1. Cell Organells ...1
2. Cell Membranes: Structure and Function ...3
3. Amino Acids: Structure and Properties ...8
4. Proteins: Structure and Function ...17
5. Enzymology-I ...27
6. Enzymology-II: Iso-Enzymes and Clinical Enzymology ...40
7. Methods of Separation & Purification of Biological Compounds, Methods of Study of Metabolism...47
8. Carbohydrates-I:Chemistry, Digestion and Absorption ... 52
9. Carbohydrates-II: Major Metabolic Pathways of Glucose, Glycolysis, Gluconeogenesis, Glycogen Metabolism ...61
10. Carbohydrates-III: Regulation of Blood Sugar, Insulin and Diabets Mellitus ...70
11. Carbohydrates-IV: Other Metabolic Pathways (HMP Shunt Pathway, Fructose, Galactose, Glucuronic Acid, Alcohol)...81
Contents
Contents
Contents
Contents
Contents
12. Lipids-I: Chemistry, Digestion and
Absorption of Lipids ...88
13. Lipids-II: Metabolism of Fatty acids, Fatty acid oxidation, Fatty acid synthesis, Lipolysis, Ketone bodies ...95
14. Lipids-III: Cholesterol, Lipoproteins and Cardiovascular Diseases ...103
15. Lipids-IV: MCFA, PUFA and
Prostaglandins ...112
16. Lipids-V: Compound Lipids ...118
17. Amino Acid Metabolism-I: General: Digestion, Absorption,
Transamination, Urea...122
18. Amino Acid Metabolism-II: Simple, Hydroxy and Sulfur Containing Amino Acids Glycine, Serine, Alanine, Threonine, Methionine, Cysteine...128
19. Amino Acid Metabolism-III: Acidic, Basic and Branched Chain Amino Acids, Glutamic acid, Glutamine, Aspartic acid, Asparagine, Lysine, Nitric Oxide, Valine, Leucine, Isoleucine....132
20. Amino Acid Metabolism-IV: Aromatic Amino Acids: Phenyl alanine, Tyrosine, Tryptophan, Histidine ...137
21. Amino Acid Metabolism-V: Inter-relations of Amino Acid Metabolisms, One Carbon
Contents 3
22. Citric Acid Cycle ...144
23. Electron Transport Chain ...147
24. Free Radicals and Anti-oxidants ...151
25. Plasma Proteins ...153
26. Immunochemistry ...156
27. Specialised Proteins: Collagen, Myosin ...158
28. Heme Synthesis and Breakdown ...160
29. Haemoglobins ...165
30. Vitamin-I: Fat Soluble Vitamins: A, D, E and K ...169
31. Vitamin-II: Water soluble vitamins ...174
32. Mineral Metabolism ...189
33. Energy Metabolism and Nutrition ...195
34. Detoxification and Biotransformation of Xenobiotics...199
35. Biochemical Aspects of Environmental Pollution ...201
36. Acid Base Balance and pH ...203
37. Electrolyte and Water Balance ...207
38. Molecular Biology-I: Nucleotides, Chemistry and Metabolism ...209
39. Molecular Biology-II: DNA structure and
Replication ...216
40. Molecular Biology-III: Transcription and Translation ...220
41. Molecular Biology-IV: Molecular Genetics and Control of Gene Expression ...227
42. Molecular Biology-V: Recombinant DNA Technology and Gene Therapy ...229
43. Biochemistry of AIDS ...233
44. Biochemistry of Cancer...235
45. Applications of Radio-isotopes in Medicine ...238
46. Body Fluids ...240
47. Hormones-I: Mechanism of Action of Hormones ...242
48. Hormones-II: Pituitary Hormones ...244
49. Hormones-III: Steroid Hormones ... 246
50. Hormones-IV: Thyroid Hormones ...248
51. Clinical Biochemistry-I: ...250
52. Clinical Biochemistry-II: Liver and Gastric Function Tests...252
53. Clinical Biochemistry-III: Kidney Function Tests...256
Cell Organelles 1
Cell Organelles
Q. What is the function of Golgi complex?
A. Maturation and processing of nascent proteins, glycosylation of proteins, secretion newly
synthesised proteins (Page 3).
Q. What is the function of endoplasmic reticulum? (Page 3)
A. Biosynthesis of proteins, drug metabolism, desaturation of fatty acids.
Q. What is the marker enzyme for endoplasmic reticulum?
A. Glucose-6-phosphatase (Page 3, Table 1.3).
Q. Where does protein synthesis take place?
A. On the walls of endoplasmic reticulum and also
in cytosol (Page 3).
Q. What are cathepsins?
A. They are intracellular proteolytic enzymes (Page 4).
Q. What is the function of lysosomes?
A. They are bags of hydrolytic enzymes that bring about degradation of macromolecules (Page 4).
Q. What is lysozyme?
A. It is an enzyme present in external secretions (Page 35).
Q. What are peroxisomes?
A. They contain peroxidase and catalase, necessary for destroying the unwanted free radicals. (Page 4)
Q. What are the important metabolic events taking place in cytoplasm?
A. Glycolysis (Embden-Meyerhof pathway), HMP shunt pathway, glycogen metabolism, Fatty acid synthesis, Synthesis of nucleotides, Degradation of amino acids (See Page 5, Table 1.4).
Q. What is the function of mitochondria?
A. Generation of ATP (See page 5).
Q. What are the important metabolic events taking place in mitochondria?
A. TCA cycle, electron transport chain, beta oxida-tion of fatty acids and urea cycle (Page 5).
Cell Membranes; Structure and Function 3
Q. What are ecto-enzymes?
A. They are enzymes seen on the outer part of cell membrane (Page 6).
Q. Give examples of ecto-enzymes.
A. Alkaline phosphatase, 5’nucleotidase (Page 6).
Q. How do you describe the structure of cell mem-brane?
A. Fluid mosaic model (Page 6).
Q. What are the characteristics of fluid mosaic model?.
A. Membrane is composed of lipid bilayer. Phospho-lipids are arranged in bilayers with a hydropho-bic core (Page 6).
Q. What do you mean by fluidity of the membrane?
A. The lipid bilayer shows free lateral movement of its components; but flip-flop movement is re-stricted (Page 6).
Cell Membranes:
Q. What are the components of membrane that al-ter the fluidity?
A. Cholesterol and unsaturated fatty acids (Page 6).
Q. What are the different types of transport mecha-nisms?
A. Passive and active. Passive type is subclassified as simple diffusion and facilitated diffusion (Page 8).
Q. What are the salient features of facilitated diffu-sion?
A. It is carrier mediated. It does not require energy directly (Page 8).
Q. Can you give an example of facilitated transport?
A. Glucose transporters (Page 8).
Q. What are ion channels?
A. They are special devices for quick transport of electrolytes (Page 9).
Q. Give some examples of ion channels.
A. Ion channels specific for calcium, potassium and chloride (Page 9).
Q. What are ionophores?
A. They are transport antibiotics which increase the permeability of membrane to ions, e.g. valinomy-cin, gramicidin (Page 10).
Q. What are the salient features of active transport?
A. It requires transporters. It requires energy. Trans-port is generally unidirectional (Page 10).
Cell Membranes; Structure and Function 5 Q. Give examples of active transport systems.
A. Sodium pump, calcium pump (Page 10).
Q. What is the importance of sodium pump?
A. Cell has low intracellular sodium; but concentra-tion of potassium inside the cell is high; this is maintained by sodium pump. About 40% of the total energy expenditure in a cell is used for this active transport system (Page 10).
Q. How does sodium pump work?
A. It is called sodium-potassium activated ATPase. Hydrolysis of one molecule of ATP can result in expulsion of 3 sodium ions and influx of 2 potas-sium ions (Page 10).
Q. What is its clinical significance?
A. Digoxin increases the contractility of the cardiac muscle, by inhibiting the sodium pump (Page 10).
Q. What is a uniport?
A. It carries single solute across the membrane (Page 10).
Q. Give examples of uniport.
A. Glucose transporter (GluT2) operating in most of the cells is an example. Calcium pump is another example (Page 10).
Q. What is co-transport?
A. If transfer of one molecule depends on simulta-neous or sequential transfer of another molecule, it is called co-transport system (Page 10).
