Clinical Chemistry I Lecture: Proteins PDF

MLS 3101: CLINICAL CHEMISTRY I LECTURE PROTEINS Mrs. Arlene Laquian, RMT, MBio 1st Semester A.Y. 2024-2025 OUTLINE I. Amino Acids IV. Miscellaneous a. Major Groups...

MLS 3101: CLINICAL CHEMISTRY I LECTURE PROTEINS Mrs. Arlene Laquian, RMT, MBio 1st Semester A.Y. 2024-2025 OUTLINE I. Amino Acids IV. Miscellaneous a. Major Groups Proteins of Amino Acids V. Measurement of II. Proteins Proteins a. Functions of protein b. Protein Structure c. Classification III. Specific Plasma Proteins I. AMINO ACIDS Building blocks of proteins Are molecules containing an amine group, a carboxylic acid group, and a side chain that varies between different amino acids. II. PROTEINS These molecules contain the key The word protein comes from the Greek word proteis meaning elements of carbon, hydrogen, oxygen, first rank of importance. and nitrogen Synthesized by the liver and secreted by the hepatocyte into Amino Acid Structure the circulation. ○ Amino acids differ from one Most abundant macromolecule (a molecule with a molecular another by the chemical mass of several thousand or more) composition of their R groups They are polymers which are constructed from common 20 (side chain) amino acids joined by peptide bonds. A typical protein contains 200 to 300 amino acids. Product of genes and considered as an active molecule in all cells. The basic building block of the body. Ordinarily, a balance exists between protein anabolism (synthesis) and catabolism (breakdown). Normally, this turnover totals about 125 to 220 grams of protein each day, with the rate of individual proteins widely varying. Nitrogen balance Functions of amino acids a. Negative nitrogen balance ○ Building blocks of protein When protein catabolism exceeds ○ Important in many other anabolism. biological molecules, such as Seen in burns, wasting disease, high forming parts of coenzymes fever, starvation (e.g. S-adenosyl methionine) b. Positive nitrogen balance ○ Precursors for the biosynthesis When anabolism is greater than of molecules such as heme catabolism. Seen in growth, pregnancy, and repair Divisions of Amino Acids process. a. Essential Amino Acids ○ Those that cannot A. FUNCTIONS OF PROTEINS be produced in 1. Protein as enzyme our body and Responsible for almost every chemical reaction that therefore have to takes place in the body be obtained from 2. Transportation and storage food sources. Transport substances across cell membranes that b. Non-Essential amino acids other molecules can’t penetrate ○ Amino acids 3. Cell and Tissue Growth synthesized by Continuous supply of amino acids needed by the the body from body in order to build the proteins that create the carbon, nitrogen, tissue. hydrogen, 4. Mechanical Support oxygen, and Collagen, the most abundant protein found in the sulfur. human body is a structural protein that is fibrous in nature. 5. Coordination and Motion A. 4 MAJOR GROUPS OF AMINO ACIDS Proteins are major components in muscle 1. Non-Polar or Hydrophobic contraction ○ Amino acids that are not soluble in water, which 6. Immune Protection makes them hydrophobic. Antibodies are highly specific proteins that are ○ Examples: Alanine, Valine, Glycine, Leucine, responsible for detecting a foreign substance Isoleucine, Proline, Methionine, Phenylalanine, otherwise known as antigen Tryptophan 7. Nerve Generation and Impulses 2. Uncharged polar The nervous system is responsible for keeping the ○ Polar amino acids have hydrophilic side chain, body in balance which forms hydrogen bonds with water. 