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Ion Exchange Chromatography

  1. In ion exchange chromatography, proteins interact with the stationary phase by charge-charge interactions. Proteins with a net positive charge at a given pH adhere to beads with negatively charged functional groups such as carboxylates or sulfates (cation exchangers). Similarly, proteins with a net negative charge adhere to beads with positively charged functional groups, typically tertiary or quaternary amines (anion exchangers). Proteins, which are polyanions, compete against monovalent ions for binding to the support—thus the term "ion exchange.
  2. " For example, proteins bind to diethylaminoethyl (DEAE) cellulose by replacing the counter-ions (generally Cl or CH3COO) that neutralize the protonated amine.
  3. Bound proteins are selectively displaced by gradually raising the concentration of monovalent ions in the mobile phase.
  4. Proteins elute in inverse order of the strength of their interactions with the stationary phase.

Heme Metabolism

Biosynthesis of Heme
The complex heme molecule is synthesized from two simple precursor, glycine and succinyl coenzyme
  1. Disorder of Heme Biosynthesis
    1. Porphyrias are genetic defect in enzymes of heme biosynthesis.
      1. Porphyrias result in excessive accumulation of intermediates in heme biosynthesis that cannot be further metabolized by the body.
      2. Symptoms of porphyrias include abdominal pain, dermatologic problems, neurologic abnormalities, and photosensitivity.
    2. Lead poisoning affects heme biosynthesis. Two enzymes, ALA dehydratase and ferrochelatase, are inactivated by lead.
Summary of Major Findings in the Porphyrias1
Enzyme Involved2
Type, Class, and OMIM
Major Signs and
Results of Laboratory
1. ALA synthase (erythroid form) X-linked sideroblastic
anemia3 (erythropoietic)
(OMIM 301300)
Anemia Red cell counts and
hemoglobin decreased
2. ALA dehydratase ALA dehydratase
deficiency (hepatic)
(OMIM 125270)
Abdominal pain, 
Urinary ALA and
coproporphyrin III
3.  Uroporphyrinogen I
4.  synthase4
Acute intermittent
porphyria (hepatic) (OMIM
Abdominal pain, 
Urinary ALA and PBG
4. Uroporphyrinogen III synthase Congenital erythropoietic (erythropoietic) (OMIM 263700) Photosensitivity Urinary, fecal, and red cell
uroporphyrin I increased
5. Uroporphyrinogen decarboxylase Porphyria cutanea
tarda (hepatic)
(OMIM 176100)
Photosensitivity Urinary uroporphyrin I
6. Coproporphyrinogen oxidase Hereditary
(hepatic) (OMIM
abdominal pain, 
neuropsychiatric symptoms
Urinary ALA, PBG, and
coproporphyrin III and
fecal coproporphyrin
III increased
7. Protoporphyrinogen oxidase Variegate porphyria
(hepatic) (OMIM
abdominal pain, 
Urinary ALA, PBG, and
coproporphyrin III and
fecal protoporphyrin
IX increased
8. Ferrochelatase Protoporphyria
(OMIM 177000)
Photosensitivity Fecal and red cell protoporphyrin IX increased

There are total 6 porphyrias out of which 5 are dominant and 1 recessive. the only recessive is congenital erythroporphyria. out of 5 dominant 4 affects while only 1 affects bone marrow and that is hereditary protoporphyria.
  1. Heme Degradation
    1. Conversion of heme to biliverdin is catalyzed by heme oxygenase.
    2. Biliverdin is reduced by (NADPH) to bilirubin, which is transported to the liver bound to serum albumin.
    3. In the liver, bilirubin is conjugated with glucuronic acid by enzyme Uridine diphosphate (UDP)-glucuronyl
    4. transferase. The bilirubin diglucuronide that is formed is soluble and is secreted into the bile. 
    5. Bilirubin diglucuronide is hydrolyzed to free bilirubin in the bowel, their it is converted urobilinogens and stercobilinogens, which are excreted in the urine and faces.

