Proteins Lecture 5 Introduction - PowerPoint Presentation

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Proteins

Lecture 5

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Introduction

Proteins are polymers of amino acids produced by living cells in all forms of life.

A large number of proteins exist with diverse functions, sizes, shapes and structures but each is composed of essential and non-essential amino acids in varying numbers and sequences.

The number of distinct proteins within one cell is estimated at 3,000 - 5,000The most abundant organic molecule in cells (50-70% of cell dry weight)

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Size

A typical protein contains 200-300 amino acids, but some are much smaller and some are much larger

Proteins range in molecular weight from 6,000 Daltons (insulin) to millions of Daltons (structural proteins)

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Peptide bond

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Protein Structure

Primary structure

Sequence of AA

In order to function properly, proteins must have the correct sequence of amino acids.e.g when valine is substituted for glutamic acid in the



chain of

HbA

,

HbS

is formed, which results in sickle-cell anemia.

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Protein Structure

Secondary structure

A regularly repeating structures stabilized by hydrogen bonds between the amino acids within the protein

Initial helical folding (α

helix)

Beta pleated sheet

Held together by Hydrogen bonding

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Protein Structure

Tertiary Structure

The overall shape, or conformation, of the protein molecule

Chain folds back on itself to form 3D structure

Interaction of R groups (

hydrogen bonds, and disulfide bonds)

Responsible for the biological activity of the molecule

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Protein Structure

Quaternary structure

The shape or structure that results from the interaction of more than one protein molecule, or protein subunits, held together by

noncovalent forces such as hydrogen bonds and electrostatic interactions

2 or more polypeptide chains binding together

eg

. Hemoglobin

Hemoglobin has 4 subunits

Two

 chains

Two  chains

Many enzymes have quaternary structures

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Protein Structure

Disruption of bonds holding 2

o

, 3o or 4o structure together is called denaturation and can cause loss of function of the proteinThese Bonds that hold the protein together are weak It is important in the clinical lab to note that excessive heat, freeze thaw cycle or vigorous mixing can break these bonds and denature the protein

An enzyme can loose its activity, Ag can loose its antigenicity

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Protein Charges

Proteins contain many ionizable groups on the side chains of their amino acids as well as their N- and C-terminal ends

A protein can bear positive and/or negative charges on each molecule, due to amino acid composition

pH dependent

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Protein Charges

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No net charge at pH 2.8,

but a net negative charge at pH 9.47

No net charge at pH 9.47,

but a net positive charge at acid pH

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Protein Charges

Isoelectric point (

pI

) – The pH at which an amino acid or protein has no net charge

The point at which the number of positively charged groups equals the number of negatively charged groups in a protein

When the pH >

pI

, a protein has a net negative charge

When the pH <

pI

, a protein has a net positive charge

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Solubility

Soluble proteins have a charge on their surfaces.

A protein has its lowest solubility at its isoelectric point

Without a net charge, protein-protein interactions and precipitation are more likely The solubility of proteins in blood requires a pH in the range of 7.35-7.45. Differences in solubility can be used to separate major plasma fractions from each other

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Classification by Protein Structure

Simple Proteins

(contain only amino acids) are classified by shape as –

Globular proteins: compact, tightly folded and coiled chainsMajority of serum proteins are globularFibrous proteins: elongated, high viscosity (hair, collagen)

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Classification by Protein Structure

Conjugated proteins

contain non-amino acid groups

Amino acid portion is called apoprotein and non-amino acid portion is called the prosthetic group

The prosthetic group may be lipid, carbohydrate, porphyrins, metals, and others

It is the prothetic groups that define the characteristics of these proteins.

Name of the conjugated protein is derived from the prosthetic group

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Conjugated Proteins

Classification

Prosthetic group

Example

Lipoprotein

Lipid

HDL

Glycoprotein

Carbohydrates

Immunoglo-bulins

Phosphoprotein

Phosphate

Casein of milk

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Functions of proteins

Generally speaking, proteins do everything in the living cells

Functional classification of plasma proteins is useful in understanding the changes that occur in disease:

Tissue nutrition Proteins of immune defense

Antibodies

Acute phase proteins

Proteins associated with inflammation

Transport proteins (albumin, transferrin)

Proteins used to bind and transport

Hemostasis

Proteins involved in forming clots and acting very closely with complement

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Functions of proteins

Regulatory

( receptors, hormones )

Catalysis, enzymesOsmotic forceMaintenance of water distribution between cells and tissue and the vascular system of the body

Acid-base balance

Participation as buffers to maintain pH

Structural, contractile, fibrous and keratinous

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Catabolism & Nitrogen Balance

Most proteins in the body are constantly being repetitively synthesized and then degraded

Balance exists between protein anabolism (synthesis) and catabolism (breakdown)

 Nitrogen Balance = 

Nitrogen

 intake - 

Nitrogen

 loss

Turnover totals about 125-220 g of protein each day

Normal, healthy adults are generally in nitrogen balance,

with intake and excretion being equal

Pregnant women, growing children, and adults recovering from major illness

are often in positive nitrogen balance

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Catabolism & Nitrogen Balance

Conditions in which there is excessive tissue destruction, such as burns, wasting diseases, continual high fevers, or starvation.

more nitrogen is excreted than is incorporated into the body,

an individual is in negative nitrogen balance20

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Plasma Proteins

About 500 proteins have been identified in plasma

The plasma proteins include the immunoglobulins, enzymes, and enzyme inhibitors.

