1. Proteins
Lecture 5

2. 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
The most abundant organic molecule in cells (50-70% of cell dry weight)

3. Size
A typical protein contains 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)

4. Peptide bond

5. 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.

6. 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

7. 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

8. 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

9. Protein Structure
Disruption of bonds holding 2o, 3o or 4o structure together is called denaturation and can cause loss of function of the protein
These 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

10. 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

11. Protein Charges No net charge at pH 2.8, No net charge at pH 9.47,
but a net negative charge at pH 9.47
No net charge at pH 9.47,
but a net positive charge at acid pH
If pH values greater than the pKa, the deprotonated form predominate

12. 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
In general terms, as the pH of a solution increases, deprotonation of the acidic and basic groups on proteins occurs

13. 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
Differences in solubility can be used to separate major plasma fractions from each other

14. Classification by Protein Structure
Simple Proteins (contain only amino acids) are classified by shape as –
Globular proteins: compact, tightly folded and coiled chains
Majority of serum proteins are globular
Fibrous proteins: elongated, high viscosity (hair, collagen)

15. 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

16. Conjugated Proteins Classification Prosthetic group Example
Lipoprotein
Lipid
HDL
Glycoprotein
Carbohydrates
Immunoglo-bulins
Phosphoprotein
Phosphate
Casein of milk

17. 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

18. Functions of proteins Regulatory Catalysis, Osmotic force
( receptors, hormones )
Catalysis,
enzymes
Osmotic force
Maintenance 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

19. 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 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

20. 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 balance

21. 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.

22. 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

23. 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)

24. 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 disease
Increased level found in:
Steroid treatment
Alcoholism
Chronic renal failure (glomerular filtration rate decreased)
Anabolic steroids, androgens, prednisolone, and high-dose non-steroidal anti-inflammatory medications can increase prealbumin concentrations.
The decrease of such proteins may be used as markers of inflammation.
In acute alcohol intoxication, a leakage of proteins from damaged hepatic cells may cause a rise in the prealbumin level

25. 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 protein
Albumin also exists in the extravascular (interstitial) space
Two primary functions
Colloidal osmotic pressure (80%)
Bind and transport of numerous substances
Bilirubin, steroids, fatty acids, Ca++, Mg++,
salicylic acid & other medications

26. Albumin Decreased Albumin Malnutrition & muscle wasting diseases
Liver diseases – inability to synthesize
GI loss due to inflammation or mucosal lining diseases
Loss 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.
genetically inherited metabolic defect characterised by an impaired synthesis of serum albumin

27. Albumin Increased levels of Albumin Seen in dehydration
After excessive albumin infusion
Bisalbuminemia or alloalbuminemia is an inherited or acquired, rarely encountered serum protein anomaly, characterized by the occurrence of bicuspid electrophoretic pattern in the albumin fraction detected on serum electrophoresis

28. Globulins
The globulin group of proteins consists of α1, α2, β, and γ fractions.
Each fraction consists of a number of different proteins with different functions.

29. 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 α1-antitrypsin can also accumulate in the liver and can cause cirrhosis
Emphysema is a type of chronic obstructive pulmonary disease (COPD) involving damage to the air sacs (alveoli) in the lungs

30. α1-Antitrypsin Increased levels in: Inflammatory reactions Pregnancy
Contraceptive use
Since major component of α1 band – changes in levels apparent on protein electrophoresis
Quantitative methods used to confirm electrophoresis findings are radial immunodiffusion

31. 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
In neural tube defects, the skin of the fetus is not intact and so larger amounts of AFP is measured in the mother's blood. In Down syndrome, the AFP is decreased in the mother's blood, presumably because the yolk sac and fetus are smaller than usual.
In spina bifida, the fetal spinal column doesn't close completely.

32. α1-Fetoprotein (AFP) In adults level is high in:
80% of hepatocellular carcinoma
Gonadal tumors in adults
The methods commonly used for AFP determinations are radioimmunoassay (RIA) and enzyme-labeled immunoassay (EIA)
Gonadal : An organ in animals that produces gametes, especially a testis or ovary.

33. 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

34. radial immunodiffusion

35. Anemia present is not due to RBC breakdown
Haptoglobin (α2)
Synthesized in the liver
Considered an acute-phase protein
Binds free hemoglobin
Prevents loss of Hemoglobin & Iron into urine
Used 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
RBC destruction may be occurring in organs such as the spleen and liver 3
Anemia present is not due to RBC breakdown
When the haptoglobin and hemoglobin attach, the reticuloendothelial cells (mainly in the spleen) remove the haptoglobin–hemoglobin complex from circulation
2- Because the freed hemoglobin is not released into the bloodstream, the haptoglobin is not used up and so is normal.