Q. How are co-transport systems classified?
A. The cotransport system may be symport or antiport (Page 10).
Q. What is symport?
A. In symport, the transporter carries two solutes in the same direction across the membrane (Page 10).
Q. Give examples of symport. (Page 10)
A. Sodium dependent glucose transporter (SgluT) (Fig.8.31). Phlorhizin, an inhibitor of sodium-de-pendent cotransport of glucose, especially in the proximal convoluted tubules of kidney, produces renal damage and results in renal diabetes. Amino acid transport is another example for symport.
Q. What is antiport system?. (Page 10)
A. The antiport system carries two solutes or ions in opposite direction.
Q. Give examples of antiport. (Page 10)
A. Sodium pump (Fig.2.7) or chloride-bicarbonate exchange in RBC (Fig.29.4).
Q. What is endocytosis?. (Page 11)
A. It is the mechanism by which cells internalise ex-tracellular macromolecules.
Q. What is pinocytosis?. (Page 11)
A. It is receptor mediated. Low Density Lipoprotein (LDL) binds to the LDL receptor and the complex is later internalised. These vesicles are coated with Clathrin. .
Cell Membranes; Structure and Function 7 Q. What is phagocytosis. (Page 11)
A. It is the engulfment and internalisation of large particles such as bacteria by macrophages and granulocytes.
Q. What is respiratory burst. (Page 11)
A. During phagocytosis, there is an increase in oxy-gen consumption with formation of the superox-ide ion.
Amino Acids:
Structure and
Properties
Q. How do you classify amino acids? (Page 12)
A. Based on the structure, amino acids are classified into: Simple amino acids, Branched chain amino acids, Hydroxy amino acids, Sulfur containing amino acids, Amino acids with amide group, Acidic amino acids, Basic amino acids, Aromatic amino acids, Heterocyclic amino acids, Imino acid and Derived amino acids.
Q. What are branched chain amino acids?(Page 12)
A. Valine, leucine and isoleucine.
Q. What are hydroxy amino acids? (Page 12)
A. Serine and threonine.
Q. Name the Sulfur containing amino acids.
(Page 12)
A. Cysteine and methionine.
Q. Name the acidic amino acids. (Page 12)
Amino Acids: Structure and Properties 9 Q. What are the basic amino acids? (Page 12)
A. Lysine and arginine.
Q. Which amino acid has a net positive charge at
physiological pH? (Page 12)
A. Arginine and lysine.
Q. Amino acid containing a thio-ether bond is. (Page 12)
A. Methionine.
Q. Give examples of amino acids with
hydropho-bic side chains. (Page 12)
A. Valine, leucine, isoleucine.
Q. Give the names of aromatic amino acids.
(Page 12)
A. Phenylalanine and tyrosine.
Q. What are heterocyclic amino acids? (Page 12)
A. Tryptophan and histidine.
Q. Give an example of an imino acid. (Page 12)
A. Proline.
Q. Give examples of derived amino acids.(Page 12)
A. Hydroxy proline, hydroxy lysine, ornithine, cit-rulline, homocysteine.
Q. Arginine contains which special group?
(Page 12)
A. Guanidinium group (-NH-CNH-NH2).
Q. Benzene group is present in which amino acid? (Page 12)
Q. Phenol group is present in which amino acid? (Page 12)
A. Tyrosine.
Q. Tryptophan contains what special group? (Page 12)
A. Indole group.
Q. Which special group is present in Histidine? (Page 12)
A. Imidazole group.
Q. Name some hydrophobic amino acids.(Page 12)
A. Valine, leucine and isoleucine.
Q. Pyrrolidine group is present in which amino
acid? (Page 12)
A. Proline.
Q. Hydrophobic bonds are formed in protein be-tween which amino acids?
A. Valine, leucine and isoleucine residues.(Page 12)
Q. What is the basis of classification of amino acids into ketogenic and glucogenic?
(Page 12 and 13)
A. Ketogenic amino acids enter into the metabolic pathway of fats, while glucogenic amino acids enter the pathway of glucose metabolism.
Q. Name a purely ketogenic amino acid.
(Page 12)
A. Leucine.
Q. Name some glucogenic amino acids. (Page 13)
Amino Acids: Structure and Properties 11 Q. Which amino acid is synthesised after it gets
in-corporated into the protein?
A. Hydroxyproline (Page 12).
Q. What are essential amino acids? (Page 13)
A. They cannot be synthesized in the body; and so, they are to be provided in the diet.
Q. How many amino acids are essential? (Page 13)
A. Eight amino acids are essential; two are semi-es-sential and the rest 10 are non-essemi-es-sential.
Q. Are non-essential amino acids necessary for the body?
A. They are also necessary for protein synthesis, but they can be synthesized by the body and need not be essentially present in the diet (Page 14).
Q. Name any three essential amino acids. (Page 13)
A. Isoleucine, leucine, threonine.
Q. Is phenyl alanine an essential amino acid? (Page 13)
A. Yes.
Q. What about Tyrosine? (Page 13)
A. Tyrosine is non-essential, it is synthesized from phenyl alanine.
Q. Name the semi-essential amino acids. (Page 14)
A. Histidine and arginine.
Q. Why are they called semi-essential? (Page 14)
A. Because growing children require them in food. But they are not essential for the adult individual.
Q. What is iso-electric point?. (Page 14)
A. The pH at which the molecule carries no net charge is called iso-electric point.
Q. What are the characteristic features of
iso-elec-tric pH. (Page 14)
A. At iso-electric point the amino acid will carry no net charge, there is no mobility in electrical field, solubility will be minimum, the tendency for pre-cipitation will be maximum
Q. What is the speciality of Histidine? (Page 14)
A. The pK value of Histidine is 6.1, and therefore effective as a buffer at the physiological pH of 7.4. The buffering capacity of plasma proteins and hemoglobin is mainly due to histidine residue.
Q. Which is the amino acid having maximum buff-ering capacity at physiological pH? (Page 14)
A. Histidine.
Q. Which amino acid is optically inactive?
(Page 15)
A. Glycine.
Q. What are the isomers of amino acids? (Page 16)
A. D and L varieties.
Q. What are natural amino acids? (Page 16)
A. Only L amino acids are seen in large quantities in nature.
Amino Acids: Structure and Properties 13 Q. Can you name some substances where D-amino
acids are seen?
A. D-amino acids are seen in cell walls of micro-or-ganisms and as constituents of certain antibiotics such as gramicidin-S, polymyxin, actinomycin-D
and valinomycin (Page 16).
Q. What is meant by decarboxylation of an amino
acid? (Page 16)
A. The carboxyl group is removed from the amino acids to form the corresponding amine (Fig.3.8).
Q. Give examples of decarboxylation reactions. (Page 16)
A. Histidine to histamine; tyrosine to tyramine; tryp-tophan to tryptamine.
Q. What is produced when Glutamic acid is
decar-boxylated? (Page 16)
A. Gamma amino butyric acid or GABA.
Q. What is glutamine? (Page 16)
A. That is the amide of glutamic acid.
Q. What is an amide? (Page 16)
A. The extra carboxyl group (other thanÿalpha car-boxyl) can combine with ammonia to form the cor-responding amide.
Q. How asparagines is produced? (Page 16)
A. Aspartic acid + ammonia will form asparagine.
Q. What is transamination? (Page 16)
A. The alpha amino group of amino acid can be trans-ferred to alpha keto acid to form the correspond-ing new amino acid and alpha keto acid (Fig.3.10).
Q. Give an example of transamination reaction. (Page 16)
A. Glutamic acid + pyruvic acid alpha keto glutarate + alanine.
Q. What is the product of transamination reaction of pyruvate with glutamate?
A. Alanine and alpha keto glutarate (Page 16).
Q. What is the biological significance of transami-nation reaction?
A. These are important for the interconversion of amino acids. Non-essential amino acids are
syn-thesized by this process (Page 16).
Q. What is the clinical significance of
transami-nases? (Page 16)
A. Transaminases in blood are elevated in liver and heart diseases.
Q. What is the significance of SH groups in
pro-teins? (Page 17)
A. The SH group of cysteine can form a disulfide (S-S) bond with another cysteine residue. The two cysteine residues can connect two polypeptide chains by the formation of interchain disulfide bonds.
Q. Glutathione is made up of which amino acids? (Page 17)
A. Glutamic acid, cysteine and glycine.
Q. Phosphorylation is taking place on which amino acid residue?
Amino Acids: Structure and Properties 15 Q. What is ninhydrin reaction? (Page 18)
A. All amino acids when heated with ninhydrin will give a pink colour.
Q. What is the importance of ninhydrin reaction? (Page 18)
A. It is used for qualitative test and quantitative es-timation of amino acids. It is often used for detec-tion of amino acids in chromatography.