Synthesis of nutritionally non essential amino acids
  • Nutritionally non essential amino acids, as the name suggests, are not essential in diet. This is because they are synthesized in body.
  • Glutamate is synthesized from a-ketoglutarate by enzyme glutamate dehydrogenase.
  • Glutamine is synthesized from glutamate by enzyme glutamine synthase.
  • Alanine is synthesized from pyruvate by transamination.
  • Aspartate is synthesized from oxaloacetate by transamination.
  • Asparagine is synthesized from aspartate by enzyme asparagine synthase.
  • Serine is synthesized from 3-phosphoglycerate (a glycolytic intermediate). It can also be synthesized from glycine as conversion of serine to glycine is reversible.
  • Proline is synthesized from glutamate
  • Cysteine is synthesized from methionine and serine
  • Tyrosine is synthesized from phenylalanine.
  • Selenocysteine is considered as 21 st standard amino acid. It is present at the active site of some enzymes that catalyze redox reactions, e.g. thioredoxin reductase, glutathione peroxidase, and the deiodinase (converts thyroxin to triiothyronine). Biosynthesis of selenocysteine requires cysteine, serine, ATP and a specific t-RNA
Extra Edge
  1. Proteins that assist folding include protein disulfide isomerase, proline-cis, trans isomerase, and the chaperones that participate in the folding of over half of mammalian proteins. Chaperones shield newly synthesized polypeptides from solvent and provide an environment for elements of secondary structure to emerge and coalesce into molten globules.
  2. Techniques for study of higher orders of protein structure include x-ray crystallography, NMR spectroscopy, analytical ultracentrifugation, gel filtration, and gel electrophoresis
  3. Prions-proteins particles that lack nucleic acid-cause fatal transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, scrapie, and bovine spongiform encephalopathy. Prions diseases involve an altered secondary-tertiary structure of a naturally occurring protein, PrPc. When PrPc interacts with its pathologic isoform PrPSc, its conformation is transformed from a predominantly ?-helical structure to the ?-sheet structure characteristic of PrPSc
  4. Myoglobin is monomeric; hemoglobin is a tetramer of two subunit types (?2ß2 in HbA). Despite having different primary structures, Myoglobin and the subunits of hemoglobin have nearly identical secondary and tertiary structures.
  5. Restriction endonucleases facilitate diagnosis of genetic diseases by revealing restriction fragment length polymorphisms.
  6. Recombinant fusion proteins such as His-tagged or GST fusion enzyme are readily purified by affinity chromatography.
  7. The substrates for most enzyme are usually present at a concentration close to Km. This facilitates passive       
  8. control of the rates of product formation in response to change in levels of metabolic intermediates.
  9. Phosphorylation by protein kinases of specific seryl, threonyl, or tyrosyl residues-and subsequent dephosphorylation by protein phosphatases-regulates the activity of many human enzymes. The protein kinases and phosphatases that participate in regulatory cascades that respond to hormonal or second messenger signals constitute regulatory networks that can process and integrate complex environmental information to produce an appropriate and comprehensive cellular response. This regulation is known as covalent regulation or covalent modification.
  10. All vertebrates can form certain amino acids from amphi­bolic intermediates or from other dietary amino acids. The intermediates and the amino acids to which they give rise are a-ketoglutarate (Glu, Gin, Pro, Hyp), oxalo­acetate (Asp, Asn), and 3-phosphoglycerate (Ser, Gly).
  11. Cysteine, tyrosine, and hydroxylysine are formed from nutritionally essential amino acids. Serine pro­vides the carbon skeleton, and methionine the sul­fur for cysteine biosynthesis. Phenylalanine hydroxy­lase converts phenylalanine to tyrosine.
