Structural proteins Storage proteins Energy Source Osmotic force Homeostasis Acid-Base Balance

Classes of Proteins based on their Functions (Catalyst)

Protein Fractions Phosphofructokinase Biological Functions Enzymes Actions An enzyme in carbohydrate metabolism that catalyzes phosphate group transfer from ATP to fructose-6-phosphate. A digestive enzyme in vertebrates that catalyzes protein hydrolysis An enzyme that catalyzes the formation of the second messenger cyclic AMP. An enzyme that catalyzes DNAdirected RNA synthesis An enzyme found in HIV catalyzing RNA directed DNA synthesis.

Trypsin Adenylatecyclase

Enzymes Enzymes

RNA polymerase Reverse transcriptase

Enzymes Enzymes

Classes of Proteins based on their Functions (Structural)

Protein Fractions Collagen Elastin Biological Functions Structural protein Structural protein Actions Fibrous proteins found in all animals Fibrous proteins found in connective tissues of the lungs and in large blood vessels such as lining of aorta A mechanically durable fibrous proteins present in outer epidural layer and its appendages such as hair, nails and feathers.

Keratin

Structural protein

Classes of Proteins based on their Functions (Defense)

Protein Fractions Anibodies (Immunoglobulin Biological Functions Defense Proteins Actions Globular proteins produced by the immune system and serves to combat foreign antigens (viral and bacterial antigen) Proteins produced by higher animals that interfere with viral replication

Interferon

Defense Proteins

Classes of Proteins based on their Functions (Transport)

Protein Fractions Hemoglobin Biological Functions Transport Actions Globular heme containing protein that carries oxygen from the lungs to other tissues of vertebrates. Component of lipoproteins such as low density lipoprotein that participate in triglyceride and cholesterol transport A transport protein that carries the degraded bilirubin A protein that carries glucose into erythrocytes.

Apolipoprotein /Lipoprotein

Transport

Albumin

Transport

Protein Permease

Transport

Classes of Proteins based on their Functions (Storage)

Protein Fractions Casein Biological Functions Structural protein Actions Protein found in milk that stores amino acids

Ferritin
Myoglobin

Structural protein
Structural protein

Protein that stores iron

A heme containing protein that binds with oxygen.

Classes of Proteins based on their Functions (Regulatory)

Protein Fractions Lac repressor Biological Functions Regulatory protein Actions A genetic switch that turn off bacterial genes involved in lactose catabolism Abnormal protein synthesized in the pancreas and acts as a signal for the fed state of carbohydrates A part of the membrane that binds insulin and sends the message inside the cell to regulate carbohydrates metabolism.

Insulin

Regulatory protein

Insulin receptor

Regulatory/receptor protein

Classes of Proteins based on their Functions (Hormones)

Protein Fractions Vasopressin Biological Functions Hormones Actions Increases blood pressure and decreases kidney water retention. Abnormal protein synthesized in the pancreas and acts as a signal for the fed state of carbohydrates Initiates child birth labor, causes mammary gland secretes milk, affects kidney excretion of water and Sodium. Stimulates release of hormones from the pituitary gland.

Insulin

Regulatory protein

Oxytocin

Hormones

Thyrotrophic hormones

releasing Hormones

Classes of Proteins based on their Functions (Movement)

Protein Fractions Actin

Biological Functions Movement

Actions Component of skeletal muscle for muscle contraction and relaxation. Protein that causes movement of sperm and protozoa by their flagella and cilia.

Dynein

Movement

Classes of Proteins according to nutritional values

Complete or adequate proteins contains all essential amino acids. Examples of these proteins from animal sources are proteins in milk, fish, seafood shellfish, poultry, cheese , meat, and eggs, while examples from vegetable sources are quinoa, buckwheat, hempseed, and soybeans Examples of incomplete or inadequate proteinsare gelatin, grains, nuts,beans, seeds,peas, corns and flour.An incomplete protein is any protein that lacks one or more essential amino acids in correct proportions.