Q. Do proteins give a color with ninhydrin?
(Page 18)
A. Proteins do not give a true color reaction; but N-terminal end amino group of protein will react with ninhydrin, to produce a blue color.
Q. What is biuret reaction? (Page 18)
A. Cupric ions in alkaline medium form a violet colour with peptide bond nitrogen.
Q. Will amino acids give a positive biuret test? (Page 18)
A. No. This needs a minimum of two peptide bonds.
Q. What is the use of biuret reaction? (Page 18)
A. This reaction can be used for qualitative identifi-cation and quantitative estimation of proteins.
Q. What is biuret? (Page 18)
A. The name is derived from the compound biuret, a condensation product of two urea molecules, which also gives a positive color test.
Q. What is the basis of xanthoproteic test?
(Page 18)
A. The ring systems in phenyl alanine, tyrosine and tryptophan will answer this test.
Q. The protein which does not answer the aldehyde
test is. (Page 18)
Proteins: Structure and Function 17
Q. How proteins are made up of? (Page 19)
A. Proteins are made by polymerisation of amino acids through peptide bonds.
Q. What is a peptide bond? (Page 19)
A. Alpha carboxyl group of one amino acid reacts with alpha amino group of another amino acid to form a peptide bond or CO-NH bridge (Fig. 4.1).
Q. What is a dipeptide? (Page 19)
A. Two amino acids are combined to form a dipep-tide.
Q. How many peptide bonds are present in a
trip-eptide? (Page 19)
A. A tripeptide is a combination of three amino ac-ids; so there are two peptide bonds.
Q. What is a polypeptide? (Page 19)
A. A combination of 10 to 50 amino acids is called as a polypeptide.
Proteins: Structure
and Function
Q. What is the difference between a polypeptide
and a protein? (Page 19)
A. A combination of 10 to 50 amino acids is called a polypeptide. By convention, chains containing more than 50 amino acids are called proteins.
Q. What are the levels of organizations of proteins? (Page 19)
A. Proteins have primary, secondary, tertiary and quaternary levels of organisation.
Q. What is meant by primary structure of a protein? (Page 19)
A. It denotes the number and sequence of amino ac-ids in the protein.
Q. What is the force that maintains the primary
structure? (Page 19)
A. The primary structure is maintained by the cova-lent bonds of the peptide linkages (Fig. 4.2).
Q. What are the salient features of a peptide bond? (Page 19)
A. The peptide bond is a partial double bond. The C-N bond is ‘trans’ in nature and there is no free-dom of rotation because of the partial double bond character.
Q. What is the N-terminal end of a protein?
(Page 20)
A. In a protein, at one end there will be one free al-pha amino group. This end is called the amino terminal (N-terminal) end and the amino acid con-tributing the ?-amino group is named as the first amino acid.
Proteins: Structure and Function 19 Q. What are the names for the end amino acids of
proteins. (Page 20)
A. The end where there is a free alpha amino group is called the amino terminal (N-terminal) end. The other end of the polypeptide chain is called the carboxy terminal end (C-terminal) where there is a free alpha carboxyl group.
Q. Can you give an example of a pseudopeptide? (Page 20)
A. Glutathione (gamma-glutamyl-cysteinyl-glycine). The pseudopeptide a peptide bond formed by carboxyl group, other than that of alpha position.
Q. What are the salient structural features of
insu-lin? (Page 20)
A. It has two polypeptide chains. These chains are held together by disulfide bridges. Insulin has total 51 amino acids.
Q. What is pro-insulin? (Page 20)
A. Insulin is synthesised by the beta cells of pancreas as a prohormone, proinsulin is a single poly-pep-tide chain with 86 amino acids.
Q. What is mutation? (Page 20)
A. Amino acid change in the linear sequence is called a mutation.
Q. Can you give an example? (Page 20)
A. sickle cell anemia due to Haemoglobin S,
Q. What is the defect in HbS? (Page 20)
A. Normally the 6th amino acid in the beta chain is glutamic acid, this is replaced by valine in the HbS molecule.
Q. Which are the forces that maintain the second-ary, tertiary and quaternary structures of a
pro-tein? (Page 21)
A. Hydrogen bonds, Electrostatic bonds, Van der Waal’s forces and Hydrophobic bonds.
Q. What are the salient features of alpha structure
of proteins? (Page 22)
A. It is a right-handed spiral structure; each turn is formed by 3.6 amino acid residues; it is major structural motif in globular proteins.
Q. Which will inhibit the formation of alpha helix? (Page 22)
A. Proline.
Q. What is meant by secondary structure of a
pro-tein? (Page 22)
A. Secondary structure denotes the configurational relationship between residues which are about 3-4 amino acids apart. In other words, secondary level defines the organisation at immediate vicin-ity of amino acids.
Q. What is meant by tertiary structure of a protein? (Page 22)
A. The tertiary structure denotes three dimensional structure of the whole protein. It defines the steric relationship of amino acids which are far apart from each other in the linear sequence.
Q. What is meant by a domain of a protein?
(Page 22)
A. It is the term used to denote a compact unit of a protein. It generally represents a functional unit.
Proteins: Structure and Function 21 Q. What is meant by quaternary structure of a
pro-tein? (Page 22)
A. Certain polypeptides will aggregate to form one functional protein. This is referred to as the qua-ternary structure.
Q. Give some examples of proteins having
quater-nary structure. (Page 23)
A. Hemoglobin, lactate dehydrogenase, immunoglo-bulin.
Q. What are the reagents that are used for identify-ing the first amino acid in a protein? (Page 24)
A. Fluoro dinitro benzene, dansyl chloride, phenyl iso thio cyanate.
Q. Protein chains may be separated by what
re-agent? (Page 24)
A. 8 molar urea.
Q. What is meant by Ingram’s technique?(Page 24)
A. Protein digestion by trypsin, followed by two di-mensional chromatography.
Q. It is otherwise known as what? (Page 24)
A. Finger printing of proteins or peptide mapping.
Q. Secondary structure of protein can be studied by
what methods? (Page 25)
A. X-ray diffraction study, optical rotatory disper-sion, and nuclear magnetic resonance (NMR).
Q. What is iso-electric point of a protein? (Page 25)
A. At the iso-electric point, the number of anions and cations present on the protein molecule will be equal and the net charge is zero.
Q. What are the characteristic features of
iso-elec-tric point? (Page 25)
A. At the pI value, the proteins will not migrate in an electrical field; solubility, buffering capacity and viscosity will be minimum and precipitation will be maximum.
Q. What is the iso-electric pH of human albumin? (Page 25)
A. It is 4.7.
Q. How proteins are precipitated from solution? (Page 25)
A. Any factor which neutralises the charge or re-moves water of hydration will cause precipita-tion of proteins.
Q. How albumin is precipitated? (Page 25)
A. By full saturation of ammonium sulfate or 28 % sodium sulfate.
Q. What will be precipitated by half-saturation of
ammonium sulfate? (Page 25)
A. Globulins are precipitated by half-saturation of ammonium sulfate.
Q. Give an example of precipitation at iso-electric
point. (Page 26)
A. Casein is precipitated when the solution is brought to iso-electric pH.
Q. What is the iso-electric pH of casein? (Page 26)
Proteins: Structure and Function 23 Q. Give some examples of anionic precipitating
agents. (Page 26)
A. Tungstic acid, phosphotungstic acid, trichloro acetic acid, picric acid, sulphosalicylic acid and tannic acid are protein precipitating agents.
Q. What are the features of denaturation?(Page 26)
A. The secondary, tertiary and quaternary structures are lost, but primary structure is preserved. The functional activity is lost. The denature proteins are insoluble and easily precipitated.
Q. What are the usual agents that cause denaturation
of proteins? (Page 26)
A. Brief heating, urea, X-ray, ultraviolet ray, high pressure, vigorous shaking.
Q. What is heat coagulation? (Page 26)
A. When heated at iso-electric point, some proteins will denature irreversibly to produce thick float-ing conglomerates called coagulum. This is called heat coagulation.
