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Amino acids and proteins


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aminoacids and proteins
by sunil shah (the great)

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Amino acids and proteins

  2. 2. BASIC STRUCTURES  An amino acid contains:  a central carbon bonded to a carboxyl group (-COOH),  an amino group (NH2), an  R-group, and  a hydrogen.  The R-group is what varies between the 20 amino acids and gives them unique characteristics.  The covalent bonds between amino acids are called peptide bonds.  It is a bond between the carboxyl group of one amino acid and the amino group of another amino acid.
  3. 3. Abbre Full Name Side chain type Remarks v. Very abundant and versatile. It behaves fairly neutrally and can be located in both hydrophilicA Ala Alanine hydrophobic regions on the outside of the protein and hydrophobic interior areas. The sulfur atom binds readily to heavy metal ions. Under oxidizing conditions, two cysteines can be joined together by a disulfide bond to form the amino acid cystine. When cystines are components of a protein, they enforce tertiary structure and makes hydrophobic (Nagano, the protein more resistant to unfolding andC Cys Cysteine 1999) denaturation; disulfide bridges are therefore common in proteins that have to function in harsh environments, digestive enzymes (e.g., pepsin and chymotrypsin), structural proteins (e.g., keratin), and proteins too small to hold their shape on their own (eg. insulin). Behaves similarly to glutamic acid. Carries a hydrophilic acidic group with strong negative charge. Aspartic Usually is located on the outer surface of the protein,D Asp acidic acid making it water-soluble. Binds to positively-charged molecules and ions, often used in enzymes to fix the
  4. 4. Side chainAbbrev. Full Name Remarks type Phenylalanine, tyrosine, and tryptophan contain large rigid aromatic group on the side chain. These are the biggest Phenylala*F Phe hydrophobic amino acids. Like isoleucine, leucine and valine, they are nine hydrophobic and tend to orient towards the interior of the folded protein molecule. Because of the two hydrogen atoms at the α carbon, glycine is not optically active. It is the smallest amino acid, rotates easily,G Gly Glycine hydrophobic and adds flexibility to the protein chain. It is able to fit into the tightest spaces (e.g., the triple helix of collagen). In even slightly acidic conditions, protonation of the nitrogen occurs, changing the properties of histidine and the polypeptide as a whole. It is used by many proteins as a*H His Histidine basic regulatory mechanism, changing the conformation and behavior of the polypeptide in acidic regions such as the late endosome or lysosome, enforcing conformation change in enzymes. Isoleucine, leucine and valine have large aliphatic hydrophobic side chains. Their molecules are rigid, and their mutual hydrophobic interactions are important for the correct folding*I Ile Isoleucine hydrophobic of proteins, as these chains tend to be located inside of the protein molecule.
  5. 5. Side chainAbbrev. Full Name Remarks type Behaves similarly to arginine. Contains a long flexible side- chain with a positively-charged end. The flexibility of the chain makes lysine and arginine suitable for binding to molecules with many negative charges on their surfaces. (e.g., DNA-*K Lys Lysine basic binding proteins have their active regions rich with arginine and lysine.) The strong charge makes these two amino acids prone to be located on the outer hydrophilic surfaces of the proteins.*L Leu Leucine hydrophobic Behaves similar to isoleucine and valine. Always the first amino acid to be incorporated into a protein; sometimes removed after translation. Like cysteine, it contains sulfur, but with a methyl group instead of hydrogen.*M Met Methionine hydrophobic This methyl group can be activated, and is used in many reactions where a new carbon atom is being added to another molecule.N Asn Asparagine hydrophilic Neutralized version of aspartic acid.
  6. 6. Side chainAbbrev. Full Name Remarks type Contains an unusual ring to the N-end amine group, which forces the CO-NH amide sequence into a fixed conformation. Can disrupt protein folding structures like α helix or β sheet,P Pro Proline hydrophobic forcing the desired kink in the protein chain. Common in collagen, where it undergoes a posttranslational modification to hydroxyproline. Uncommon elsewhere. Neutralized version of glutamic acid. Used in proteins and asQ Gln Glutamine hydrophilic a storage for ammonia.*R Arg Arginine Basic Functionally similar to lysine. Serine and threonine have a short group ended with a hydroxyl group. Its hydrogen is easy to remove, so serineS Ser Serine Hydrophilic and threonine often act as hydrogen donors in enzymes. Both are very hydrophilic, therefore the outer regions of soluble proteins tend to be rich with them.