Most plasma proteins, with the notable exception of immunoglobulins, are synthesized in the liver.Plasma proteins circulate in the blood and between the blood and the extracellular tissue spaces.

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Plasma Proteins

Prealbumin

α2 Macroglobulin

Albumin

Transferrin (

Siderophilin

)

β

Globulins α1, α2, β, and γ

Hemopexin

α1-Antitrypsin

Acute phase proteins (CRP)

α1-Fetoprotein

Immunoglobulins

α1-Acid Glycoprotein

Myoglobin

Haptoglobin

(α2)

Troponin

Ceruloplasmin

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Prealbumin (Transthyretin)

Migrates just ahead of Albumin

When a diet is deficient in protein, hepatic synthesis of proteins is reduced

Indicator of nutritional status (very short half life- 2 days)Transport of thyroid hormones & it also binds with retinol-binding protein to form a complex that transports retinol (vitamin A)

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Prealbumin (Transthyretin)

Low levels found in:

Hepatic damage

Acute phase responses (-ve acute phase reactant)Nutritional deficit – short half-life means decrease seen early in diseaseIncreased level found in:Steroid treatmentAlcoholism

Chronic renal failure (glomerular filtration rate decreased)

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Albumin

Synthesized in the liver from 585 amino acids

at the rate of 9–12 grams per day with no reserve or storage

Highest concentration plasma proteinAlbumin also exists in the extravascular (interstitial) spaceTwo primary functions

Colloidal osmotic pressure (80%)

Bind and transport of numerous substances

Bilirubin, steroids, fatty acids, Ca

++

, Mg

++

,

salicylic acid & other medications

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Albumin

Decreased Albumin

Malnutrition & muscle wasting diseases

Liver diseases – inability to synthesizeGI loss due to inflammation or mucosal lining diseasesLoss in urine due to renal disease

Genetic

analbuminemia

(Mutation causes absence of albumin)

Bisalbuminemia

results from two copies of different albumin genes, resulting in different charges.

Albumin

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Albumin

Increased levels of Albumin

Seen in dehydration

After excessive albumin infusion

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Globulins

The globulin group of proteins consists of α

1

, α2, β, and γ fractions. Each fraction consists of a number of different proteins with different functions.

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Globulins (α1-Antitrypsin)

Major component (90%) of α1 fraction

Acute phase reactant, synthesized in the liver

Most important function the inhibition of the protease neutrophil elastase

Neutrophil elastase is released from leukocytes to fight infection, but it can destroy alveoli

Mutations in the

SERPINA1

gene cause:

α1-antitrypsin deficiency which is

associated with

Severe degenerative emphysema

Abnormal form of α

1

-antitrypsin can also accumulate in the liver and can cause cirrhosis

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α1-Antitrypsin

Increased levels in:

Inflammatory reactions

PregnancyContraceptive useSince major component of α1 band – changes in levels apparent on protein electrophoresisQuantitative methods used to confirm electrophoresis findings are radial immunodiffusion

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α1-Fetoprotein (AFP)

AFP synthesized by fetal yolk sac & later by parenchymal cells of liver

Peaks in fetus at 13 weeks – decreases gradually by birth

Acts like a fetal “albumin”Maternal serum testing used to screen fetus for birth defects

Neural tube defects & twins

Increase risk of Down’s syndrome

& trisomy 18

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α1-Fetoprotein (AFP)

In adults level is high in:

80% of hepatocellular carcinoma

Gonadal tumors in adultsThe methods commonly used for AFP determinations are radioimmunoassay (RIA) and enzyme-labeled immunoassay (EIA)

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α1-Acid Glycoprotein (AAG)

Acute phase reactant, synthesized in the liver

Regulates immune responses

Increased in: inflammation, cancer,

pneumonia,

Rheumatoid arthritis (RA).