  12. Neither dietary hydroxyproline nor hydroxylysine is incorporated into proteins because no codon or tRNA dictates their insertion into peptides.
  13. Peptidyl hydroxyproline and hydroxylysine are formed by hydroxylation of peptidyl proline or lysine in reac­tions catalyzed by mixed-function oxidases that re­quire vitamin C as cofactor. The nutritional disease scurvy reflects impaired hydroxylation due to a defi­ciency of vitamin C.
  14. Selenocysteine, an essential active site residue in sev­eral mammalian enzymes, arises by co-translational insertion from a previously modified tRNA. Thus selenocysteine is referred as 21st amino acid.
  15. Biliverdin is an early product of catabolism and on reduction yields bilirubin. The latter is transported by albumin from peripheral tissues to the liver, where it is taken up by hepatocytes. The iron heme and the amino acids of globin are conserved and reutilized
  16. The major components of the extra cellular matrix are the structural proteins collagen, elastin, and fibrillin; a num­ber of specialized proteins (eg, fibronectin and lami­nin); and various proteoglycan.
  17. Collagen is the most abundant protein in the animal kingdom; approximately 25 types have been isolated. All collagens contain greater or lesser stretches of tri­ple helix and the repeating structure (Gly-X-Y).
  18. The biosynthesis of collagen is complex, featuring many posttranslational events, including hydroxyla­tion
  19. Diseases associated with impaired synthesis of collagen include scurvy, osteogenesis imperfecta, Ehlers­ Danlos syndrome (many types), and Menkes disease.
  20. Elastin confers extensibility and elastic recoil on tis­sues. Elastin lacks hydroxylysine, Gly-X-Y sequences, triple helical structure, and sugars but contains des­mosine and isodesmosine cross-links not found in collagen.
  21. Fibrillin is located in microfibrils, mutations in the gene for fibrillin cause Marfan syndrome
  22. Many cases of malignant hyperthermia in humans are due to mutations in the gene encoding the Ca2+ release channel.
  23. Smooth muscle, unlike skeletal and cardiac muscle, does not contain the troponin system; instead, phosphorylation of myosin light chains initiates contraction
  24. Nitric oxide is a regulator of vascular smooth muscle; blockage of its formation from arginine causes an acute elevation of blood pressure, indicating that regulation of blood pressure is one of its many functions.
  25. Two major types of muscle fibers are found in humans: white (anaerobic) and red (aerobic). The former are particularly used in sprints and the latter in prolonged aerobic exercise. During a sprint, muscle uses creatine phosphate and glycolysis as energy sources; in the marathon, oxidation of fatty acids is of major importance during the latter phases
  26. Ceruloplasmin contains substantial amounts of copper, but albumin appears to be more important with regard to its transport. Both Wilson disease and Menkes disease, which reflect abnormalities of copper metabolism, have been found to be due to mutations in genes encoding copper-binding P-type ATPases.
  27. α1-Macroglobulin is a major plasma protein that neutralizes many proteases and targets certain cytokines to specific organs
  28. Xenobiotics are chemical compounds foreign to the body, such as drugs, food additives, and environmental pollutants; more than 200,000 have been identified
  29. Xenobiotics are metabolized in two phases. Phase I reaction is hydroxylation catalyzed by a variety of mono oxygenases, known as the cytochrome P450. In phase 2, the hydroxylated species are conjugated with a variety of hydrophilic compounds such as glucuronic acid, sulfate, or glutathione. The combined operation of these two phases renders lipophilic compounds into water-soluble compounds that can be eliminated from the body.