Q. Give examples of proteins that coagulate easily. (Page 26)
A. Albumin is easily coagulated, and globulins to a lesser extent.
Q. How proteins are classified? (Page 27)
A. They may be classified depending on the func-tion or based on the physicochemical characteris-tics or based on their nutritional value.
Q. What is the functional classification of proteins? (Page 27)
A. 1. Catalytic proteins, 2. Structural proteins, 3. Con-tractile proteins, 4. Transport proteins, 5. Regula-tory proteins or hormones, 6. Genetic proteins, and 7. Protective proteins.
Q. Based on physiochemical properties, how are
they classified? (Page 27)
A. Simple proteins, conjugated proteins and derived proteins.
Q. Give examples of simple proteins. (Page 27)
A. Albumins, globulins, protamines, prolamines, lectins, scleroproteins.
Q. Give examples of scleroproteins. (Page 27)
A. Collagen of bone, cartilage and tendon, keratin of hair, horn, nail and hoof.
Q. What are conjugated proteins? (Page 27)
A. Combinations of protein with a non-protein part is called prosthetic group.
Q. How are conjugated group subclassified? (Page 27)
A. Glycoproteins, lipoproteins, nucleoproteins, chro-moproteins, phospho-proteins and metallo-pro-teins.
Q. Give some examples of chromoproteins.
(Page 27)
Proteins: Structure and Function 25 Q. Give examples of phosphoproteins.
(Page 27)
A. Casein of milk and vitellin of egg yolk.
Q. Where is this phosphate attached to proteins? (Page 27)
A. The phosphoric acid is added to the hydroxyl groups of serine and threonine residues of pro-teins.
Q. What are lectins? (Page 27)
A. Plant proteins having specific carbohydrate bind-ing site.
Q. Give an example of a nutritionally rich protein (first class protein). (Page 28)
A. Casein.
Q. Some proteins are called as poor proteins; why? (Page 28)
A. They lack in many essential amino acids and a diet based on these proteins will not even sustain the body weight.
Q. Give an example of nutritionally poor protein. (Page 28)
A. Zein from corn lacks tryptophan and lysine.
Q. Which method of protein estimation is depen-dent on the intact peptide bond?
Q. What is the advantage of biuret method?
(Page 28)
A. The biuret method is simple one step process, and is the most widely used method for plasma pro-tein estimations.
Q. What is the disadvantage of biuret method? (Page 28)
A. The sensitivity of the method is less and is un-suitable for estimation of proteins in milligram or microgram quantities.
Q. What is the basis of Lowry’s method of protein
estimation? (Page 28)
A. This is based on the reduction of folin-ciocalteau phenol reagent (phosphomolybdic acid and phos-photungstic acid) by the tyrosine and tryptophan residues of protein.
Q. Which component of the protein absorb UV light
at 280 nm? (Page 28)
A. Indole ring of tryptophan.
Q. What is nephelometry? (Page 29)
A. Nephelometry is defined as the detection of light scattered by turbid particles in solution.
Enzymology-I 27
Enzymology-I
Q. How are enzymes classified? (Page 30)
A. They are classified into five major classes.
Q. What are those classes? (Page 30)
A. Oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
Q. What is the function of oxidoreductases? (Page 30)
A. Transfer of hydrogen.
Q. Give an example of oxidoreducatase. (Page 30)
A. Alcohol dehydrogenase.
Q. What is the function of transferases? (Page 31)
A. Transfer of groups other than hydrogen.
Q. Give an example of transferase. (Page 31)
A. Hexokinase.
Q. What is the function of hydrolases? (Page 31)
Q. Give an example of a hydrolase. (Page 31)
A. Acetyl choline esterase.
Q. Peptidases are classified under which class of
enzyme? (Page 31)
A. Hydrolases.
Q. What is the function of lyases? (Page 31)
A. Cleave bond without adding water.
Q. Which enzyme will add water to a double bond, without breaking the bond?
A. Hydratase. (Page 31)
Q. Give an example of lyase. (Page 31)
A. Aldolase.
Q. Give an example of isomerase. (Page 31)
A. Triose phosphate isomerase.
Q. What is the function of ligases? (Page 31)
A. ATP dependent condensation of two molecules.
Q. What is the difference between synthase and
syn-thetase? (Page 31)
A. Synthetases are ATP-dependent enzymes catalysing biosynthetic reactions; they belong to Ligases. Synthases are enzymes catalysing biosyn-thetic reactions; but they do not require ATP di-rectly; they belong to classes other than Ligases.
Q. Give examples of synthetases. (Page 31)
A. Carbamoyl phosphate synthetase, arginino suc-cinate synthetase, PRPP synthetase, glutamine synthetase.
Enzymology-I 29 Q. Give examples of synthases. (Page 31)
A. Glycogen synthase, ALA synthase, IMP synthase.
Q. What are co-enzymes? (Page 31)
A. Enzyme may contain a non-protein part, the co-enzyme. The co-enzyme is essential for the bio-logical activity of the enzyme. A co-enzyme is a low molecular weight organic substance, without which the enzyme cannot exhibit any reaction. Co-enzyme accepts one of the products of the reac-tion; and so act as a co-substrate.
Q. What is holo-enzyme? (Page 31)
A. When apo-enzyme and co-enzymes are added, holo-enzyme is produced. Fully active enzyme is called Holo-enzyme.
Q. How are co-enzymes classified? (Page 31)
A. (a) Those taking part in reactions catalysed by oxi-doreductases by donating or accepting hydrogen atoms or electrons. (b) Those co-enzymes taking part in reactions transferring groups other than hydrogen.
Q. Give some examples of co-enzymes involved in
oxidoreductases. (Page 31)
A. NAD, NADP, FAD.
Q. What is the full form of NAD? (Page 32)
A. Nicotinamide adenine dinucleotide.
Q. What is FAD? (Page 32)
Q. Give some examples of co-enzymes involved in reactions other than hydrogen transfer.
(Page 32)
A. Thiamine pyrophosphate, pyridoxal phosphate, biotin, co-enzyme A, ATP.
Q. What is the full form of ATP? (Page 32)
A. Adenosine triphosphate.
Q. What is the function of ATP? (Page 32)
A. It is the energy currency in the body. During the oxidation of food stuffs, energy is released, a part of which is stored as chemical energy in the form of ATP. Other reaction requiring energy are coupled with ATP.
Q. Name the enzymes containing copper.(Page 33)
A. Superoxide dismutase, tyrosinase, cytochrome oxidase.
Q. Which metal is required for the action of Kinases? (Page 33)
A. Magnesium.
Q. Chloride ions activate which enzyme? (Page 33)
A. Amylase.
Q. Which enzyme contains molybdenum?
(Page 33)
A. Xanthine oxidase.
Q. Name some iron containing enzymes. (Page 33)
A. Cytochrome oxidase, catalase, peroxidase, xan-thine oxidase.
Enzymology-I 31 Q. What is Michaelis-Menten Theory ? (Page 33)
A. It is otherwise called enzyme-substrate complex theory. The enzyme combines with the substrate, to form an enzyme-substrate complex, which im-mediately breaks down to the enzyme and the product.
Q. What is Fischer’s theory? (Page 34)
A. It states that the three dimensional structure of the active site of the enzyme is complementary to the substrate. Thus, enzyme and substrate fit each other like a key and its lock.
Q. What is Koshland’s induced fit theory?
(Page 34)
A. The substrate induces conformational changes in the enzyme, such that precise orientation of cata-lytic groups is effected.
Q. What is active site of an enzyme? (Page 35)
A. That area of the enzyme where catalysis occurs is referred to as active site or active center.
Q. What is meant by serine proteases? (Page 35)
A. Proteases (proteolytic enzymes) having a serine residue at its active center.
Q. Give an example of a serine protease. (Page 35)
A. Trypsin, chymotrypsin, thrombin.
Q. Thermodynamically, how reactions are
classi-fied? (Page 36)
A. Exothermic, isothermic and endothermic reac-tions.
Q. What is exothermic reaction? (Page 36)
A. Here energy is released from the reaction, and therefore reaction essentially goes to completion, e.g. urease enzyme, converting urea to ammonia + CO2 + energy.
Q. What is endergonic reaction? (Page 36)
A. Energy is consumed and external energy is to be supplied for these reactions. In the body this is usually accomplished by coupling the endergonic reaction with an exergonic reaction, e.g. Hexoki-nase reaction, Glucose + ATP ®
Glucose-6-Phos-phate + ADP.