  7. 7. Side chainAbbrev. Full Name Remarks type*T Thr Threonine hydrophilic Behaves similarly to serine. Behaves similarly to isoleucine and leucine.*V Val Valine hydrophobic See isoleucine. Behaves similarly to phenylalanine and*W Trp Tryptophan hydrophobic tyrosine (see phenylalanine). Precursor of serotonin. Behaves similarly to phenylalanine andY Tyr Tyrosine hydrophobic tryptophan (see phenylalanine). Precursor of melanin, epinephrine, and thyroid hormones.
  8. 8. GENERALITIESClassification  Neutral• Neutral • Aliphatic • Aromatic• Acidic • Heterocyclic• Basic • S-containing AA
  9. 9. GENERALITIES - NEUTRALA. Aliphatic  Hydroxy Amino Acids – they contain OH group in the side chains1. Glycine (Gly) (G) – alpha– amino acetic acid  6. Serine (Ser) (S) or alpha – amino β – hydroxy propionic acid2. Alanine (Ala) (A) – alpha –  7. Threonine (Thr) (T) or alpha – amino propionic acid amino β – hydroxybutyric acid3. Valine (Val) (V) – alpha– amino – iso – valeric acid4. Leucine (Leu) (L) – alpha – amino – iso – caproic acid5. Isoleucine (Ile) (l) – alpha – amino – B – methyl valeric acid Above = Simple monoamino monocarboxylic acids
  10. 10. GENERALITIES B. Aromatic  C. Heterocyclic AA 8. Phenylalanine (Phe) (F)  10. Tryptophan (Trp) (W) – or alpha – amino – β – alpha – amino β – 3 – indole phenyl propionic acid propionic  - often considered 9. Tyrosine (Tyr) (y) or as aromatic AA since it has parahydroxy aromatic ring in its structure. phenylalanine or alpha –  11. Histidine (His) (H) – amino β – parahydroxy alpha – amino – β - phenylpropionic acid imidazole propionic acid  Histidine is basic in solution on account of the imidazole ring and often considered as Basic AA
  11. 11. GENERALITIES D. Imino Acids  E. „S‟ containing Amino Acids 12. Proline (Pro) (P) or  - contains 2 sulfur containing AA Pyrrolidone – 2 – carboxylic Acid  14. Cysteine (Cys) (C) or alpha – 13. Hydroxyproline (Hyp) or 4 – amino – β – mercaptopropionic Hydroxy pyrrolidone – 2 – acid carboxylic acid  S – S linkage is called a Disulfide bridge Proline & Hydroxyproline do  15. Methionine (Met) (M) or alpha not have a free _NH2 group but – amino y – methylthio - - butyric only a basic pyrrolidone ring in acid which the Nitrogen of the Imino group is in ring but can still function in the formation of peptides. Are called Imino Acids.
