Proteins Introduction n Proteins are polymers of amino

Proteins

Introduction n Proteins are polymers of amino acids produced by living cells in all forms of life. n 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. n The number of distinct proteins within one cell is estimated at 3, 000 - 5, 000 ¨ The most abundant organic molecule in cells (50 -70% of cell dry weight) 2 Dr. M. Alzaharna Clini. Chem. 2015

Size n A typical protein contains 200 -300 amino acids, but some are much smaller and some are much larger n Proteins range in molecular weight from 6, 000 Daltons (insulin) to millions of Daltons (structural proteins) 3 Dr. M. Alzaharna Clini. Chem. 2015

Peptide bond 4 Dr. M. Alzaharna Clini. Chem. 2015

Protein Structure Primary structure n Sequence of AA n In order to function properly, proteins must have the correct sequence of amino acids. n e. g when valine is substituted for glutamic acid in the chain of Hb. A, Hb. S is formed, which results in sickle-cell anemia. 5 Dr. M. Alzaharna Clini. Chem. 2015

Protein Structure Secondary structure n A regularly repeating structures stabilized by hydrogen bonds between the amino acids within the protein n Initial helical folding (α helix) n Beta pleated sheet n Held together by Hydrogen bonding 6 Dr. M. Alzaharna Clini. Chem. 2015

Protein Structure Tertiary Structure n n The overall shape, or conformation, of the protein molecule Chain folds back on itself to form 3 D structure Interaction of R groups (hydrogen bonds, and disulfide bonds) Responsible for the biological activity of the molecule Dr. M. Alzaharna Clini. Chem. 2015 7

Protein Structure Quaternary structure n 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 n 2 or more polypeptide chains binding together ¨ eg. Hemoglobin n Hemoglobin has 4 subunits ¨ ¨ n Two chains Many enzymes have quaternary structures Dr. M. Alzaharna Clini. Chem. 2015 8

Protein Structure n Disruption of bonds holding 2 o, 3 o or 4 o structure together is called denaturation and can cause loss of function of the protein n These Bonds that hold the protein together are weak n 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 9 Dr. M. Alzaharna Clini. Chem. 2015

Protein Charges n Proteins contain many ionizable groups on the side chains of their amino acids as well as their N- and Cterminal ends n A protein can bear positive and/or negative charges on each molecule, due to amino acid composition n p. H dependent n Aspartic acid has: n no net charge at p. H 2. 8, but a net negative charge at p. H 9. 47 Lysine has: n n no net charge at p. H 9. 47, but a net positive charge at acid p. H Dr. M. Alzaharna Clini. Chem. 2015 10

Protein Charges 11 Dr. M. Alzaharna Clini. Chem. 2015

Protein Charges n Isoelectric point (p. I) – The p. H 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 n When the p. H > p. I, a protein has a net negative charge n When the p. H < p. I, a protein has a net positive charge 12 Dr. M. Alzaharna Clini. Chem. 2015

Solubility n Soluble proteins have a charge on their surfaces. n A protein has its lowest solubility at its isoelectric point n Without a net charge, protein-protein interactions and precipitation are more likely n The solubility of proteins in blood requires a p. H in the range of 7. 35 -7. 45. n Differences in solubility can be used to separate major plasma fractions from each other 13 Dr. M. Alzaharna Clini. Chem. 2015

Classification by Protein Structure n Simple Proteins (contain only amino acids) are classified by shape as – ¨ Globular proteins: compact, tightly folded and coiled chains n Majority of serum proteins are globular ¨ Fibrous proteins: elongated, high viscosity (hair, collagen) 14 Dr. M. Alzaharna Clini. Chem. 2015

Classification by Protein Structure n Conjugated proteins contain non-amino acid groups ¨ Amino acid portion is called apoprotein and nonamino 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 15 Dr. M. Alzaharna Clini. Chem. 2015