8. Fluid balance/Buffer ○ Most have at least one atom (nitrogen, oxygen, or Proteins have the ability to regulate the amount of sulfur) with electron pairs available for hydrogen fluid within the cell bonding to water and other molecules. ○ Examples: Serine, Threonine, Cysteine, B. PROTEIN STRUCTURE Asparagine, Glutamine, Tyrosine Protein structures range in size from tens to several thousands 3. Acidic residues. ○ Negatively charged (overall charge of -1), amino Protein may undergo reversible changes in performing its acids whose side chains can ionize as a weak acid biological function. ○ Examples: Aspartic acid, Glutamic acid 4. Basic 1. Primary structure: ○ Positively charged (overall charge of +1), the side Refers to number and sequence of chains of the basic amino acids contain an amino amino acids in the protein chain group that can ionize as a weak base. The unique amino acid sequence ○ Examples: Lysine, Arginine, Histidine 2. Secondary Structure: Branched-chain amino acid Refers to the regular recurring ○ Refers to the amino acids having aliphatic side arrangement of the amino acid chains that are non-linear. chain into a coil or pleated ○ Examples: Leucine, Isoleucine and Valine sheet Proline Describes protein molecule as ○ Is the only proteinogenic amino acid whose side alpha-helix or beta-pleated group links to the alpha-amino group. sheet. ○ Is also the only proteinogenic amino acid containing a secondary amine Tumalaytay 1  3. Tertiary Structure: ○ Has the capacity to bind with trypsin and inactivate Refers to the specific folding it (acts as a protease inhibitor and neutrophil and bending of the coils into elastase inhibitor) specific layers or fibers. ○ Glycoproteins that rise in response to acute 3D configuration of the protein inflammation. 4. Quaternary Structure: ○ Major component of the fraction of serum proteins Made up of more than 1 that migrate electrophoretically immediately after polypeptide chain. albumin Occurs when several protein ○ Clinical significance: units combine to form a more Increased in: acute inflammatory complex unit. process, pregnancy, and contraceptive Overall arrangement of use. polypeptide chains/subunits Decreased in: liver disease, emphysematous pulmonary disease Reference value: 145-270 mg/dL ○ Measurement: C. CLASSIFICATION OF PROTEIN Radial immunodiffusion According to COMPOSITION: Electrophoresis 1. Simple Immunonephelometric assay Contains peptide chain on which hydrolysis yields Immunofixation for phenotyping only amino acids. Globular proteins such as albumin, Alpha-1-antichymotrypsin globulin, albuminoids, histones, and ○ An alpha globulin that is a member of the serine protamines. protease inhibitor (serpin) family. Fibrous proteins such as collagen and ○ It inhibits the activity of the enzymes cathepsin G, troponin pancreatic elastase, mast cell chymase, and 2. Conjugate chymotrypsin by cleaving them into a different Comprised of a protein moiety (apoprotein) and shape (conformation). non-protein moiety (prosthetic group) ○ Produced in the liver; it is an acute phase reactant Lipoproteins ○ Deficiency has been associated with liver disease Metalloproteins Glycoproteins Inter-alpha-trypsin inhibitor Nucleoproteins ○ A family of serine protease inhibitors, assembled According to SHAPE: from two precursor proteins. 1. Globular ○ Plays a particular role in inflammation and Spherical in shape with mobile and dynamic carcinogenesis. function. ○ Elevations are seen in inflammatory disorders Ex: hormones, enzymes, hemoproteins 2. Fibrous Alpha-2 macroglobulin (AMG) Elongated in shape which are for structural ○ Largest non-immunoglobulin protein in plasma, purposes. synthesized in the hepatocytes. Ex: keratin, myosin ○ Increased in nephrotic syndrome, diabetes, liver disease According to SOLUBILITY: ○ Measurements: 1. Albumin: soluble in water and high concentration salt solution a. Immunonephelometry, radial 2. Globulin: insoluble in water and concentrated salt solutions, immunodiffusion, ultracentrifugation, soluble in weak salt solution. latex agglutination, ELISA 3. Albuminoids: insoluble in most common reagents Haptoglobin ❖ Reference value: Total Protein (TP): 60-70 g/L ○ Migrates in the alpha-2 region ○ Function is to bind free hemoglobin by its alpha III. SPECIFIC PLASMA PROTEINS chain thus prevents the loss of hemoglobin and its A. MAJOR COMPONENTS constituent iron into the urine. ○ Used to evaluate degree of intravascular hemolysis 1. Prealbumin/Transthyretin/TBPA that has occurred in transfusion reaction or HDN ○ Migrates faster than albumin towards the anode in ○ Half