Proteins

1.      Protein

1. Any of a group of complex organic compounds that contains carbon, hydrogen, oxygen, nitrogen and usually sulfurs (the characteristic element being nitrogen) and is distributed widely in plants and animals.  In plasma they are either albumin or globulins.

2.      Albumin

2. A small globular protein with molecular mass of 66.3 kDa.  It is a most abundant protein found in plasma from mid-gestation until death, accounting for approximately the plasma protein mass because of its high plasma concentration and relatively small size, albumin is also a major protein component of the most extra vascular body fluids, including 1.CSF, 2.Intestitual fluid, 3.urine and 4.amniotic fluid. Approximately 60%of the total body albumin is in the extra vascular space. It has no carbohydrate side chains but is highly soluble in water due to its high net negative charge at physiological pH.

3     Globulin

3. Globular (spherical) protein with a compact morphology that is soluble in water or salt solution.

4.      Immunoglobulin;

4. A class of proteins also known as antibodies synthesized by the B cells of the immune system in response to a specific antigen and containing a region that binds to this antigen (Antigen-binding site); there are five classes of immunoglobulins (IgA, IgD, IgE, IgG and IgM)

5.      Amino Acids:

5. The basic structural units of proteins. Their measurement in physiological fluids provides important information for the diagnosis of many pathological and inherited conditions.

6.      What is Protein Metabolism?

6. Protein metabolism is the set of processes involved in either anabolism, which is synthesis of proteins, or catabolism, which is breakdown of proteins.

 

Protein Sources

Proteins are composed of amino acids in a specific sequence. The source for most proteins is synthesis within the liver cells.  For immunoglobulins, this occurs in the plasma cells (mature B lymphocytes).  However some amino acids, the building block of proteins, must first be supplied by our diet because they are not synthesized and these are termed essential amino acids.  For example, the amino acid, phenylalanine, is essential and cannot be synthesized in the liver.

 

Protein Metabolism

Anabolism:  Synthesis of Proteins involves transcription of the genetic code within the nucleus and translation, which direct the formation of amino acids in the ribosomes.  Proteins are composed of amino acids in a specific sequence.

 

Transcription: Before the synthesis of a protein begins in a cell, the corresponding RNA molecule is produced by RNA transcription. One strand of the DNA double helix is used as a template by the RNA polymerase to synthesize a messenger RNA (mRNA). This mRNA migrates from the nucleus to the cytoplasm. During this step, mRNA goes through different types of maturation including one called splicing when the non-coding sequences are eliminated. The coding mRNA sequence can be described as a unit of three nucleotides called a codon.

 

Translation and Peptide Synthesis: The ribosome binds to the mRNA that is recognized only by the transfer RNA (tRNA). The ribosome proceeds to the synthesis phase of protein synthesis. During this stage, complexes, composed of an amino acid linked to tRNA, sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the tRNA. The ribosome moves from codon to codon along the mRNA. Amino acids are added one by one, translated into polypeptidic sequences dictated by DNA and represented by mRNA. At the end, a release factor stops translation and releases the complete polypeptide from the ribosome.

 

Catabolism: There is a continuous process of breakdown and re-synthesis of all cellular proteins. Adults breakdown over 1-2% (125-220g) of their total body protein daily and 75-80% of liberated amino acids are reutilized for protein synthesis. This protein breakdown occurs primarily in the digestive tract, kidneys, and liver with the nitrogen from the protein used to form urea and other waste products. These protein waste products are commonly excreted by the kidney.

 

Four Stages of Protein Structure

The four stages are:

·         Primary

·         Secondary

·         Tertiary

·         Quaternary

 

Primary Structure is made of sequences of amino acids bound by the peptide bond:

Tertiary Bonds: weaker bonds between amino acids causing folding or three-dimensional shape.

Quaternary Bonds: weak bonds between several amino acids.  Example is the haemoglobin molecule with 4 polypeptide chains.

Properties of Proteins based on their Structures

·         Molecular Mass of proteins is large and forms the principle of methods for separation.  This is also a reason for proteins being good antigens or stimulators of antibody production.

·         Solubility varies with pH, ionic strength, temperature and dielectric constant (electrical charge), which forms the principle of methods of separation by electrophoresis.

·         Electrical Charge:  Each protein has a unique pH in which is electrically neutral and above or below this point changes the electrical charge of the protein.

·         Adsorption:  proteins react with large surface areas by this interaction based on either hydrophobic, absorptive, ionic or hydrogen bonding.

·         Specific binding to other molecules is common in proteins due to their tertiary and quaternary structure.  For example, enzymes and immunoglobulins exhibit specific binding.

·         Denaturation:  the process of unfolding quaternary, tertiary and sometimes secondary bonding of protein structure due to exposure to extreme temperature, pH, ionic strength and physical forces such as shaking.

 

 

Functions of individual Proteins

Transportation:

·         Albumin transports unconjugated bilirubin, drugs, minerals, hormones and other non-water soluble molecules

·         Transferrin transports iron

·         Ceruloplasmin transports copper

·         Haptoglobin transports free haemoglobin

·         Transthyretin (pre-albumin) transports thyroid hormones

 

Colloid Osmotic Pressure: Albumin’s most important function is in maintaining Colloid Osmotic Pressure (COP), which maintains equilibrium between vascular and extravascular spaces to regulate fluid levels.