  12. 12. GENERALITIES - ACIDIC II. Acidic AA - AA with 2 _COOH groups and 1 _NH2 group - monoaminodicarboxylic Acids 16. Aspartic Acid (Asp) (P) or alpha – amino succinic acid Asparagine (Asn) (N) or delta amide of alpha – amino succinic acid 17. Glutamic Acid (Glu) (E) or alpha aminoglutaric acid Glutamine (Gln) (Q) – amide of glutamic acid or 8 – amide of alpha – amino glutaric acid
  13. 13. GENERALITIES - BASIC  III. Basic AA  - AA with 1 – COOH group and 2 – NH2 groups  - Diamino monocarboxylic acids  18. Arginine (Arg) (R) or alpha – amino – 8 – guanidino - valeric acid  19. Lysine (Lys) (K) or alpha – E diamino  8 – hydroxy - - valeric acid  As already mentioned Histidine – is also classified as Basic AA
  14. 14. GENERALITIES Classificationof Amino Acids based on polarity of the R group: • 4 groups • Polarity  reflects the functional role of AA in protein structure
  15. 15. GENERALITIES 1. Non-polar AA • hydrophobic (water hating) • No charge on the ‘R’ group • Examples are: Alanine Methionine Leucine Phenylalanine Isoleucine Tryptophan
  16. 16. GENERALITIES  2. Polar AA with (+) ‘R’ group • carries (+) charge • Examples: Histidine Arginine Lysine  3. Polar AA with (-) ‘R’ group • carries (-) charge • Examples: Glutamic Acid Aspartic Acid
  17. 17. GENERALITIES  4. Polar AA with no charge  on ‘R’ group • no charge on the ‘R’ group • possess groups  hydroxyl sulfhydryl amide • participate in hydrogen bonding of • protein structure • Examples: Asparagine Glycine Cysteine Tyrosine Serine Threonine
  18. 18. GENERALITIES  Zwitterion or dipolar ion:  Zwitter   from German word – means “hybrid”  Zwitter ion (or dipolar ion)   a hybrid molecule containing (+) and (-) ionic groups
  19. 19. GENERALITIES AA rarely exist in a neutral form with free carboxylic (-COOH) and free Amino (-NH2) groups Strongly acidic pH (low pH)  AA (+) charged (cation) Strongly alkaline pH (high pH)  AA (-) charged (anion) Each AA has a characteristic pH (e.g. Leucine, pH – 6.0), at which it carries both (+) and (-) charges and exist as zwitterion
  20. 20. GENERALITIES  Isoelectric pH (symbol pI) the pH at which a molecule exists as a Zwitterion or dipolar ion and carries no net charge Molecule is electrically neutral
  21. 21. GENERALITIES Tryptophan Histidine Arginine Leucine ValinePhenylalanine PVT TIM HALL Lysine Threonine Methionine Isoleucine Essential Amino Acids
  22. 22. Essential Amino Acids (Body cannot make these amino acids, they must comefrom food or amino acid supplements.) Min. Dail Acid y Base # of pathways - One Generate Works Amino Acid Sym Abbr mg Neu. Pathway s With Augments 1 - threonine -->1 Isoleucine* I Ile 10 n - - muscles isoleucine blood, 1 - keto-isovalerate --2 Leucine* L Leu 14 n - - muscle, > leucine hormone herpes,3 Lysine K Lys 12 B 2 - asparate --> lysine - calcium triglycerides 7 - homoserine --> seleniu hair, skin,4 Methionine M Met 13 n cysteine methionine m, zinc chelator Phenylalanin 3 - chorismate -->5 F Phe 14 n tyrosine B6 depression e phenylalanine collagen, 3 - aspartate --> glycine,6 Threonine T Thr 7 N - tooth threonine serine enamel 1 - chorismate --> niacin,7 Tryptophan W Trp 3.5 n - depression tryptophan seratonin8 Valine* V Val 10 n 1 - pyruvate --> valine - - muscles Food Sources: Fish - meat - poultry - cottage cheese - peanuts - lentils A= acid, B= base, N= Neutral, n= non-polar
  23. 23. Non-Essential Amino Acids (The body can make these amino acids from the above essential aminoacids.) Min. Acid Daily Base # of pathways - One Amino Acid Sym. Abbr mg. Neu. Pathway Generates Works With Augments 4 - valine pyruvate -->1 Alanine A Ala - n - - - alanine immune, polyamines,2 Arginine* R Arg - B 4 - citrulline --> arginine zinc (lysine) healing, creatine muscles3 Asparagine N Asn - N 3 - asparate and ammonia - - CNS Aspartic4 D Asp - A 3 - glutamate --> aspartate - - CNS, brain Acid homocysteine5 Cysteine C Cys - N 4 - serine --> cysteine B6, Vit.E skin, hair , taurine Glutamic 3 - ketoglutarate --> glutamine,6 E Glu - A B6 brain Acid glutamate GABA7 Glutamine Q Gln - N 2 - glutamate --> glutamine - - brain GABA,8 Glycine G Gly - N 2 - serine --> glycine glutathione body protein taurine blood, allergy,9 Histidine* H His - B 1 - histidinol --> histidine histamine - sex hydroxyprolin collagen,10 Proline P Pro - n 4 - l-glutamine --> proline Vitamin C e elastin 1 - phosphoglycerate --> cysteine,11 Serine S Ser - N choline blood sugar serine glycine 2 - phenylalanine --> thyroxin,12 Tyrosine Y Tyr - N B6 thyroid tyrosine melanin * = These are Essential for infants, since their bodies cannot produce them yet.