Conjugated Proteins Classification Example Lipoprotein Prosthetic group Lipid Glycoprotein Carbohydrates Phosphoprotein Phosphate Immunoglobulins Casein of milk HDL 16 Dr. M. Alzaharna Clini. Chem. 2015

Functions of proteins n Generally speaking, proteins do everything in the living cells n Functional classification of plasma proteins is useful in understanding the changes that occur in disease: ¨ Tissue nutrition ¨ Proteins of immune defense n Antibodies ¨ Acute phase proteins n Proteins associated with inflammation ¨ Transport proteins (albumin, transferrin) n Proteins used to bind and transport ¨ Hemostasis n Proteins involved in forming clots and acting very closely with complement 17 Dr. M. Alzaharna Clini. Chem. 2015

Functions of proteins ¨ Regulatory n ( receptors, hormones ) ¨ Catalysis, n enzymes ¨ Osmotic n force Maintenance of water distribution between cells and tissue and the vascular system of the body ¨ Acid-base n balance Participation as buffers to maintain p. H ¨ Structural, contractile, fibrous and keratinous 18 Dr. M. Alzaharna Clini. Chem. 2015

Catabolism & Nitrogen Balance n Most proteins in the body are constantly being repetitively synthesized and then degraded n Balance exists between protein anabolism (synthesis) and catabolism (breakdown) n Nitrogen Balance = Nitrogen intake - Nitrogen loss n Turnover totals about 125 -220 g of protein each day n Normal, healthy adults are generally in nitrogen balance, n with intake and excretion being equal Pregnant women, growing children, and adults recovering from major illness ¨ ¨ are often in positive nitrogen balance Dr. M. Alzaharna Clini. Chem. 2015 19

Catabolism & Nitrogen Balance n 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 20 Dr. M. Alzaharna Clini. Chem. 2015

Plasma Proteins n About 500 proteins have been identified in plasma n The plasma proteins include the immunoglobulins, enzymes, and enzyme inhibitors. n Most plasma proteins, with the notable exception of immunoglobulins, are synthesized in the liver. n Plasma proteins circulate in the blood and between the blood and the extracellular tissue spaces. 21 Dr. M. Alzaharna Clini. Chem. 2015

Plasma Proteins n Prealbumin n Albumin n Globulins α 1, α 2, β, and γ • α 2 Macroglobulin • Transferrin (Siderophilin) β • Hemopexin • Acute phase proteins (CRP) • Immunoglobulins • α 1 -Antitrypsin • α 1 -Fetoprotein • α 1 -Acid Glycoprotein n Myoglobin • Haptoglobin (α 2) n Troponin • Ceruloplasmin 22 Dr. M. Alzaharna Clini. Chem. 2015

Prealbumin (Transthyretin) n Migrates just ahead of Albumin n When a diet is deficient in protein, hepatic synthesis of proteins is reduced ¨ Indicator of nutritional status (very short half life- 2 days) n Transport of thyroid hormones & it also binds with retinol-binding protein to form a complex that transports retinol (vitamin A) 23 Dr. M. Alzaharna Clini. Chem. 2015

Prealbumin (Transthyretin) n Low levels found in: ¨ Hepatic damage ¨ Acute phase responses (-ve acute phase reactant) ¨ Nutritional deficit – short half-life means decrease seen early in disease n Increased level found in: ¨ Steroid treatment ¨ Alcoholism ¨ Chronic renal failure (glomerular filtration rate decreased) 24 Dr. M. Alzaharna Clini. Chem. 2015

Albumin n Synthesized in the liver from 585 amino acids at the rate of 9– 12 grams per day with no reserve or storage n Highest concentration plasma protein n Albumin also exists in the extravascular (interstitial) space n Two primary functions ¨ Colloidal osmotic pressure (80%) ¨ Bind and transport of numerous substances n n Bilirubin, steroids, fatty acids, Ca++, Mg++, salicylic acid & other medications Dr. M. Alzaharna Clini. Chem. 2015 25