Q. What are the salient features of enzyme
kinet-ics? (Page 36)
A. Enzymes lower activation energy. They increase the chemical reaction, but do not alter equilibrium of the reaction.
Q. What are the factors influencing enzyme
reac-tion? (Page 36)
A. Enzyme concentration, substrate concentration, product concentration, temperature, pH and pres-ence of activators or inhibitors.
Q. What is Km value? (Page 37)
A. Substrate concentration (expressed in moles/L) at half-maximal velocity is the Km value.
Q. What does it indicate? (Page 37)
A. It denotes that 50% of enzyme molecules are bound with substrate molecules at that particu-lar substrate concentration
Enzymology-I 33 Q. What is its significance? (Page 37)
A. Km is independent of enzyme concentration. Km value is thus constant for an enzyme. It is the char-acteristic feature of a particular enzyme for a spe-cific substrate. Km denotes the affinity of enzyme to substrate. Thus, the lesser the numerical value of Km, the affinity of the enzyme for the substrate is more.
Q. What is the use of assessing the Km value of an enzyme? What is the application? (Page 38)
A. Determination of Km value is also useful to un-derstand the natural substrate of an enzyme. Study of Km value will also differentiate the competi-tive and non-competicompeti-tive inhibitions.
Q. What is the effect of temperature on enzyme
ve-locity? (Page 39)
A. The velocity of reaction increases when tempera-ture is increased, reaches a maximum and then falls (Bell-shaped curve)
Q. Why it falls? (Page 39)
A. when temperature is more than 50ºC, heat dena-turation and consequent loss of tertiary structure of protein occurs.
Q. What is the effect of pH on the activity of an
en-zyme? (Page 39)
A. Each enzyme has an optimum pH, on both sides of which the velocity will be drastically reduced. The graph will show a bell-shaped curve.
Q. What is the explanation for the effect of pH? (Page 39)
A. The pH decides the charge on the amino acid resi-dues at the active site. The net charge on the en-zyme protein would influence substrate binding and catalytic activity.
Q. What is the optimum pH of usual enzymes? (Page 39)
A. Usually enzymes have the optimum pH between 6 and 8.
Q. Are there any important exceptions for this
gen-eral rule? (Page 39)
A. Pepsin (optimum pH 1-2), alkaline phosphatase (optimum pH 9-10) and Acid phosphatase (4-5).
Q. What is zymogen? (Page 39)
A. It is otherwise called pro-enzyme. Inactive zy-mogen is activated by removal of a piece of the pro-enzyme.
Q. Give an example of zymogen is activated? (Page 39)
A. By splitting a single peptide bond, and removal of a small polypeptide from trypsinogen, the ac-tive trypsin is formed. This results in unmasking of the active centre.
Enzymology-I 35 Q. What is the significance of zymogen activation? (Page 39)
A. Gastro-intestinal enzymes are synthesised in the form of pro-enzymes, and only after secretion into the alimentary canal, they are activated. This pre-vents autolysis of cellular structural proteins. Co-agulation factors are seen in blood as zymogen form, their activation takes place only when ne-cessity arises. This prevents intravascular coagu-lation.
Q. What are the different types of inhibitions of
enzyme activity? (Page 39)
A. Competitive inhibition, non-competitive inhibi-tion, suicide inhibiinhibi-tion, and allosteric regulation.
Q. What are salient features of competitive
inhibi-tion? (Page 40)
A. Competitive inhibitor is a structural analogue. 2. It is reversible. 3. Km is increased. 4. Vmax is not changed.
Q. Give examples of competitive inhibition. (Page 40)
A. Malonate inhibits succinate dehydrogenase.
Q. Give examples of clinical application of
competi-tive inhibition. (Page 40)
A. Sulfonamide inhibits PABA incorporation in bac-teria, and so acts as an antibacterial agent. Meth-otrexate inhibits folate reductase system, dicoumarol inhibits vitamin K.
Q. What is the immediate treatment for methanol
poisoning? (Page 41)
A. Methanol is oxidised by alcohol dehydrogenase to formaldehyde which causes the acute toxicity. Antidote to methanol poisoning is ethanol which is the natural substrate for alcohol dehydrogenase. So ethanol is preferentially utilised.
Q. What are the salient features of non-competitive
inhibition? (Page 41)
A. Non-competitive inhibitor has no structural simi-larity with the substrate. 2. It is generally not re-versible 3. Km is not changed. 4. Vmax is reduced.
Q. Give examples of non-competitive inhibition. (Page 41)
A. Di-isopropyl fluoro phosphate inhibits trypsin, fluoride inhibits and enolase.
Q. Iodo-acetate inhibits enzyme by reacting with which group at the active site of the enzyme?
(Page 41)
A. Sulfhydryl group.
Q. What is the mechanism of inhibitory action of Di-isopropyl fluoro phosphate? (Page 41)
A. It inhibits enzymes with serine in their active cen-tres, e.g. acetylcholine esterase.
Q. What is suicide inhibition? (Page 42)
A. In suicide inhibition, the structural analogue is converted to a more effective inhibitor with the help of the enzyme to be inhibited. The inhibitor makes use of the enzyme’s own reaction mecha-nism to inactivate it.
Enzymology-I 37 Q. What is the other term for suicide inhibition?
(Page 42)
A. Mechanism based inactivation.
Q. Give examples for suicide inhibition. (Page 42)
A. Ornithine decarboxylase (ODC) is inhibited by difluro methyl ornithine (DFMO). Another ex-ample is Allopurinol which is oxidised by xan-thine oxidase to alloxanxan-thine that is a strong in-hibitor of xanthine oxidase.
Q. What is allosteric inhibition? (Page 42)
A. Allosteric enzyme has one catalytic site where the substrate binds and another separate allosteric site where the modifier binds.
Q. What are the salient features of allosteric
inhibi-tion? (Page 43)
A. (1) The inhibitor is not a substrate analogue. (2) It is partially reversible when excess substrate is added. (3) Km is usually increased. (4) Vmax is reduced. (5) Most allosteric enzymes possess qua-ternary structure. They are made up of subunits.
Q. Give examples for allosteric inhibition.
(Table 5.7)
A. ALA synthase, aspartyl trans-carbamoylase, HMG CoA reductase
Q. What is covalent modification? (Page 43)
A. It means, either addition of a group to the enzyme protein by a covalent bond; or removal of a group by cleaving a covalent bond.
Q. Give some examples of covalent modification. (Page 44)
A. Glycogen synthase is inactive, in the phosphory-lated state, whereas glycogen phosphorylase is active when phosphorylated.
Q. What is meant by induction? (Page 44)
A. Induction is effected at the level of DNA. The in-ducer will relieve the repression on the operator site and will remove the block on the biosynthe-sis of the enzyme molecules.
Q. Give an example of induction. (Page 44)
A. Induction of lactose-utilising enzymes in the bac-teria when the media contains lactose in the ab-sence of glucose. In humans, Tryptophan pyrrolase and transaminases are induced by glu-cocorticoids. Glucokinase is induced by glucose. ALA synthase is induced by barbiturates.
Q. What are constitutive enzymes? (Page 44)
A. Enzymes whose concentration in a cell is inde-pendent of inducer are called constitutive en-zymes.
Q. What is repression? (Page 44)
A. Repression acts at the gene level, the number of enzyme molecules is reduced in the presence of repressor molecule.
Q. Give an example of repression. (Page 44)
A. The key enzyme of heme synthesis, ALA synthase is autoregulated by the heme by means of repres-sion.
Enzymology-I 39 Q. Give examples of multi-enzyme complexes.
(Page 44)
A. Fatty acid synthase, pyruvate dehydrogenase, and alpha keto glutarate dehydrogenase.
Q. What are the types of specificity shown by
en-zymes? (Page 45)
A. Absolute specificity, group specificity and streospecificity.
Q. Give an example for absolute specificity.
(Page 45)
A. Urea is the only substrate for urease.
Q. Give an example for group specificity. (Page 45)
A. trypsin can hydrolyse peptide bonds formed by carboxyl groups of arginine or lysine residues.
Q. What are iso-enzymes? (Page 46)
A. They are physically distinct forms of the same enzyme activity. They have identical catalytic properties, but differ in structure.
Q. How to differentiate iso-enzymes. (Page 46)
A. Electrophoresis, heat stability, km value, inhibi-tor specificity, and tissue localization.
Q. Which is a functional enzyme in plasma? (Page 46)
A. They are actively secreted into plasma, and have some functions in the blood. For example, en-zymes of blood coagulation.