  24. 24. Other Amino Acid Factors Lysine, carnitine --> fat- Carnitine - Car - - methionin Vitamin C butyrobetaine --> metabolism e argenine --> citrulline- Citrulline - Cit - - Ornithine zinc urea cycle --> ornithine Hydroxy Hy proline -->- - - - - Vitamin C collagen proline p hydroxyproline Or argenine --> citrulline- Ornithine - - - - - urea cycle n --> ornithine methionine --> GABA,- Taurine - Tau - - B6 heart, bile cysteine --> taurine glycine The above are precursors for, or important products of, the 20 "true" amino acidslisted above.
  25. 25. AMINO ACID SYNTHESIS  ALL ARE SYNTHESIZED FROM COMMON METABOLIC INTERMEDIATES  NON-ESSENTIAL  Transamination of -KETOACIDS that are available as common intermediates  All except tyrosine are derived from one of the following common intermediates: pyruvate, oxalacetate, -KG, 3-phosphoglycerate  ESSENTIAL  Their -KETOACIDS are not common intermediates (Enzymes needed to form them are lacking)
  26. 26. Amino Acids Glucogenic Glucogenic and KetogenicA Ketogenicm Non-Essential Alanine Tyroxinei AA Asparagineno AspartateA Cysteinec Glutamateid GlutamineM Glycinee Prolineta Serinebo Essential AA Arginine Isoleucine Leucinel Histidine Phenylalanine Lysineis Methionine Tryptophanm Threonine ValineGlucogenic – amino acids that give rise to pyruvate andcitric acid cycle intermediates that can be turned intoglucoseKetogenic – amino acids that give rise to acetoacetate and
  29. 29. ALKAPTONURIA  DEFECTIVE PROCESS: TYROSINE DEGRADATION  DEFECTIVE ENZYME: HOMOGENTISATE- 1,2-DIOXYGENASE • Original “inborn error of metabolism • Darkening of urine at long standing • OCHRONOSIS – generalized pigmentation of tissues and arthritislike degeneration
  30. 30. PHENYLKETONURIA DEFECTIVE PROCESS: CONVERSION OF PHENYLALANINE TO TYROSINE DEFECTIVE ENZYME: PHENYLALANINE HYDROXYLASE (Phenylalanine-4-mono-oxygenase) An autosomal recessive trait – urine has a musty odor Compounds seen in both urine and blood: Phenylpyruvic acid – primary catabolite Phenyllactic acid – product of deaminzation Phenylacetylglutamine – produced from oxidation of phenylpyruvic acid
  31. 31. PHENYLKETONURIA  NOT RESPONSIVE TO DIET: DEFICIENCY OF ENZYME S NEEDED FOR THE REGENERATION AND SYNTHESIS OF TETRAHYDROBIOPTERIN (BH4) • High phenylalanine and deficiency of production of neurotransmitters from tyrosine and tryptophan • Administration of L-dopa and 5-OH tryptophan  SCREENING: GUTHRIE BACTERIAL INHIBITION ASSAY – B. subtilis + β2-thienylalanine • Semi-qualitative method: phenylalanine >2-4 mg/dL will result to bacterial growth indicative of a POS (+) result • Microfluorometric assay – based on the fluorescence complex formed of phenylalanine-ninhydrin copper in the presence of dipeptides (L-leucyl-L- alanine) • Quantitative method: filter paper is pretreated with trichloroacetic acid added with ninhydrin, succinate and leucylalanine in the presence of copper tartrate and read at excitation and emission wavelengths of 360nm and 530nm  HPLC – reference method (1.2 -3.4 mg/dL)
  32. 32. MAPLE SYRUP DISEASE DEFECTIVE PROCESS: METABOLISM OF THREE ESSENTIAL BRANCHED-CHAINAMINO ACIDS (LEUCINE, ISOLEUCINE AND VALINE) DEFECTIVE ENZYME: BRANCHED CHAIN α-KETO ACID DECARBOXYLASE COMPLEX• Burnt sugar odor of urine, breath and skin• Screening: Modified Guthrie test – metabolic inhibitor of B. subtilis which is 4-azaleucine is impregnated in the medium • POS (+) for MSUD = bacterial growth• Microfluorometric assay using leucine dehydrogenase • Filter paper specimen is treated with methanol and acetone to denature hemoglobin • The NADH fluorescence produced is measured at 450nm; excitation wavelength at 360nm• Confirmed diagnosis is based on finding increased plasma and urinary levels of the three branched-chain amino acids and their ketoacids with LEUCINE (highest: above 4mg/dL)• Prenatal diagnosis: measuring decarboxylase enzyme concentration in cells cultured from amniotic fluid