Albumin n 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 n results from two copies of different albumin genes, resulting in different charges. 26 Dr. M. Alzaharna Clini. Chem. 2015

Albumin n Increased levels of Albumin ¨ Seen in dehydration ¨ After excessive albumin infusion 27 Dr. M. Alzaharna Clini. Chem. 2015

Globulins n The globulin group of proteins consists of α 1, α 2, β, and γ fractions. n Each fraction consists of a number of different proteins with different functions. 28 Dr. M. Alzaharna Clini. Chem. 2015

Globulins (α 1 -Antitrypsin) n n n Major component (90%) of α 1 fraction Acute phase reactant, synthesized in the liver Most important function the inhibition of the protease neutrophil elastase n n Neutrophil elastase is released from leukocytes to fight infection, but it can destroy alveoli Mutations in the SERPINA 1 gene cause: deficiency which is associated with n Severe degenerative emphysema ¨ Abnormal form of α 1 -antitrypsin can also accumulate in the liver and can cause cirrhosis ¨ α 1 -antitrypsin 29 Dr. M. Alzaharna Clini. Chem. 2015

α 1 -Antitrypsin n Increased levels in: ¨ Inflammatory reactions ¨ Pregnancy ¨ Contraceptive use n Since major component of α 1 band – changes in levels apparent on protein electrophoresis n Quantitative methods used to confirm electrophoresis findings are radial immunodiffusion 30 Dr. M. Alzaharna Clini. Chem. 2015

α 1 -Fetoprotein (AFP) n AFP synthesized by fetal yolk sac & later by parenchymal cells of liver n Peaks in fetus at 13 weeks – decreases gradually by birth n Acts like a fetal “albumin” n Maternal serum testing used to screen fetus for birth defects Neural tube defects & twins n Increase risk of Down’s & trisomy 18 n syndrome 31 Dr. M. Alzaharna Clini. Chem. 2015

α 1 -Fetoprotein (AFP) n In adults level it is high in: ¨ 80% of hepatocellular carcinoma ¨ Gonadal tumors in adults n The methods commonly used for AFP determinations are radioimmunoassay (RIA) and enzyme-labeled immunoassay (EIA) 32 Dr. M. Alzaharna Clini. Chem. 2015

α 1 -Acid Glycoprotein (AAG) n Acute phase reactant, synthesized in the liver n Regulates immune responses ¨ Increased n n n in: inflammation, cancer, pneumonia, Rheumatoid arthritis (RA). The analytic methods used most commonly for the determination of AAG are radial immunodiffusion, immunoturbidity, and nephelometry 33 Dr. M. Alzaharna Clini. Chem. 2015

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Haptoglobin (α 2) n Synthesized in the liver n Considered an acute-phase protein n Binds free hemoglobin n Prevents loss of Hemoglobin & Iron into urine n Used to detect and evaluate hemolytic anemia and to distinguish it from anemia due to other causes Haptoglobin Reticulocytes 1 Decreased Increased Hemolytic anemia 2 Normal Increased RBC destruction may be occurring in organs such as the spleen and liver 3 Normal Anemia present is not due to RBC breakdown Dr. M. Alzaharna Clini. Chem. 2015 35

Haptoglobin (α 2) n Increased in ¨ Inflammations, burns & nephrotic syndrome due to fluid losses n 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 n Radial immunodiffusion has been used for the quantitative determination of haptoglobin 36 Dr. M. Alzaharna Clini. Chem. 2015

Ceruloplasmin (α 2) n Synthesized in the liver n acute-phase reactant n Copper carrying protein n 90% of serum copper is bound to it n 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. 37 Dr. M. Alzaharna Clini. Chem. 2015

Ceruloplasmin n Increased levels ¨ Pregnancy, inflammatory processes, ¨ malignancies, oral estrogens & contraceptives n Low levels ¨ Malabsorption ¨ Severe Liver Disease ¨ Nephrotic syndrome ¨ Menkes’ Syndrome (decreased Cu absorption) n Most assays today use immunochemical methods, including radial immunodiffusion and nephelometry 38 Dr. M. Alzaharna Clini. Chem. 2015