Q. What is non-functional enzymes in plasma? (Page 46)
A. They are coming out from cells due to normal wear and tear.
Enzymology-II
Iso-Enzymes and
Enzymology-II 41 Q. What is their clinical significance? (Page 46)
A. Their normal levels in blood are very low, but are drastically increased during cell death (necro-sis) or disease. Therefore, assays of these enzymes are very useful in diagnosis of diseases.
Q. Lactate dehydrogenase has how many
polypep-tide subunits? (Page 47)
A. Four. It is a tetramer.
Q. Lactate dehydrogenase has how many
iso-en-zymes? (Page 47)
A. Five
Q. What are they? (Page 47)
A. H4, H3M, H2M2, M3H and M4 varieties, forming five iso-enzymes. All these five forms are seen in all persons.
Q. How do you separate LDH iso-enzymes in
labo-ratory? (Page 47)
A. By cellulose acetate electrophoresis at pH 8.6.
Q. LDH level in blood is increased in which
condi-tions? (Page 47)
A. Myocardial infarction, hemolytic anemias, mus-cular dystrophy, carcinomas, leukemias, and any condition which causes necrosis of body cells.
Q. How do you further investigate for myocardial
infarction? (Page 47)
Q. What is flipped pattern? (Page 47)
A. Normally LDH-2 (H3M1) concentration in blood is greater than LDH-1 (H4), but this pattern is re-versed in myocardial infarction, this is called flipped pattern.
Q. What are the serum enzymes helpful in the di-agnosis of myocardial infarction?
A. Lactate dehydrogenase (LDH) H4 iso-enzyme, creatine kinase (CK) CK MB iso-enzyme and as-partyl transaminase (AST).
Q. Creatine kinase (CK) level in serum is increased in which conditions?
A. Myocardial infarction, muscular dystrophies. (Page 48)
Q. What is the advantage of CK estimation over LDH estimation to identify myocardial
infarc-tion? (Page 48)
A. The CK level starts to rise within three hours of infarction. Therefore, CK estimation is very use-ful to detect early cases, where ECG changes may be ambiguous. The CK level is not increased in hemolysis or in congestive cardiac failure; and therefore CK has an advantage over LDH.
Q. What are the iso-enzymes of CK? (Page 48)
A. CK is a dimer, the subunits are called B for brain and M for muscle. Therefore, three iso-enzymes are possible.
Enzymology-II 43 Q. What are the origins of the CK iso-enzymes?
(Page 48)
A. Eighty percent of molecules in circulation are MM (CK3) variety of skeletal origin, five percent in circulation are MB (CK2) from heart, one percent from brain (BB or CK1) and fifteen percent CKmt from mitochondria.
Q. When do you estimate total CK and the
iso-en-zyme? (Page 48)
A. Estimation of total CK is employed in muscular dystrophies and CK-MB iso-enzyme is estimated to identify myocardial infarction.
Q. What is the advantage of cardiac troponin I over other parameters to identify the myocardial
inf-arction? (Page 48)
A. Cardiac Troponin I is released into the blood within four hours after the onset of cardiac symp-toms, peaks at 12-16 hours and remains elevated for 5-9 days post-infarction. Therefore, CTI is very useful as a marker at any time interval after the heart attack. It is 75% sensitive index for myocar-dial infarction.
Q. What is the significance of AST? (Page 48)
A. It is significantly elevated in myocardial infarc-tion and moderately elevated in liver diseases.
Q. What is the significance of ALT? (Page 49)
A. Very high values are seen in acute hepatitis. Rise in ALT levels may be noticed several days before clinical signs such as jaundice are manifested. Moderate increase may be seen in chronic liver diseases such as cirrhosis, and malignancy in liver.
Q. Alkaline phosphatase level in serum is elevated in which conditions? (Page 49)
A. Moderate increase is seen in hepatic diseases (in-fective hepatitis, alcoholic hepatitis). High levels may be noticed in obstructive jaundice or cholestasis. Very high levels are seen in bone dis-eases such as Paget’s disease, rickets, osteomala-cia, osteoblastoma, metastatic carcinoma of bone.
Q. For alkaline phosphatase, how many
iso-en-zymes are present? (Page 49)
A. Six.
Q. What is Regan iso-enzyme? (Page 49)
A. It is the iso-enzyme of alkaline phosphatase, in-hibited by phenylalanine. It is of placental origin. It is elevated in about 15% cases of carcinoma of lung, liver and gut and then named as Regan iso-enzyme or carcinoplacental iso-iso-enzyme.
Q. It is said that nucleotide phosphatase (NTP) is a better index of obstructive liver disease than al-kaline phosphatase (ALP), why? (Page 49)
A. ALP level is increased in both liver and bone dis-eases, but NTP is only in liver diseases.
Q. Estimation of gamma glutamyl transferase is use-ful to detect which condition? (Page 50)
A. Alcohol abuse.
Q. What are the enzymes useful in diagnosing liver
pathology? (Page 50)
Enzymology-II 45 Q. Give the clinical implications of these enzymes. (Page 50)
A. In infective hepatitis, ALT level is increased; in alcohol abuse, GGT level is increased; in obstruc-tive jaundice, ALP level is increased.
Q. Serum acid phosphatase level is increased in
which condition? (Page 50)
A. Prostate carcinoma.
Q. Total acid phosphatase may increase in some other conditions also; what are they? (Page 50)
A. Prostate carcinoma, secondary metastasis in bones, per rectal examination, intravascular hemolysis.
Q. In such conditions, iso-enzyme study is helpful
or not? (Page 50)
A. Yes, tartarate labile iso-enzyme is specific for pros-tate carcinoma.
Q. What is the advantage of prostate specific
anti-gen? (Page 50)
A. PSA is very specific for prostate carcinoma.
Q. What are the enzymes useful as tumour
mark-ers? (Page 50)
A. Regan iso-enzyme of ALP for lung tumour; tartarate labile iso-enzyme of ACP and Prostate specific antigen (PSA) for prostate carcinoma; Neuron specific enolase (NSE) for cancers of neuro-endocrine origin.
Q. Pseudo-cholinesterase deficiency is manifested
as what? (Page 50)
A. Succinyl choline apnoea; prolonged apnea when succinyl choline is given as anesthetic drug.
Q. Which enzyme deficiency is inherited as
X-linked? (Page 50)
A. Glucose-6-phosphate-dehydrogenase.
Q. How the deficiency of GPD is manifested? (Page 51)
A. Drug induced hemolytic anemia.
Q. Acute pancreatitis can be diagnosed by
estimat-ing which enzymes? (Page 51)
A. Amylase and lipase.
Q. Name some enzymes that are used as
therapeu-tic agents. (Page 52)
A. Asparaginase for leukemia, streptokinase to dis-solve clots, and pepsin for indigestion.
Methods of Separation & Purification of Biological Compounds, Methods of Study of Metabolism 47
Q. What is meant by electrophoresis? (Page 53)
A. The term refers to the movement of charged par-ticles through an electrolyte when subjected to an electric field.
Q. What are the factors affecting the mobility in
elec-trophoresis? (Page 53)
A. Net charge on the particles (pI of proteins), mass and shape of the particles, the pH of the medium, strength of electrical field, and properties of the supporting medium.
Q. What are the types of electrophoresis? (Page 53)
A. Horizontal and vertical types.
Q. What are the supporting media used? (Page 53)
A. Filter paper, cellulose acetate, agar gel, agarose gel, starch gel and polyacrylamide gel.
Methods of Separation
and Purification of
Biological Compounds,
Methods of Study of
Metabolism
Q. Electrophoresis is commonly employed for what purpose in laboratory?
A. For serum electrophoresis and to see abnormali-ties in serum protein concentrations.
Q. What is the advantage of polyacrylamide gel? (Page 53)
A. It has a molecular sieving effect and so separa-tion is very efficient.
Q. What is immuno-electrophoresis? (Page 54)
A. Here electrophoretic separation is followed by an antigen-antibody reaction.
Q. What is the principle of adsorption
chromatog-raphy? (Page 54)
A. separation is based on differences in adsorption at the surface of a solid stationary medium.
Q. What is the principle of partition
chromatogra-phy? (Page 55)
A. the components of the mixture to be separated are partitioned between the two phases depend-ing on the partition co-efficient (solubility) of the particular substances.