  33. 33. CYSTINURIA  DEFECTIVE PROCESS: AMINO ACID TRANSPORT SYSTEM RATHER THAN A METABOLIC ENZYME DEFICIENCY • 20 – 30 fold increase in the urinary excretion of cystine as a result of genetic defect in the renal resorptive mechanism • Other amino acids excreted: ornithine, lysine and arginine • Cystine being relatively insoluble and once accumulated will tend to precipitate in the kidney tubules forming calculi • Remedy: high fluid intake and alkalinizing the urine: penicillin • Diagnosis: Cyanide-nitroprusside (red-purple color)
  34. 34. ARGININOSUCCINIC ACIDURIA ANDCITRULLINEMIA PROCESS: UREA CYCLE  DEFECTIVE  DEFECTIVE ENZYME: ARGININOSUCCINIC ACID LYASE • Decrease in activity of ASA synthetase  Citrullinemia • Citrulline is elevated in MS/MS • Argininosuccinic aciduria – milder elevations • Citrullinemia – dramatic elevations • * Ornithine and arginine are seen increased in older infants
  35. 35. ISOVALERIC ACIDEMIA DEFECTIVE PROCESS: DEGRATIVE PATHWAY OF LEUCINE DEFICIENCY ENZYME: ISOVALERYL-CoA DEHYDROGENASE • “Sweaty feet” odor urine • Elevations of glycine conjugate: isovaleric acid and isovalerylglycine
  37. 37. FUNCTIONS OF PROTEINS  1. Enzymatic catalysis  2. Transport and storage  3. Coordinated motion  responsible for movements in the body (muscles)  4. Mechanical support  support of body - bones and muscles  5. Generation and transmission of impulses  6. Control of growth and differentiation  AA - simplest form of proteins  20 AA
  38. 38. CHARACTERISTICS OF PROTEINS  1. Have more members  2. Fundamental component of protoplasm  3. Elements in protein  C, H, O, N (I & Fe)  4. Proteins are considered macromolecules, composed of a number of AA joined together by peptide bond or linkage  5. Only foodstuff when absent in the diet will cause death  6. Utilize in the body for growth
  39. 39. PROTEIN METABOLISM GASTRIC • Digestion of protein begins in the stomach where the enzyme pepsin is secreted by the chief cells as proenzyme or zymogen (inactive form) • Pesinogen – activated by HCL secreted by the parietal cells • By autoactivation – pepsin itself stimulates its own activation
  40. 40. PROTEIN METABOLISM  PANCREATIC • Once protein reaches the duedenum, exocrine pancreatic secretion: trypsin,chymotrypsin, elastase and carboxypeptidase in their inactive forms are release • Endopeptidase – cleave protein in the internal sites • Exopeptidase – cleave one amino acid from the carboxyl terminus of the polypeptide Secretin stimulates the pancreas to produce a protein-free electrolyte solution rich in bicarbonates
  41. 41. PROTEIN METABOLISM INTESTINAL• This mediated by peptidase produced by the mucosal cells• Amino peptidases and dipeptidases hydrolyze the residual peptides• The end products of protein digestion are amino acids and some short peptides
  42. 42. AMINO ACID POOL Primarily for the  Also for the synthesis of synthesis of body nonprotein nitrogen- proteins: containing compounds• Plasma • Purines• Intracellular proteins • Pyrimidines• Structural proteins • Creatine • Porphyrins • Histamines