Classes of Proteins According to shape

Examples of Globular and Fibrous Proteins
Examples 1. Hemoglobin 2. Myoglobulin 3. Ribonuclease 4. Lysozyme 5. Cytochrome C 6. Immunoglobulin 7. Actin 8. Collagen 9. Keratin 10. Myosin 11. Elastin Classes of Proteins Globular Globular Globular Globular Globular Globular Globular Fibrous Fibrous Fibrous Fibrous Functions Oxygen transport Oxygen storage Enzyme for RNA hydrolysis Enzyme for bacterial wall synthesis Electron transport Antibodies Movement (muscle protein) Structural protein Structural protein Movement (muscle protein) Elasticity

Classification by Protein Structure

1.

2.

Simple Proteins Conjugated Proteins

Classification by Protein Structure

1.

Simple Proteins

Contain peptide chains composed of only amino acids. May be globular (albumin and Igs) or fibrous (connective tissues, tendons, bone and muscle)

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 1-antitrypsin e. Nucleoproteins Nucleic acids attached Chromatin

Classification by Protein Structure

1.

2.

Simple Proteins Conjugated Proteins

a. b. c. d.

Metalloprotein Lipoprotein Mucoprotein and glycoprotein Nucleoprotein

Plasma Proteins
A.

B.

Albumin Globulin
1. 2.

3.
4.

1-Globulins 2- Globulins -Globulins -Globulins

1 1

Alb

2 2

Plasma Proteins
Function
Prealbumin Albumin
Indicator of nutrition Binds thyroid hormones (T3, T4) Binds retinol-binding protein

Binds bilirubin, steroids, fatty acids Major contributor to oncotic pressure

Plasma Proteins
1-Globulins
1 - Antitrypsin

Function
Acute phase reactant Protease inhibitor Principal fetal protein spina bifida, - Down syndrome

1 - Fetoprotein

1 - Acid glycoprotein Acute phase reactant

Plasma Proteins
1-Globulins
1 Lipoprotein 1 - Antichymotrypsin Inter--trypsin inh. Gc-globulin

Function
Transport lipids (HDL) Inhibits serine proteinases

Inhibits serine proteinases

Transports Vit. D and binds actin

Plasma Proteins
1-Globulins
1 - Antitrypsin 1 - Fetoprotein 1 - Acid glycoprotein 1 Lipoprotein 1 - Antichymotrypsin Inter--trypsin inhibitor Gc-globulin

Plasma Proteins
2-Globulins
Haptoglobins Ceruloplasmin 2 - Macroglobulin

Function
Acute Phase reactant, Binds Hgb. Contains copper - Wilsons disease. Menkes synd. Inhibits protease

Plasma Proteins
-Globulins
Pre--lipoprotein -Lipoprotein Trasferrin Hemopexin 2-Microglobulin

Function
Transports lipids (VLDL triglyceride) Transports lipids (LDL cholesterol) Transport Iron,-IDA, Hemochromatosis Acute phase reactant, Binds heme Component of HLA molecules

Plasma Proteins
-Globulins
C4, C3, C1q complement Fibrinogen

Function
Immune response Precursor of fibrin clot Acute phase reactants Promotes phagocytosis

C-reactive protein

Plasma Proteins
-Gamma-Globulins
Immunoglobulin G Immunoglobulin A Immunoglobulin M Immunoglobulin E Immunoglobulin E

Function
Antibodies Antibodies in secretions Antibodies in early response Antibodies (reagen, allergy) Surface antibody

Plasma Proteins
A.

B.

Albumin Globulin
1. 2.

3.
4.

1-Globulins 2- Globulins -Globulins -Globulins

1 1

Alb

2 2

Other Proteins
Function
Myoglobin Troponin (cTn)
Oxygen carrier in muscles Cardiac marker (AMI) 2-3 hrs of onset, peak at 8-12 hrs

Cardiac marker for acute coronary syndrome Cellular interaction Fibronectin Placental adherence to the uterus Fetal fibronectin (fFN) - Preterm labor and delivery

Other Proteins
Function
Cross-Linked C-Telopeptides
Proteolytic fragment of collagen I Marker of bone resorption Syn: Prostaglandin D synthase Marker for CSF leakage Cysteine proteinase inhibitor Marker for kidney function (GFR)