Q. What are the common types of partition
chroma-tography? (Page 55)
A. Paper chromatography and thin layer chromatog-raphy.
Q. What is the advantage of TLC over paper
chro-matography? (Page 55)
A. TLC needs lesser time, and separation is more ef-fective.
Methods of Separation & Purification of Biological Compounds, Methods of Study of Metabolism 49
Q. What is Rf value? (Page 56)
A. It is the ratio of the distance travelled by the sub-stance (solute) to the disub-stance travelled by the sol-vent. The Rf value is a constant for a particular solvent system at a given temperature.
Q. What is the basic principle of ion-exchange
chro-matography? (Page 56)
A. Here, the separation is based on electrostatic at-traction between charged molecules to oppositely charged groups on the ion exchange resins.
Q. What is the principle of gel filtration
chromatog-raphy? (Page 56)
A. The separation is effected on the basis of the size of the molecules. It is otherwise called molecular sieving.
Q. Give the principle of affinity chromatography. (Page 56)
A. The technique is based on the high affinity of spe-cific proteins for spespe-cific chemical groups.
Q. Give an example of affinity chromatography. (Page 57)
A. Separation and quantitation of glycated hemoglo-bin.
Q. What is the quickest method for separation of
proteins? (Page 58)
Q. What is the principle of ultracentrifugation? (Page 58)
A. Large molecules can be sedimented at high cen-trifugal forces whereas small molecules cannot. Rate of sedimentation depends on the size, shape and density of solute particles.
Q. What is Svedberg unit? (Page 57)
A. Sedimentation constant is expressed in Svedberg (S) units.
Q. What are the uses of ultracentrifugation?
A. 1. Separation of subcellular organelles. 2. Separa-tion of lipoproteins. 3. DeterminaSepara-tion of molecu-lar weight of proteins.
Q. What are the methods used to determine the molecular weight of proteins?
A. (1) Ultracentrifugation, (2) Gel filtration and (c) PAGE (poly acrylamide gel electrophoresis).
(Page 57)
Q. What is the advantage of radio-immuno assay? (Page 57)
A. Very small quantities of substances could be ac-curately measured.
Q. What is the radio-active label used for RIA? (Page 58)
A. Iodine-125.
Q. What is the half life of Iodine-125? (Page 58)
Methods of Separation & Purification of Biological Compounds, Methods of Study of Metabolism 51 Q. What are the disadvantages of RIA, when
com-pared to ELISA? (Page 58)
A. 1. Since radio-isotopes are used, only approved laboratories could take up the assay. 2. The shelf life of the reagent is short.
Q. What are the enzymes commonly used in ELISA
technique. (Page 58)
A. Alkaline phosphatase (ALP) and horse radish per-oxidase (HRP).
Q. How carbohydrates are classified? (Page 61)
A. Based on the number of the sugar units available, they are classified as monosaccharides, disaccha-rides, oligosacchadisaccha-rides, and polysaccharides.
Q. What is a monosaccharide? (Page 61)
A. Molecules having only one actual or potential sugar group are called monosaccharides.
Q. What is a polysaccharide? (Page 61)
A. They contain more than 10 sugar units.
Q. How are they combined together? (Page 61)
A. Through glycosidic linkages.
Q. How are monosaccharides further classified? (Page 61)
A. Sugars having aldehyde group are called aldoses and sugars with keto group are ketoses.
Carbohydrates-I:
Chemistry, Digestion
and Absorption
Carbohydrates-I 53 Q. Name some important monosaccharides.
(Page 61)
A. Glucose, fructose, galactose, mannose.
Q. What are pentoses? (Page 61)
A. Monosaccharides with five carbon atoms.
Q. Name a few pentoses. (Page 61)
A. Arabinose, Xylose, Ribose.
Q. Which is the reference carbon atom in sugars? (Page 62)
A. Penultimate carbon atom.
Q. What is the difference between D and L sugars? (Page 62)
A. They are mirror images with reference to penultimate carbon atom.
Q. Which isomer is common in nature? (Page 62)
A. D variety of sugars are common in nature.
Q. Which is the most common monosaccharide in the body?
A. Glucose. (Page 62)
Q. What is the difference between glucose and
ga-lactose? (Page 62)
A. They are different with regard to the H and OH groups at the 4th carbon atom. Galactose is the 4th epimer of glucose (Fig.8.3).
Q. Galactose is present in which food? (Page 62)
A. Lactose is present in milk. Lactose contains ga-lactose and glucose.
Q. What is epimerism? (Page 62)
A. When sugars are different from one another, only in configuration with regard to a single carbon atom (other than the reference carbon atom), they are called epimers.
Q. Give an example. (Page 62)
A. For example, glucose and mannose are an epimeric pair which differ only with respect to carbon atom 2. Similarly, galactose is the 4th epimer of glucose. (Fig. 8.3).
Q. Anomerism is produced with reference with
which carbon atom? (Page 62)
A. Anomers are produced by the spatial configura-tion with reference to the first carbon atom in al-doses and second carbon atom in ketoses. (Fig. 8.4).
Q. How alpha and beta forms of sugars are
pro-duced? (Page 62)
A. These are anomers. The difference lies in the spa-tial configuration with reference to the first car-bon atom in aldoses and second carcar-bon atom in ketoses. (Fig. 8.4).
Q. What is the basis of mutarotation? (Page 62)
A. It is due to the anomeric carbon atom.
Q. What is the difference between glucose and fruc-tose?
A. Glucose is an aldohexose, and fructose is a ketohexose.
Carbohydrates-I 55
Q. Name a ketose. (Page 63)
A. Fructose.
Q. What is the principle of Benedict’s test?
(Page 64)
A. In alkaline medium, sugar will cause reduction of cupric ions, to form red coloured precipitate.
Q. What is the composition of Benedict’s reagent. (Page 64)
A. It contains sodium carbonate, copper sulfate and sodium citrate. In the alkaline medium provided by sodium carbonate, the copper remains as cu-pric hydroxide. Sodium citrate acts as a stabilising agent to prevent precipitation of cupric hydrox-ide.
Q. Benedict’s test is commonly employed for what? (Page 64)
A. To detect the presence of glucose in urine.
Q. Name a few reducing sugars. (Page 64)
A. Glucose, fructose, mannose.
Q. Keto group is non-reducing, but fructose reduces Benedict’s solution, what is the cause for this
anomaly? (Fig. 8.10)
A. In alkaline medium, ketone group is converted to aldehyde, through enediol formation.
Q. In the case of sugars, which of the properties go hand in hand?
A. Reducing property, osazone formation and
Q. Glucose and fructose will form identical osazones, why?
A. The difference in glucose and fructose is depen-dent on the first and second carbon atoms, and this is masked by the osazone formation.
(Page 64)
Q. On oxidation of glucose, what are produced? (Page 64)
A. Glucuronic acid, gluconic acid and glucos-accharic acid.
Q. Reduction of glucose produces what? (Page 64)
A. Sorbitol.
Q. Name some deoxy sugars. (Page 66)
A. Deoxy ribose, fucose (deoxy galactose).
Q. Which is the stain used to identify deoxysugar? (Page 66)
A. Feulgen staining.
Q. Name some important disaccharides. (Page 67)
A. Sucrose, lactose, maltose.
Q. What is the glycosidic linkage in lactose? (Page 67)
A. Beta 1-4 linkage.
Q. What is the glycosidic linkage in sucrose? (Page 67)
Carbohydrates-I 57 Q. Which disaccharide has no free aldehyde or
ke-tone group?
A. Sucrose. (Page 67)
Q. Glucose and fructose are reducing sugars, but sucrose (containing glucose and fructose) is a non-reducing sugar, why? (Page 67)
A. Because the glycosidic linkage in sucrose involves 1st carbon of glucose and 2nd carbon of fructose, so both reducing groups are masked.