36. Haptoglobin (α2) Increased in Decreased in
Inflammations, burns & nephrotic syndrome due to fluid losses
Decreased in
with transfusion reactions, HDN
Thus, 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
Increases are also seen in conditions such as burns and nephrotic syndrome when large amounts of fluid and lower-molecular-weight proteins have been lost

37. Ceruloplasmin (α2) Synthesized in the liver Acute-phase reactant
Copper carrying protein
90% of serum copper is bound to it
Ordered 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.
Wilson's disease, an autosomal recessive inherited disorder
In Wilson disease, the processes of incorporation of copper into ceruloplasmin and excretion of excess copper into bile are impaired

38. Ceruloplasmin Increased levels Low levels
Pregnancy, inflammatory processes,
malignancies, oral estrogens & contraceptives
Low levels
Malabsorption
Severe Liver Disease
Nephrotic syndrome
Menkes’ Syndrome (decreased Cu absorption)
Most assays today use immunochemical methods, including radial immunodiffusion and nephelometry
Ceruloplasmin is a weak, late reacting Acute phase protein (APP). Levels are increased significantly by estrogens as in cases of pregnancy and in oral contraception

39. α2 Macroglobulin Tetramer of four identical subunits
Synthesized by liver
major component of the α2 band in protein electrophoresis
Primarily intravascular spaces due to size
Inhibits proteases trypsin, pepsin & plasmin

40. α2 Macroglobulin Increased levels Decreased levels
Nephrosis (Large size prevents loss)
Oral contraceptives (high estrogens)
Decreased levels
Pancreatitis
The analytic methods that have been used for the assay of this protein are radial immunodiffusion, ELISA, and latex agglutination immunoassay.
protease inhibitor and therefore counteract the proteases released by the inflamed pancreas

41. Transferrin (Siderophilin)
The major component of the -globulin.
Glycoprotein synthesized by liver
Carries 2 ferric iron molecules
Normally 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

42. Transferrin (Siderophilin)
Transferrin is abnormally high in iron deficiency anemia
Decreased in
General protein deficiencies
Liver disease
Malnutrition
Inflammations (Negative acute phase protein)
Hereditary Disorders
The analytic method used for the quantitation of transferrin is immunodiffusion

43. 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 heart failure.
This disease can be effectively treated by plasma infusions of transferrin.

44. Hemopexin Beta globulin acute-phase reactant
Purpose is to remove circulating Heme
Breakdown product of Hemoglobin & myoglobin
Carried to liver – broken down
Increased in
pregnancy,
Some malignancies
Low hemopexin levels are diagnostic of a hemolytic anemia
Hemopexin can be determined by radial immunodiffusion

45. 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 bacteria
Opsonization (protein-coating process to enhance phagocytosis)
It is elevated in acute rheumatic fever, bacterial infections, myocardial infarction, rheumatoid arthritis, etc…
CRP received its name because it precipitates with the C substance, a polysaccharide of pneumococci.

46. Immunoglobulins IgA, IgD, IgE, IgG, IgM
Consist of two identical heavy (H) and two identical light (L) chains
Decreases seen in general protein deficiencies
Increases
Chronic inflammatory processes - polyclonal
Malignancy (multiple myeloma) - monoclonal

47. Myoglobin Heme protein of skeletal & cardiac muscle
Single polypeptide chain transports oxygen to muscle tissue
Released into blood when striated muscle is damaged
Cardiac injury (AMI)
Trauma or crush injuries
Latex agglutination, ELISA

48. Troponin
Complex of 3 proteins that bind to filaments of striated muscle (cardiac & skeletal)
Troponin T (TnT)
Troponin I (TnI)
Troponin C (TnC)
Regulate muscle contractions
Calcium release attaches troponin
Troponin T binds the troponin components to tropomyosin, troponin I inhibits the interaction of myosin with actin, and troponin C contains the binding sites for Ca2+ that helps initiate contraction
Increase indicative of myocardial injury
Cardiac troponins can be measured by ELISA

49. 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 burns
Excessive catabolism
Burns, trauma, shock
Liver disease
Primary site of protein
The normal range is 6.0 to 8.3 gm/dL 

50. Hyperproteinemia Dehydration Monoclonal increases Polyclonal increases
Relative due to fluid decrease
Decreased intake or increased loss
All fractions remain normal ratios
Monoclonal increases
Multiple Myeloma or related malignancies
Polyclonal increases
Chronic inflammatory diseases