  30. Cytochrome P450 catalyze reactions that introduce one atom of oxygen derived from molecular oxygen into the substrate, yielding a hydroxylated product. NADPH and NAPDH- cytochrome P450 reductase are involved in the complex reaction mechanism
  31. All cytochrome P450 are hemoproteins and generally have a wide substrate specificity, acting on many exogenous and endogenous substrates. They represent the most versatile biocatalyst known.
  32. Xenobiotics can produce a variety of biologic effects, including pharmacologic responses, toxicity, immunologic reactions, and cancer.
  33. Human subjects degrade 1-2% of their body protein daily at rates that vary widely between proteins and with physiologic state. Key regulatory enzymes often have short half-lives.
  34. Proteins are degraded by both ATP-dependent and ATP-independent pathways. Ubiquitin targets many intracellular proteins for degradation. Liver cell sur­face receptors bind and internalize circulating asialoglycoprotein destined for lysosomal degradation.
  35. Ammonia is highly toxic. Fish excrete NH3 directly; birds convert NH3 to uric acid. Higher vertebrates convert NH3 to urea.
  36. Transamination channels ?-amino acid nitrogen into glutamate. L-Glutamate dehydrogenase (GDH) oc­cupies a central position in nitrogen metabolism. GDH is a enzyme of aerobic deamination.
  37. Glutamine synthase converts NH3 to nontoxic glutamine. Glutaminase releases NH3 for use in urea synthesis.
  38. NH3, CO2, and the amide nitrogen of aspartate pro­vide the atoms of urea.
  39. Hepatic urea synthesis takes place in part in the mi­tochondrial matrix and in part in the cytosol. Inborn errors of metabolism are associated with each reac­tion of the urea cycle.
  40. Changes in enzyme levels and allosteric regulation of carbamoyl phosphate synthase by N-acetylglutamate regulate urea biosynthesis.
  41. Tandem mass spectrometry is the technique of choice for screening neonates for over two dozen metabolic diseases.
  42. Excess amino acids are catabolized to amphibolic in­termediates used as sources of energy or for carbohy­drate and lipid biosynthesis.
  43. Transamination is the most common initial reac­tion of amino acid catabolism. Subsequent reac­tions remove any additional nitrogen and restruc­ture the hydrocarbon skeleton for conversion to oxaloacetate, a-ketoglutarate, pyruvate, and acetyl­ CoA.
  44. Metabolic diseases associated with glycine catabo­lism include glycinuria and primary hyperoxaluria.
  45. Two distinct pathways convert cysteine to pyruvate. Metabolic disorders of cysteine catabolism include cystine-Lysinuria, cystine storage disease, and the homocystinuria.
  46. Threonine catabolism merges with that of glycine after threonine aldolase cleaves threonine to glycine and acetaldehyde.
  47. Following transamination, the carbon skeleton of tyrosine is degraded to fumarate and acetoacetate. Metabolic diseases of tyrosine catabolism include tyrosinosis, Richner-Hanhart syndrome, neonatal tyrosinemia, and alkaptonuria.
  48. Metabolic disorders of phenylalanine catabolism in­clude phenylketonuria (PKU) and several hyperphenylalaninemia.
  49. Lysine does not undergo transamina­tion. Metabolic diseases of lysine catabolism include periodic and persistent forms of hyperlysinemia ammonemia.
  50. The catabolism of leucine, valine, and isoleucine presents many analogies to fatty acid catabolism. Metabolic disorders of branched-chain amino acid catabolism include hypervalinemia, maple syrup urine disease, intermittent branched-chain ketonuria.
  51. Glycine participates in the biosynthesis of heme, pu­rines, and creatine and is conjugated to bile acids and to the urinary metabolites of many drugs, specially benzoic acid to hippuric acid.
  52. S-Adenosylmethionine, the methyl group donor for many biosynthetic processes, also participates directly in/spermine and spermidine biosynthesis.
  53. Glutamate and ornithine form the neurotransmitter???aminobutyrate (GABA)