Q. Hydrolysis of maltose will give rise to what ? (Page 67)
A. Two glucose units.
Q. Which is the sugar found in milk? (Page 67)
A. Lactose.
Q. What are the component monosaccharides of
lac-tose? (Page 67)
A. Galactose and glucose.
Q. Sucrose consists of what monosaccharides? (Page 67)
A. Glucose + fructose.
Q. Name reducing disaccharides. (Page 67)
A. Lactose and maltose.
Q. How polysaccharides are classified? (Page 68)
A. Homopolysaccharides (homoglycans) and heteropolysaccharides (heteroglycans).
Q. What is a homopolysaccharide? (Page 68)
A. They are composed of single kind of monosac-charides.
Q. Give examples of homopolysaccharides.
(Page 68)
A. Starch, and glycogen.
Q. What are heteropolysaccharides? (Page 68)
A. They are composed of two or more different monosaccharides.
Q. What are the characteristics of glycogen?
(Page 69)
A. It is composed of glucose units. It is the stored form of carbohydrate in animal kingdom. It has a highly branched structure.
Q. What is the reserve carbohydrate in plant
king-dom? (Page 69)
A. Starch.
Q. What is the end product of action of pancreatic
amylase on starch? (Page 69)
A. Maltose.
Q. Cellulose and starch are polysaccharides made of glucose, but cellulose cannot be digest by
human beings, why? (Page 69)
A. Cellulose contains beta 1,4 linkages, which can-not be digested by human enzymes.
Q. What is inulin? (Page 69)
A. It is a homopolysaccharide, composed of fructose units.
Q. What is the use of inulin? (Page 69)
A. It is used to find renal clearance and glomerular filtration rate.
Carbohydrates-I 59 Q. Give examples of heteropolysaccharides.
(Page 70)
A. Agar, hyaluronic acid, heparin, chondroitin sul-fate.
Q. What are mucopolysaccharides? (Page 70)
A. They contain uronic acid and amino sugars.
Q. Which heteropolysaccharide does not contain
uronic acid? (Page 70)
A. Keratan sulfate.
Q. Hyaluronic acid is seen in which tissues? (Page 70)
A. Connective tissue, synovial fluid, tendons, vitre-ous humor.
Q. What is the difference between glycoprotein and
mucoprotein? (Page 71)
A. If the carbohydrate content is less than 10%, it is called a glycoprotein. If the carbohydrate content is more than 10% it is a mucoprotein.
Q. The rate of absorption of sugars in intestine is highest for which monosaccharide? (Page 71)
A. Absorption rate of galactose is more than glucose, while fructose is absorbed at a lesser rate than glucose.
Q. Glucose is absorbed at the luminal side of gastro intestinal cells by which mechanism?
(Page 72, and Fig. 8.30)
A. Carrier mediated co-transport with sodium, named as sodium dependent glucose transporter (SGluT).
Q. How glucose is released from intestinal cells into the blood stream?
A. Glucose transporter type 2 (GluT2) (Fig.8.30).
Q. How glucose is taken up by cells from blood
stream? (Page 72)
A. In tissues GluT2 is involved in absorption of glu-cose from blood.
Q. What is the importance of GluT4? (Page 72)
A. It is the glucose transporter present in muscle and adipose tissues. Insulin induces these transport-ers. In diabetes mellitus, entry of glucose into muscle is decreased, because GluT4 is reduced in insulin deficiency.
Q. What is the glucose sensor in the beta cells of
pancreas? (Page 72)
A. GluT2 acts as the glucose sensor mechanism, for the controlled supply of insulin into blood stream.
Carbohydrates-II 61
Carbohydrates-II:
Major Metabolic
Pathways of Glucose,
Glycolysis, Gluconeogenesis,
Glycogen Metabolism
Q. What is glycolysis? (Page 73)
A. In this pathway, glucose is converted to pyruvate or lactate, along with production of a small quan-tity of energy.
Q. In which condition pyruvate is produced, and
when lactate? (Page 73)
A. In aerobic condition pyruvate is produced. When oxygen is lacking, lactate is produced.
Q. What is the significance of Glycolysis?(Page 73)
A. It is the only pathway that is taking place in all the cells of the body. Glycolysis is the only source of energy in erythrocytes. Moreover, anaerobic glycolysis forms the major source of energy in actively contracting muscles.
Q. What is hexokinase? (Page 73)
A. Hexokinase is the first step in the glycolysis path-way. It phosphorylates glucose to glucose-6-phos-phate.
Q. What is glucokinase? (Page 73)
A. The reaction is similar to hexokinase. But glucoki-nase is present only in liver, acts specifically on glucose, and is active when glucose level in blood is increased after a food.
Q. Which tissues prefer anaerobic glycolysis? (Page 73)
A. RBCs, exercising muscle, and cancer cells.
Q. What is the importance of phospho fructokinase? (Page 74)
A. It is the key enzyme (rate limiting enzyme) of the pathway. It is an irreversible reaction.
Q. What is the substrate for aldolase reaction? (Page 74)
A. Fructose-1,6-bisphosphate.
Q. During glycolysis, energy is produced during
which steps? (Page 74, 75)
A. Step 5, glyceraldehyde-3-phosphate to 1,3-bisphospho glycerate, Step 6, 1,3-bis phospho glycerate to 3-phospho glycerate, and Step 9, Phospho enol pyruvate to pyruvate.
Q. Fluoride ions inhibit which enzyme? (Page 75)
Carbohydrates-II 63 Q. What is the importance of the above inhibition? (Page 75)
A. Fluoride is used to prevent glycolysis, as preser-vative for blood before glucose estimation.
Q. NAD is reduced to NADH in which reaction of
glycolysis? (Page 75)
A. Glyceraldehyde-3-phosphate dehydrogenase re-action.
Q. NADH is oxidised to NAD in which reaction of
glycolysis? (Page 76)
A. Lactate dehydrogenase reaction.
Q. What are substrate level phosphorylations in
gly-colysis? (Page 75, 76)
A. 1,3-bisphospho glycerate kinase (step 6) and pyru-vate kinase (step 9).
Q. What is the purpose of lactic acid production under anaerobic conditions?
A. NADH generated in the 5th step has to be oxidised to NAD+. This can be done by oxygen. But when oxygen is lacking, the 5th step has to be coupled with the 10th step for regeneration of NAD.
(See Fig. 9.11)
Q. As the end product of glycolysis, pyruvate and NADH are formed. During anaerobiasis, this NADH is reconverted to NAD+ by what
mecha-nism? (Page 76)
Q. As the end product of glycolysis, pyruvate and NADH are formed. During aerobic conditions, this NADH is reconverted to NAD+ by what
mechanism? (Page 76)
A. Oxygen.
Q. What is Cori’s cycle? (Fig.9.13)
A. During exercise, lactate is produced in muscle. This lactate diffuses into the blood. Lactate then reaches liver, where it is oxidised to pyruvate. It is then taken up through gluconeogenesis path-way, and becomes glucose. This glucose can en-ter into blood and then taken to muscle. This cycle is called Cori’s cycle, or lactic acid cycle.
Q. What is the purpose of Cori’s cycle? (Page 77)
A. By this means, the lactate is efficiently reutilised by the body.
Q. Why lactate is transported from muscle to liver? (Page 77)
A. Oxygen is limited in muscle, so lactic acid could not be made to pyruvate in muscle. So, it is trans-ported to liver, where it is made to pyruvate and then to glucose.
Q. What are the inhibitors of phosphofructokinase? (Page 77)
A. ATP, Citrate, Glucocorticoids.
Q. What are the activators of phospho fructo kinase? (Page 77, 78)
A. AMP, Fructose-2,6-bisphosphate, Fructose-6-phosphate.
Carbohydrates-II 65 Q. What are key glycolytic enzymes? (Page 77, 78)
A. Glucokinase, Phospho fructo kinase, Pyruvate ki-nase.
Q. What is the action of insulin on glycolysis? (Page 77, 78)
A. Insulin stimulates glycolysis.
Q. What is the net yield of ATP from one glucose molecule during anaerobic glycolysis?
(Page 78 and Table 9.3)
A. 2 ATP.
Q. In aerobic glycolysis, the net yield from one glu-cose molecule is how much?
(Page 78 and Table 9.4)
A. 8 ATP.
Q. During complete oxidation, what is the net yield of ATP from one glucose molecule?
(Page 78 and Table 9.5)
A. 38 ATP.
Q. How many ATPs are generated per one rotation of the citric acid cycle?
A. 12 ATP.
Q. What is the function of 2,3-bisphospho glycerate? (Page 79)
A. When combined with hemoglobin, 2,3-BPG re-duces the affinity towards oxygen.
Q. What are the steps in which carbon dioxide is produced from a glucose molecule? (Page 80)
A. Pyruvate dehydrogenase, isocitrate dehydroge-nase, alpha keto glutarate dehydrogenase.