life is 4 days electrophoresis ○ Considered as an acute phase protein. ○ Tetrameric structure ○ Used to evaluate rheumatic diseases. ○ Binds with Retinol Binding Protein (RBP) to form a ○ Measured by: radial immunodiffusion, complex that transports retinol. immunonephelometry. ○ Short half-life in the circulation (2 days) ○ Clinical significance: ○ Quantitation is important marker for nutritional Increased in: ulcerative colitis, acute status. rheumatic disease, heart attack, severe ○ Easily crosses the placenta and CSF; very rich in infection, nephrotic syndrome tryptophan. Decreased in: HDN, burns, hemolytic ○ Transport protein for thyroxine and triiodothyronine transfusion reaction. ○ Reference value: 18-45 mg/dL ○ Prealbumin decreased in: Transferrin/Siderophilin a. Hepatic damage ○ Major beta-globulin protein in electrophoresis b. Acute phase inflammatory response ○ Transports ferric iron to its storage sites. c. Tissue necrosis ○ A glycoprotein synthesized in the liver. ○ Prealbumin increased in: ○ Negative acute phase protein a. Patient receiving steroids ○ Low plasma transferrin can impair hemoglobin b. Alcoholism production and lead to anemia c. Chronic renal failure ○ Reference value: Male: 215-365 mg/dL; Female: 2. Albumin 250-380 mg/dL ○ Most abundant protein in normal plasma ○ Measurement: immunodiffusion, constituting about 2/3 of the total protein immunonephelometry ○ Synthesized in the liver at a rate of 9-12 grams/day ○ Clinical significance: ○ Reference value: 32-45 g/L Increased in: iron deficiency anemia, ○ Half life: 7 days pregnancy, oral contraceptive, ○ Functions: hemochromatosis a. Regulation of oncotic pressure, Decreased in: chronic infection, maintains appropriate fluid balance in the malignancy, iron poisoning, liver disease, tissue. malnutrition, nephrotic syndrome b. Acts as mobile repository of amino acid c. Acts as a carrier protein molecule Fibrinogen Molecules transported by ○ One of the largest proteins in the blood; synthesized albumin: thyroxine, penicillin, in liver. estrogen, bilirubin, cortisol, ○ Most abundant of the coagulation factors free fatty acid responsible for the formation of fibrin clot. ○ Albumin increased in: ○ Classified as a glycoprotein because it has Dehydration considerable carbohydrate content. Prolonged tourniquet application ○ Migrates between beta and gamma fractions. ○ Albumin decreased in: ○ Reference value: 200-400 mg/dL Malnutrition, liver disease, GI ○ An acute phase protein inflammation, albuminuria, severe burns, ○ May serve as a marker for long-term prognosis of hypothyroidism, acute disease states cardiovascular disease. Mutation: Analbuminemia (absence); ○ High levels in plasma may cause elevated Bisalbuminemia (2 bands) erythrocyte sedimentation rate (ESR) 3. Globulin ○ Measurement: radial immunoassay, nephelometry ○ Group of protein which consists of alpha-1, alpha-2, ○ Clinical significance: beta, and gamma fractions Increased in: pregnancy, oral ○ Measured by subtracting albumin from total protein contraceptives, TB, inflammatory ○ Reference: 23-35 g/L disorders, septicemia, pneumonia ○ A/G ratio: 1.5 to 2.5 : 1 Decreased in: terminal liver disease, fetal death in utero, antepartal Alpha-1-antitrypsin (AAT) hemorrhage. ○ Major component of the alpha-1 globulin fraction 2  9. Beta-2-microglobulin (B2M) Gamma globulins ○ It is the light chain component of the major ○ Also known as immunoglobulins histocompatibility complex (human leukocyte or antibodies antigen [HLA]) ○ Glycoproteins composed of 82% ○ It is found on the surface of most nucleated cells to 96% protein and 4% to 18% and is present in high concentrations on carbohydrate; produced by white lymphocytes. blood cells. ○ Because of its small size (11,800 kDa), it is filtered ○ Consist of two identical heavy and by the glomerulus, but >99% is reabsorbed and two identical light chains linked by catabolized in the proximal tubules. disulfide bonds. ○ Elevated levels are seen in: ○ Five classes: IgG, IgA, IgM, IgD, IgE a. Impaired clearance by the kidney ○ Measurement: b. Overproduction