Important Point to Remember

Amino Acid Selenocysteine (LQ 2012)
  1. The amino acid Selenocysteine contains selenium instead of sulfur in its structure and is present at the active site of several eukaryotic & prokaryotic enzymes. (ex; thioredoxin reductase, glutathione peroxidase, deiodinase etc.) Unlike hydroxyproline & hydroxylysine which are formed by post-translational modification (hydroxylation) of peptidyl amino acids, selenocysteine arises by a process that precedes its incorporation into peptides.
  2. This process parallels the process for incorporation of the common amino acids.
  3. The cotranslational incorporation of selenocysteine involves a unique tRNA, tRNA sec, whose UCA anticodon normally signals “stop”. The ability of protein synthetic machinery to distinguish selenocysteine specific UGA codon from one that signals “stop” involves the selenocysteine insertion element, a stem-loop structure present in 3’ intranslated region of mRNA.
  4. Biosynthesis of charged selenocysteine –tRNAsec involves initially its aminoacylation by L-serine, a reaction catalysed by ligase that charges tRNAsec . Subsequent replacement of serine oxygen by selenium involves seleno-phosphate formed in ATP requiring reaction catalysed by selenophosphate synthase.

Important Point to Remember

  1. All collagen have a triple helix structure
  2. A striking characteristic of collagen is the occurrence of glycine residue at every third position of the triple helical portion of a-chain (as glycine is the simples aa, it can accommodate in limited space)
  3. Proline and hydroxy proline provide rigidity to collagen molecule
  4. Laminin is the major protein present in GBM of kidney
  5. Fibronectin is an glycoprotein involved in cell adhesion and migration
  6. Type I collagen is present in most connective tissues and bone
  7. Chaperones are proteins that prevent faulty folding and unproductive interactions of other proteins
  8. Abnormal folding of proteins (-amyloid), unassisted by chaperones, leads to Alzheimer's disease.
  9. Prion diseases (e.g. CJD) which are neurodegenerative diseases result from altered protein combination.
  10. Heme in Hb is present in "Hydrophobic pockets".
  11. Switch over from fetal to adult hemoglobin occurs at about 38 weeks of gestation.
  12. Level of HbA2 increased (>3.4%) in -thall. trait & megaloblastic anemia & ed in iron deficiency anemia and - thalassemia,
  13. Hemoglobin makes its appearance in intermediate normoblast stage & nucleus disappears in late normoblast stage during erythropoiesis
  14. HbF has low iron content & large size RBC
  15. HbF is resistant to alkali denaturation
  16. Iron remains in the "Ferrous" (Fe++) State in both de-oxygenated Hb & Oxy-Hb. But in Met-Hb, Ferritin, transferrin iron is in "Ferric" (Fe+++) form. Met-Hb (methemoglobin) is ineffective in oxygen transport.
  17. Oxygenation of one heme molecule enhances and accelerates oxygenation of other heme molecules. Similarly the release of one O2 molecule promotes the release of other this is k/as positive cooperativity and is responsible for sigmoid shaped of ODC of Hb.
  18. Normal Hb level in newborn:- 16 - 18 gm% (newborn is called anemic if Hb level <13 gm% in a sick newborn and <8 gm% in a clinically stable newborn).
  19. At O2 tension of 100 mmHg or more, Hb is virtually 100% saturated.
  20. In kidney upto arginine (Arginase is absent in kidney). Kidney synthesize NH3 from Glutamine
  21. Urea is the major end product of nitrogen catabolism in humans. A human subject who consumes 300g of carbohydrate, 100g fat & 100g protein excretes about 16.5 g of nitrogen each day, 95% in urine & 5% in the feces there fore the rate of urea cycle must fluctuate to accommodate the amount of ammonia to be removed.
  22. CPS-I Initiates Urea biosynthesis. CPS-I is distinct from CPS-II which is cytosolic. CPS-I is a hepatic, mitochondrial enzyme. CPS-I is the rate-limiting /pacemaker enzyme of the urea cycle & has a mandatory requirement for the allosteric activator N-acetylglutamate. This compound is synthesized by action of N-acetylglutamate synthetase, which is activated by Arginine. Acetyl CoA, Glutamate & Arginine are needed to supply intermediates/energy for the urea cycle
  23. Enzymes catalyze the reaction by ing the free energy of activation.
  24. Do not alter the equilibrium constant of reaction that they catalyze
  25. The velocity increases as the substrate concentration is increased up to a point where the enzyme is said to be saturated with substrate i.e. all the enzyme is combined with the substrate giving hyperbolic curve.
  26. Certain proteins are manufactured and secreted in the form of inactive precursor proteins called proproteins. When the proteins are enzymes the proproteins are termed proenzymes or zymogens. Conversion of a proprotein to a mature protein involves selective proteolysis. Examples includes trypsinogen to trypsin and pepsinogen to pepsin.
  27. Isoenzymes are physically distinct forms of the same catalytic activity. In clinical medicine isoenzyme has a more restricted meaning the physically distinct and separable forms of a given enzyme present in different cell types, or subcellular compartment of a human subject. Their separation by electrophoresis can help to locate a disease to the organ, cell or subcellular organelle. Since they are physically distinct they have separate electrophoretic mobilities.
  28. Many enzymes that catalyze group transfer and other reactions require in addition to their substrate a second organic molecule known as a coenzyme without which they are inactive. To distinguish them from metal ion activators & from enzymes themselves coenzymes are defined as heat stable, low molecular weight organic compounds required for the activity of enzymes. Most co-enzymes are linked to enzymes by non covalent forces.     Those which form covalent bonds to enzymes may also be termed prosthetic groups. Enzymes that require coenzymes include IUB classes 1, 2, 5, 6. Enzymes of class 3 & 4 do not require coenzymes.
Selected List Of Applications Of Enzymes
Enzyme Application
Therapeutic Applications
1 – Antitrypsin
To remove blood clots
In cancer therapy
Anti – inflammatory
Emphysema (breathing difficulty due to lungs distension)

Biochemical test For Protein
Colour reaction Quantitive assay Detection of NH2 For sequence of peptide chain
Xanthoproteic (for aromatic AA)
Millon’s (Tyrosine)
Sakaguchi (Arginine)
Sullivan (cysteine & cystine)
Lead-acetate (cysteine, cystine & methionine)
Hopkins kole aldehyde reaction (for tryptophan)
Ninhydrin reaction
Can detect no. of
amino acid
better than Ninhydrin
Edman’s (for N terminal of amino acid)

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