 

Acute Phase Reactants (APR): These are proteins associated with nonspecific response to inflammation or tissue damage.  The most important one is C-reactive Protein (CRP) Positive Acute Phase Proteins will increase in response to inflammation; for example, CRP. Negative Acute Phase Proteins: decrease in response to inflammation.

Specific Immunity: Immunoglobins (antibodies), which are produced in, mature B-lymphocytes and plasma cells to bind to specific antigens and sometimes to initiate complement release.  Examples are IgG, IgM, IgD, IgA and IgE.

 

Haemostasis and Coagulation: Fibrinogen is activated in the clotting cascade to form fibrin

 

Enzymes: These are catalysts that accelerate biochemical reactions without becoming consumed in the process.  Examples are ALAT and CPK

 

 

 

Explanation of Clinical Disorders that affect Individual Plasma Proteins             

 

Albumin:

Increased in plasma only in acute dehydration

Decreased in plasma in a variety of conditions including:

·         Mild decreased due to urinary loss with extreme physical exercise and high fever

·         Dietary deficiency:  kwashiorkor is malnutrition due to lack of protein in the diet and results in oedema

·         Renal Failure: excessive excretion of albumin in urine due to increased glomerular filtration, tubular damage, hematuria, or a combination.

·         Inflammation: Negative Acute phase response due to haemodilution, loss to extravascular space, increase consumption by cells, decreased synthesis

·         Hepatic disease: decreased plasma albumin as a result of increased immunoglobulin levels such as in severe hepatitis or cirrhosis and loss to the extravascular space and decreased synthesis in the liver due to damage often by drugs, alcohol abuse

Decreased plasma albumin is associated with oedema due to decreased colloid osmotic pressure in the tissues

 

C-Reactive Protein:

Increased in acute phase reaction due to stress, trauma, infection, inflammation, after surgery, myocardial infarction and neoplastic proliferation

·         Alpha – 1 antitrypsin (AAT):

§  Increased in acute phase reaction.  Decreased AAT activity can be due to genetic disorder or secondary to liver, pancreatic or kidney disease.  The genetic deficiency of AAT is associated with very high risk for basilar pulmonary emphysema and linked with primary liver cancer or liver cirrhosis.

·         Transferrin:

§  Decreased levels are associated with anaemia of chronic disease such as in cancers.

§  Increased levels are associated with iron deficiency anaemia such as dietary deficiency of iron or chronic blood loss.

§  Variable results in sickle cell anaemia depending on treatment with transfusions.

·         Haptoglobin:

§  Decreased levels are associated with haemolytic anaemia

§  Increased levels are associated with acute phase reaction.

·         Fibrinogen:

§  Plasma levels Increased in acute phase reaction, infection and pregnancy.

§  Plasma levels Decreased with extensive coagulation during which fibrin is consumed such as disseminated intravascular coagulopathy.

·         Immunoglobulins:

§  An increased plasma level of one specific type (monoclonal immunoglobulin) is associated with malignancy of B cells/plasma cells in multiple myeloma.  Monoclonal immunoglobulins can be excreted in large amounts in the urine.  Advanced stages cause decreased plasma levels of normal immunoglobulins while producing large amounts of monoclonal abnormal immunoglobulins.

§  Decreased plasma immunoglobulin levels are associated with specific types of immunodeficiency.

Principle of methods of Assay for Protein Profiles

 

Biuret method for total serum protein:

·         The peptide bonds of proteins react with Biuret reagent containing Cu²⁺ ions in an alkaline solution to form a violet color measured at 540 nm. 

·         Sodium potassium tartrate is a component of the reagent and functions to maintain copper in the correct valence state and in an alkaline solution, while potassium iodide is present as an antioxidant.

 

Bromocresol Green for Albumin:

Cationic portion of albumin binds to anionic dye at pH 4.2 forming a colored product with absorbance measured at 628 nm. In this method a colored product forms that absorbs at wavelengths significantly different than the reagent and from typical interfering molecules such as haemoglobin or bilirubin.

 

Separation of Serum Proteins by Electrophoresis

Proteins are amphoteric meaning they can be positively or negatively charged, based on pH. The Isoelectric point is the pH at which a protein has no net charge so that:

·         pH > Isoelectric point: protein has net negative charge and migrate towards anode (positive terminal)

·         pH < Isoelectric point: protein has net positive charge and migrate towards cathode (negative terminal)

Proteins are separated in an electrical field towards the anode or cathode at a certain voltage and temperature based on electric charge due to the pH of the buffer reagent used. The speed of migration depends largely on the degree of ionization of the protein at the pH of the buffer system.  Separation also depends on electric field strength, size and shape of molecule, temperature, and buffer characteristics. Specialized electrophoresis methods can be used to further detect individual immunoglobulins by immunoelectrophoresis.

 

Immunoassay is a general method, including latex agglutination or immunodiffusion methods that can be used for quantification of immunoglobulins, CRP, etc

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