α 2 Macroglobulin n Tetramer of four identical subunits n Synthesized by liver n major component of the α 2 band in protein electrophoresis n Primarily intravascular spaces due to size n Inhibits proteases trypsin, pepsin & plasmin 39 Dr. M. Alzaharna Clini. Chem. 2015

α 2 Macroglobulin n Increased levels ¨ Nephrosis (Large size prevents loss) ¨ Oral contraceptives (high estrogens) n Decreased levels ¨ Pancreatitis n The analytic methods that have been used for the assay of this protein are radial immunodiffusion, ELISA, and latex agglutination immunoassay. 40 Dr. M. Alzaharna Clini. Chem. 2015

Transferrin (Siderophilin) n n n n 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 41 Dr. M. Alzaharna Clini. Chem. 2015

Transferrin (Siderophilin) n Transferrin is abnormally high in iron deficiency anemia n Decreased in ¨ general protein deficiencies ¨ Liver disease ¨ Malnutrition ¨ Inflammations (Negative acute phase protein) ¨ Hereditary Disorders n The analytic method used for the quantitation of transferrin is immunodiffusion 42 Dr. M. Alzaharna Clini. Chem. 2015

Transferrin (Siderophilin) n Atransferrinemia is inherited as an autosomal recessive trait due to mutation of both transferrin genes with a resulting absence of transferrin. n It is characterized by anemia and hemosiderosis (iron deposition) in the heart and liver. n The iron damage to the heart can lead to heart failure. n This disease can be effectively treated by plasma infusions of transferrin. 43 Dr. M. Alzaharna Clini. Chem. 2015

Hemopexin n Beta globulin n acute-phase reactant n Purpose is to remove circulating Heme Breakdown product of Hemoglobin & myoglobin ¨ Carried to liver – broken down ¨ n Increased in pregnancy, ¨ Some malignancies ¨ n Low hemopexin levels are diagnostic of a hemolytic anemia n Hemopexin can be determined by radial immunodiffusion Dr. M. Alzaharna Clini. Chem. 2015 44

C-Reactive Protein n 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… 45 Dr. M. Alzaharna Clini. Chem. 2015

Immunoglobulins n Ig. A, Ig. D, Ig. E, Ig. G, Ig. M n Consist of two identical heavy (H) and two identical light (L) chains n Decreases seen in general protein deficiencies n Increases ¨ Chronic inflammatory processes - polyclonal ¨ Malignancy (multiple myeloma) - monoclonal 46 Dr. M. Alzaharna Clini. Chem. 2015

Myoglobin n Heme protein of skeletal & cardiac muscle n Single polypeptide chain transports oxygen to muscle tissue n Released into blood when striated muscle is damaged ¨ Cardiac injury (AMI) ¨ Trauma or crush injuries n Latex agglutination, ELISA 47 Dr. M. Alzaharna Clini. Chem. 2015

Troponin n Complex of 3 proteins that bind to filaments of striated muscle (cardiac & skeletal) ¨ Troponin T (Tn. T) ¨ Troponin I (Tn. I) ¨ Troponin C (Tn. C) n Regulate muscle contractions ¨ Calcium release attaches troponin n n Increase indicative of myocardial injury n Cardiac troponins can be measured by ELISA 48 Dr. M. Alzaharna Clini. Chem. 2015

Hypoproteinemia n 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 n Burns, trauma, shock ¨ Liver n disease Primary site of protein 49 Dr. M. Alzaharna Clini. Chem. 2015

Hyperproteinemia n Dehydration ¨ Relative due to fluid decrease n Decreased intake or increased loss ¨ All n fractions remain normal ratios Monoclonal increases ¨ Multiple n Myeloma or related malignancies Polyclonal increases ¨ Chronic inflammatory diseases 50 Dr. M. Alzaharna Clini. Chem. 2015