  43. 43. PROTEIN MEASUREMENT PLASMA TOTAL  GLOBULIN: PROTEIN: • 23 – 35 g/L• 60 – 70 g/L (SI units) • 2.3 – 3.5 g/dL• 6.0 – 7.0 g/dL (Conventional unit) FIBRINOGEN: • 2 – 4 g/LALBUMIN: • 0.2 – 0.4 g/dL• 32 – 45 g/L• 3.2 – 4.5 g/dL
  44. 44. DETERMINATION OF PROTEIN NITROGEN  KJELDAHL • Reference (standard) method for the determination of protein concentration • Liberation the nitrogen content from proteins is measured by oxidation with heat at 350oC and strong sulfuric acid • Catalysts used during the digestion: copper sulfate, mercuric sulfate or selenium oxychloride • Nitrogen then is converted to ammonium ion • Separated from the digestant by steam distillation • Ammonium ion is liberated and are measured by titration with an alkali, Nesslerization or
  45. 45.  NESSLERIZATION• Liberation of ammonium ions with Nessler‟s reagent – double iodide of potassium and mercury  dimercuric ammonium iodide (yellow to orange brown product• Colloidal stabilizer: gum ghattiBERTHELOT• Idophenol reaction• Ammonia is allowed to react with alkaline hypochlorite to form indophenol blue solution• Catalyst: sodium nitroprusside
  46. 46. BIURET REACTION  Based on the ability of the peptide bonds to react with copper ions to form purple “biuret” complex  Biuret reagent contains: • Copper sulfate – biuret formation • Sodium hydroxide • Rochelle salt (potassium sodium tartrate) – stabilizes the copper sulfate • Potassium iodide – keeps copper ions in cupric state • The purple complex is measure at 540 – 560 nm  Total proteins – measure by allowing to react with the biuret reagent  “Salting-out process” – precipitation of globulins with 22- 26% sodium sulfate to get the albumin content
  47. 47. FOLIN-CIOCALTEAU METHOD based on the ability of phosphotungstomolybdic acid (phenol reagent or Folin-Ciocalteau‟s reagent) to oxidize the phenolic structures of the amino acids tyrosine, phenylalanine, tryptophan and histidine.
  48. 48. LOWRY PROTEIN ASSAY • Uses biuret method followed by the Folin-Ciocalteau‟s method • Color obtained is enhance and provide a more sensitive method • Consistently obtained accurate protein determination
  49. 49. COOMASIE BRILLIANT BLUE DYE• Free from interferences• It can detect proteins as low as 1 ug in concentration
  50. 50. NINHYDRIN REACTION • Used to detect peptides and amino acids after paper chromatography • Violet color formation upon reaction to ninhydrin (tri-keto-hydrindine hydrate and amines • Results are comparable to Coomasie dye method
  51. 51. REFRACTOMETRY• Measuring refractive index of serum• Accurate levels are obtained at protein concentrations greater than 2.5 g/dL
  52. 52. SPECIFIC GRAVITY• Copper sulfate standards with known specific gravity• Measurement of hemoglobin
  53. 53. TURBIDIMETRY• Measures total proteins and globulins in urine and CSF• Proteins are precipitated by sulfosalicylic acids, trichloroacetic acid, acetic acid-potassium ferricyanide solution
  54. 54. UV ABSORPTION METHOD• Proteins absorb UV light at 280 nm• Mostly due to the presence of tryptophan, phenylalanine and tyrosine• Quantifies protein in the range of 0.5 to 1.5 mg/dL
  55. 55. ELECTROPHORESIS Sodium dodecyl sulfate polyacrilamide gel electrophoresis • Separates protein according to molecular weight and isoelectric focusing which separates proteins on the basis of isoelectric poins • After separation, proteins are quantified by densitometry (preferred method and by elution and spectrophotometry Qualitative characterization of proteins may be stained with appropriate dyes: 1. Coomasie brilliant blue 2. Ponceau S 3. Amido black 4. Silver staining - most sensitive and measures proteins in nanogram concentration