The analytic methods used most commonly for the determination of AAG are radial immunodiffusion, immunoturbidity, and nephelometry

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radial immunodiffusion

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Haptoglobin (α2)

Synthesized in the liver

Considered an acute-phase protein

Binds free hemoglobinPrevents loss of Hemoglobin & Iron into urineUsed to detect and evaluate hemolytic anemia and to distinguish it from anemia due to other causes

Haptoglobin

Reticulocytes

Diagnosis

1

Decreased

Increased

Hemolytic anemia

2

Normal

Increased

RBC destruction may be occurring in organs such as the spleen and liver

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Normal

Normal

Anemia present is not due to RBC breakdown

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Haptoglobin (α2)

Increased in

Inflammations, burns & nephrotic syndrome due to fluid losses

Decreased in with transfusion reactions, HDNThus, a low plasma haptoglobin concentration can be indicative of intravascular haemolysis.

decreased synthesis are seen in chronic liver disease

Radial immunodiffusion has been used for the quantitative determination of haptoglobin

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Ceruloplasmin (α2)

Synthesized in the liver

Acute-phase reactant

Copper carrying protein90% of serum copper is bound to itOrdered along with blood and/or urine copper tests to help diagnose Wilson's disease, decreased levels of ceruloplasmin and excess storage of copper in the liver, brain, and other organs resulting in hepatic cirrhosis and neurologic damage.

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Ceruloplasmin

Increased levels

Pregnancy, inflammatory processes,

malignancies, oral estrogens & contraceptivesLow levels

Malabsorption

Severe Liver Disease

Nephrotic syndrome

Menkes’ Syndrome (decreased Cu absorption)

Most assays today use immunochemical methods, including radial immunodiffusion and nephelometry

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α2 Macroglobulin

Tetramer of four identical subunits

Synthesized by liver

major component of the α2 band in protein electrophoresisPrimarily intravascular spaces due to sizeInhibits proteases trypsin, pepsin & plasmin

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α2 Macroglobulin

Increased levels

Nephrosis (Large size prevents loss)

Oral contraceptives (high estrogens)Decreased levelsPancreatitis The analytic methods that have been used for the assay of this protein are radialimmunodiffusion, ELISA, and latex agglutination

immunoassay.

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Transferrin (Siderophilin)

The major component of the



-globulin.Glycoprotein synthesized by liverCarries 2 ferric iron moleculesNormally 33% saturated

Prevents loss of iron through kidneys

Transports to storage sites where Iron is transferred to ferritin

Transports to bone marrow for RBC synthesis

Negative acute-phase protein

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Transferrin (Siderophilin)

Transferrin is abnormally high in iron deficiency anemia

Decreased in

General protein deficienciesLiver diseaseMalnutritionInflammations (Negative acute phase protein)

Hereditary Disorders

The analytic method used for the quantitation of transferrin is immunodiffusion

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Transferrin (Siderophilin)

Atransferrinemia

is inherited as an autosomal recessive trait due to mutation of both transferrin genes with a resulting absence of transferrin.

It is characterized by anemia and hemosiderosis (iron deposition) in the heart and liver. The iron damage to the heart can lead to heartfailure. This disease can be effectively treated by plasma infusions of transferrin.

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Hemopexin

Beta globulin

acute-phase reactant

Purpose is to remove circulating HemeBreakdown product of Hemoglobin & myoglobinCarried to liver – broken downIncreased in pregnancy,

Some malignancies

Low hemopexin levels are diagnostic of a hemolytic anemia

Hemopexin can be determined by radial immunodiffusion

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C-Reactive Protein

Acute phase reactant

CRP was so named because it precipitates with the C substance, a polysaccharide of pneumococci.

Antibody-like reactivity for many bacteriaOpsonization (protein-coating process to enhance phagocytosis)

It is elevated in acute rheumatic fever, bacterial infections, myocardial infarction, rheumatoid arthritis, etc…

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Immunoglobulins

IgA, IgD, IgE, IgG, IgM

Consist of two identical heavy (H) and two identical light (L) chains

Decreases seen in general protein deficienciesIncreasesChronic inflammatory processes - polyclonalMalignancy (multiple myeloma) - monoclonal

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Myoglobin

Heme protein of skeletal & cardiac muscle

Single polypeptide chain transports oxygen to muscle tissue

Released into blood when striated muscle is damagedCardiac injury (AMI)Trauma or crush injuries

Latex agglutination, ELISA

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Troponin

Troponin T (TnT)

Troponin I (TnI)

Troponin C (TnC)Regulate muscle contractionsCalcium release attaches troponin

Complex of 3 proteins that bind to filaments of striated muscle (cardiac & skeletal)

Increase indicative of myocardial injury

Cardiac troponins can be measured by ELISA

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Hypoproteinemia

Total protein level less than the reference interval, occurs in any condition where a negative nitrogen balance exists:

Malnutrition and/or malabsorption

Excessive loss as in renal disease, GI leakage,excessive bleeding, severe burnsExcessive catabolismBurns, trauma, shock

Liver disease

Primary site of protein

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Hyperproteinemia

Dehydration

Relative due to fluid decrease

Decreased intake or increased lossAll fractions remain normal ratiosMonoclonal increasesMultiple Myeloma or related malignancies

Polyclonal increases

Chronic inflammatory diseases

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