of the protein (e.g. RA, 1. RIA SLE) 2. ECLIA ○ In patients with HIV, a high B2M level in the 3. Nephelometry absence of renal failure indicates a large 4. Turbidimetry lymphocyte turnover rate. 5. Radial immunodiffusion ○ Measurement: immunoassay B. MINOR COMPONENTS IV. MISCELLANEOUS PROTEINS 1. Myoglobin 1. Alpha-1 fetoprotein (AFP) ○ A small heme protein found in the striated skeletal ○ Most abundant protein in fetal serum. and cardiac muscles ○ It peaks in the fetus at 13 weeks of gestation ○ Approximately 2% of the total muscle protein ○ Synthesized initially by the fetal yolk sac and then ○ Reference value: Male: 30-90 ng/mL; Female: by the fetal parenchymal cells of the liver. 100 ug/L) ○ Increased in hepatocellular carcinoma, ○ Useful marker for monitoring success or failure of teratoblastomas reperfusion. ○ Decreased in Trisomy 21 ○ Measurement: ELISA, latex agglutination, FIA ○ Measurement: RIA, EIA, immunochemical test 2. Troponins (Tn) ○ A complex of 3 proteins (regulatory proteins) that 2. Ceruloplasmin bind to the thin filaments of cardiac muscles. ○ Reference value: 23-50 mg/dL ○ For muscle contraction; regulators of actin and ○ Copper-containing serum glycoprotein which myosin. functions as a circulating oxidase (responsible for 1. Troponin T oxidizing iron from ferrous to ferric) (TnT)/Tropomyosin-binding subunit ○ It imparts a blue color to protein Specific for heart muscle ○ It is a marker for Wilson’s disease (0.1g/L of AMI: it rises within 3-4 hours ceruloplasmin) after onset of AMI, peak level ○ Increased in: inflammation, pregnancy, cancer is at 10-24 hours and return to ○ Decreased in: Wilson’s disease, hepatocellular normal in 7 days (but may disease, malnutrition, malabsorption, nephritic remain increased for 10-14 disease. days). ○ Measurement: colorimetry, immunochemical Useful in monitoring methods, nephelometry thrombolytic therapy in AMI patients 3. Lipoproteins 2. Troponin (TnI)/Inhibitory subunit or ○ Are complexes of proteins and lipids whose function Actin-binding subunit is to transport cholesterol, triglyceride, and Only found in the myocardium phospholipid in the blood. Highly specific for AMI ○ Migrate with a characteristic sharp leading edge Very sensitive indicator of and feathery trailing edge. minor amount of cardiac ○ HDL: migrates between albumin and alpha-1 necrosis. globulin zone. AMI: it rises within 3-6 hours, ○ VLDL: migrates at the beginning of the beta-globulin peak level is at 12-18 hours, region. and return to normal in 5-10 ○ LDL: appears as a separate band in the days beta-globulin region. 3. Troponin C (TnC ) ○ Reference value: 8-14% of TP Calcium-binding protein subunit that initiates the 4. Group-specific component globulin/Gc-globulin sequence of conformational ○ Exhibits high-binding affinity with vitamin D changes on the thin filament compounds and actin. 3. Bence-Jones Protein ○ Migrates in the alpha-1 and alpha-2 interzone ○ An abnormal protein found in urine of patients with during electrophoresis. possible multiple myeloma (a type of cancer ○ Increased in: pregnancy, oral contraceptives affecting the bone marrow) ○ Decreased in: severe liver disease and ○ Precipitates in acid solution and redissolves upon protein-losing syndrome. heating of the mixture ○ Heat precipitation test: a. Upon heating a urine specimen 5. Alpha-1-acid glycoprotein/orosomucoid (AAG) containing BJP, it will precipitate at 56°C ○ Binds to progesterone and quinidine (cardioactive and redissolve again at 100°C drug) and is important in transport and metabolism 4. B-type/Brain Natriuretic Peptide (BNP) ○ High carbohydrate content which minimizes its ○ Marker for congestive heart failure visualization by standard protein stains. ○ Measurement: immunoradiometric assay, ○ Useful diagnostic tool in neonates with bacterial microparticle enzyme immunoassay, and ECLIA infections. 