  56. 56. PRECIPITATION Precipitants:• Sodium sulfate• Ammonium sulfate• Methanol
  57. 57. PLASMA PROTEIN PRE-ALBUMIN• Migrates faster than albumin toward the anode• Has the ability to bind with thyroxine (TBPA) and complex with vitamin A• Very rich with tryptophan• Marker of nutritional status• Crosses the plancenta
  58. 58. ALBUMIN • Single most abundant protein in normal plasma • About 2/3 of the total plasma proteins • Regulates intravascular oncotic pressure • Loss of albumin is seen in ascites, protein losing nephropathy and protein losing enteropathy • Seen in peripheral edema  Functions of albumin: 1. Regulation of oncotic pressure 2. Amino acid reservoir 3. Transport of small molecules
  59. 59. ALBUMIN • Bounded by thyroxine, bilirubin, penicillin, cortisol, estrogen and free fatty acids • Calcium and magnesium • Analbuminemia – absence of albumin in the blood • Bisalbuminemia – two separated albumin bands after electrophoresis
  60. 60. ALPHA1-ANTITRYPSIN • Major component of the alpha1-globulin fraction • Acts as protease inhibitor • Combines with and inactivates trypsin and elastase • In lungs, elastase is released by the neutrophils during inflammatory conditions to combat the invading microorganisms • Deficiency will result to emphysema • Also an intrinsic factor in homeostatic mechanism modulating endogenous proteolysis with the body
  61. 61. ALPHA2-MACROGLOBULIN • one of the largest non-immunoglobulin proteins in the plasma • Rises tenfold or more in nephrotic syndrome when other low molecular weight proteins are lost • In nephrotic syndrome, the levels of alpha2-macroglobulin may be equal to or greater than that of albumin (2-3 g/dL)
  62. 62. HAPTOGLOBIN • Haptoglobin migrates in the alpha2-region • Combines with hemoglobin released by lysis of red blood cells in order to preserve body iron and protein stores • Hemoglobin-haptoglobin complex is then taken by the RES where the hemoglobin fraction is broken into iron and bilirubin Hemoglobinuria vs myoglobinuria: • Peroxidase acitivity using dipstick or chemstrips – urine specimen • Increased or normal free haptoglobinuria will indicate myoglobinuria or rhabdomyolysis
  63. 63. BETA-LIPOPROTEIN Low-density lipoprotein which has a characteristic sharp leading edge and a feathery trailing edge
  64. 64. TRANSFERRIN • Also known as siderophilin • Major beta-globulin protein • Normally at 200 to 400 mg/dL • Transport ferric ions from iron stores of the intracellular or mucosal ferritin to bone marrow where the red blood cells precursors and other cells have transferrin receptor on their surfaces • Measured in terms of iron-binding capacity – seen elevated in anemia
  65. 65. COMPLEMENT• Travels with beta-globulins during the electrophoresis• C3 is decreased in autoimmune diseases
  66. 66. FIBRINOGEN • Most abundant of the coagulation factors • Elevation seen in pregnancies and the use of birth control pills • Decreased during the activation of coagulation factors • Migrates between beta and gamma fractions • Measured by Parfentjev method which uses ammonium sulfate and sodium chloride
  67. 67. MINOR PROTEINS • Ceruloplasmin – Wilson‟s disease (hepatolenticular degeneration) • Gc-globulin – binds with vit D • Hemopexin – prevent excretion of heme and iron • Alpha1-acid glycoprotein – increased during pregnancy • C-reactive protein – seen in tissue necrosis; most sensitive acute phase reactant located at chromosome 1