-Trace Protein
Cystatin C Amyloid

Fibrous protein aggregates formed from alteration of pleated sheats Amyloidoses

Hypoproteinemia

TP

Albumin Globulin

Disease

N,

Hepatic Damage Cirrhosis - bridging Hepatitis -globulins Infections Acute - 1 , 2 globulins Chronic - 1, 2 , globulins

Hypoproteinemia

TP

Albumin Globulin

Disease

Inadequate diet Nephrotic syndrome 2,-globulins;-globulins

Immunodeficiency syndrome

Hypoproteinemia

TP

Albumin Globulin Disease

Dehydration
Multiple myeloma Monoclonal & Polyclonal gammopathies

Total Protein

Reference Interval 1. 6.5-8.3 g/dL (65-83 g/L) 2. 6.0-7.8 g/dL (60-78 g/L) in recumbent position

Total Protein
Method
Kjeldahl

Principle

Refractometry
Biuret Dye Binding

Reference method. Assume average nitrogen content of 16% Measurement of refractive index due to solutes in serum Formation of violet-colored chelate between Cu2+ ions and peptide bonds Protein binds to dye and causes a spectral shift in the absorbance maximum of dye

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. Nesslers reaction (double iodide of Hg and K)

Ammonia + Nesslers rgt Gum ghatti yellow solution (Ammonium dimercuric iodide)

2.

Berthelot reaction
Na nitroprusside Indophenol blue

Ammonia + alkaline hypochlorite

Refractometry

Measurement of refractive index (velocity of light in air and water) due to solutes in serum.

Biuret

Formation of violet-colored chelate between Cu2+ ions and peptide bonds (measured at 540 nm) Composition
1. 2. 3.

Cupric ions breaks the peptide bonds Tartrate salt keeps copper in solution Potassium iodide stabilizes cupric ions

Dye binding

Protein binds to dye and causes a spectral shift in the absorbance maximum of dye.
1. 2.

3.
4. 5.

Bromphenol blue Ponceau S Amido black 10B Lissamine green Coomassie briliant blue

Albumin
Method
Salt Precipitation DYE BINDING

Principle Globulins are precipitated Albumin in supernatant is quantitated by biuret reaction Nonspecific for Albumin
Many Interferences (salicylates, bilirubin) Sensitive Overestimates low albumin levels Most commonly used dye Specific, Sensitive and Precise

Methyl orange
HABA

BCG (Bromcresol green)

BCP (Bromcresol purple)

Albumin

Method
Electrophoresis

Principle Proteins separated based on electric charge densities

Electrophoresis

Cellulose acetate/agarose gel (support media) After separation, protein fractions are immersed in acid solution then stained by dyes (e.g. Coomassie blue)

Electrophoresis

The medium is placed in scanning densitometer which compute the area under the absorbance

Electrophoresis

Reference values
Albumin, 53-65% (3.5-5.0 g/dL) 1 Globulin, 2.5-5% (0.1-0.3 g/dL) 2 Globulin, 7-13% (0.6-1.0 g/dL) Globulin, 8-14% (0.7-1.1 g/dL) Globulin, 12-22% (0.8-1.6 g/dL)

Electrophoresis

Monoclonal increase

Electrophoresis

Electrophoresis

Acute phase reactant

i.
ii. iii.

iv.
v.

Fibrinogen Haptoglobin Ceruloplasmin Serum amyloid CRP

High Resolution Protein Electrophoresis

Uses higher voltage couple with a cooling system and more concentrated buffer

Total Protein
Method Principle
Turbidimetric methods (SSA, TCA, Proteins are precipitated as particles, turbidimetry is measured benzethonium chloride) spectrophotometrically

Biuret
Folin-Lowry Dye binding (Coomassie blue, Ponceau S)

Proteins is reacted with Cu2+ forms colored complex with peptide bonds Initial biuret reaction; oxidation of AA (tyrosine, etc.) residues by Folin Phenol reagent; measurement of resultant blue color. Protein binds to dye, causes shift in absorption maximum.

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