5. Fibronectin ○ Increased in: cancer, pneumonia, rheumatoid ○ A glycoprotein composed of two nearly identical arthritis, pregnancy, cell proliferation. subunits. ○ Plasma fibronectin has been used as a nutritional 6. Hemopexin marker. ○ A beta-globulin that binds with heme released from ○ Fetal fibronectin is a glycoprotein used to help degradation of hemoglobin. predict the short-term risk of premature delivery ○ Increased in: DM, melanoma ○ Fetal fibronectin is normally detectable in amniotic ○ Decreased in: hemolytic disorders, intravascular fluid and placental tissue during early pregnancy, hemolysis and is no longer detectable after 24 weeks. ○ Reference value: 50-115 mg/dL 6. Adiponectin ○ A 247-amino acid fat hormone 7. C-reactive protein ○ Exists in trimers, hexamers, and multimers in the ○ Serves as a general scavenger molecule. blood ○ Highly sensitive acute phase reactant ○ Inverse correlation between BMI and adiponectin ○ Used to monitor the progression or remission of values. autoimmune disease. 7. Beta-trace protein (BTP) ○ Synthesized in the liver and appears in blood in ○ Also known as prostaglandin D synthase. inflammatory condition; it may be undetectable in ○ A 168-amino acid, low molecular mass protein healthy individuals ○ An accurate marker of CSF leakage. ○ Increased in: rheumatic fever, MI, bacterial ○ A potential marker in detecting impaired renal infections, RA, gout function. 8. Cross-linked C-Telopeptides (CTXs) 8. Complement ○ Proteolytic fragments of collagen I formed during ○ A collective term for several proteins that participate bone resorption (turnover) in the immune reaction and serve as a link to the ○ A biochemical marker of bone resorption inflammatory response. ○ Can be detected in serum and urine ○ Circulates in the blood as nonfunctional precursors ○ Measurement: ECLIA; can also be automated ○ Increased in: inflammatory conditions ○ Decreased in: malnutrition, hemolytic anemia, DIC ○ Measurement: immunonephelometry, turbidimetry 3  V. MEASUREMENT OF PROTEINS 1. Kjeldahl Method ○ It is the reference method but not routinely used ○ It is based on the measurement of the nitrogen content of protein; end product of the method is ammonia. ○ It uses serum treated with tungstic acid, forming PFF ○ Reagent: sulfuric acid 2. Biuret Method ○ It is the most widely used method ○ It requires at least 2 peptide bonds and an alkaline medium ○ Reagents: Alkaline copper sulfate, Rochelle salt (Na K tartrate), Sodium hydroxide, Potassium iodide ○ End product: violet-colored chelate 3. Folin-Ciocalteu (Lowry) Method ○ It has the highest analytical sensitivity ○ Principle: Oxidation of phenolic compounds to give a deep blue color ○ Main reagent: Phosphotungstic-molybdic acid or phenol 4. Ultraviolet Absorption Method ○ Principle: The absorbance of proteins at 210 nm is due to the absorbance of the peptide bonds at specific wavelength. ○ Proteins absorb light at 280 nm and at 210 nm ○ Absorption at 280 nm is due to tryptophan, tyrosine, and phenylalanine 5. Serum Protein Electrophoresis (SPE) ○ Principle: migration of charged particles in an electric field. ○ Normal SPE Pattern: 1. Albumin (1st band) – fastest band 2. Alpha-1-globulin (2nd band) glycoproteins, AAT, AAG, TBG 3. Alpha-2-globulin (3rd band) haptoglobin, ceruloplasmin, AMG 4. Beta-globulin (4th band) transferrin, beta-lipoprotein, hemopexin, complement 5. Gamma globulin (5th band; slowest) immunoglobulins ○ Abnormal SPE Pattern: 1. Gamma spike plasma cell myeloma 2. Beta-gamma bridging hepatic cirrhosis 3. Alpha-2-globulin band spike nephrotic syndrome 4. Alpha-1-globulin flat curve juvenile cirrhosis 5. Spikes of αlpha-1, alpha-2, and beta globulin bands inflammation 6. Refractometry ○ It is an alternative test to chemical analysis of serum TP. ○ Based on measurement of refractive index of serum solutes. 7. Turbidimetric and Nephelometric Methods ○ These methods utilize sulfosalicylic acid and/or trichloroacetic acid. ○ Measurement depends on the formation of a uniform fine precipitate which scatters or blocks light 8. Salt Fractionation ○ Globulins can be separated from albumin by salting-out procedures using sodium salts ○ Reagent: sodium sulfate salt Compiled by: Joy G. Raso, PhD., RB John Tacardon, RMT; Daryl Jasyl L. Cañon, RMT. 4