Biochemistry- Paper II Aug 2010

I. Essay questions ( 2 x 15 = 30 )

1. Describe the separation of serum proteins by paper electrophoresis. Draw the pattern

of electrophoresis in i) multiple myeloma II) nephrotic syndrome

Ans.

The term electrophoresis refers to the movement of charged particles through an

electrolyte when subjected to an electric field. Cations(positively charged ions) move towards

cathode and anions(negative) to anode.

When a biological mixture is subjected to electrophoresis, the compounds in the

mixture move in relation to their net charge, size, molecular weight and mass and gets

separated according to these characteristics, so that the desired compound can be identified

and isolated.

Factors affecting electrophoresis: rate of migration will depend on:

Net charge of the particles (eg. more negative particles move faster than less negative)

Mass and shape of particles(larger sized particles move slowly)

pH of medium(particles move better in a pH in which they are more ionized)

strength of electrical field(eg. Higher the voltage-faster the movement)

properties of supporting medium

temperature-increased temperature solidifies the support and impairs migration)

The electrophoresis apparatus consists if a tank which contains electrodes connected to a

power supply and buffer. The pH of buffer is selected so that it imparts maximum charge to the

electrophoresed substances(eg. Proteins get separated well in a buffer pH of 8.6

Supporting medium is the surface on which electrophoresis is carried out. It may be agar

gel, agarose gel, cellulose acetate, paper, etc.,

After the run the bands are visualized using naked eye or if needed to be quantified a

densitometer can be used.

Paper electrophoresis:

Here the supporting medium is whatmann filter paper. Sample is applied in the negative

electrode region, which will move to the positive electrode. After the run the paper is dried and

visualized

fig. paper electrophoresis apparatus

Clinical applications:

1. serum protein electrophoresis:

- in nephrotic syndrome – globulin is produced more by liver in compensation of renal

loss of albumin. So alpha 2 band is prominent

- cirrhosis- albumin band is less prominent

- multiple myeloma- light chain immunoglobulins are produced more so there will be a

prominence in gamma globulin region(M band)

Fig. serum protein electrophoresis in health and disease

2. hemoglobin electrophoresis

- S band is seen in sickle cell anemia

- various hemoglobinopathies and thalasemias can be diagnosed

2. How is blood pH regulated?

Ans.

Normal blood pH is btween7.38-7.42. it is maintained by:

I. Role of buffers in body fluids:

Buffers resist changes in pH when small quantities of an acid or an alkali are added. Various

buffers in body are:

1. Bicarbonate buffer system

it is the most important buffer in plasma and is formed by (NaHCO3/H2CO3)

the base HCO3- is the metabolic component as it is regulated by kidney and the acid

H2CO3 iss called respiratory component since it is regulated by the lungs.

The normal bicarbonate level in plasma is 24mmol/L. It has a pKa of 6.1-so it is a poor

buffer. But the high blood concentration and the ratio of base to salt is high(20:1), which

makes it a effective buffer.

When acid(H+) is added

o H+ + HCO3- H2CO3 H20 + CO2 excreted by lungs and kidney.

When alkali is (HCO3-) added

o H+ + HCO3- H2CO3 H20 + CO2 excreted by lungs and kidney.

2. phosphate buffer system

It is made of NaHPO4/NaH2PO4. It has a pKa of 6.8.

In acidosis

NaHPO4 + H+ NaH2PO4 - excreted by the kidneys

In alkalosis

NaH2PO4 - NaHPO4 + H+

3. Protein buffer system

The Histidine molecules in albumin acts as a buffer.

In acidosis H+ + Pr- - HPr

In alkalosis HPr H+ + Pr-

II. Kidneys regulate acid base balance by:

1. Excretion of H+ -In PCT cells CO2 combines with water to form carbonic acid using carbonic

anhydrase. Then it becomes H+ and HCO3- . this H+ is then excreted into lumen in exchange for

Na+ .

2. reabsorption of HCO3- - sodium bicarbonate in the lumen becomes sodium and bicarbonate.

Sodium is taken up by PCT cell in exchange of hydrogen ions. H+ combines with HCO3- to form

carbonic acid, which forms CO2 and water and both are reclaimed into the cell and converted

back to carbonic acid and again to H+ and HCO3- . HCO3

- is taken into blood with sodium.

fig Excretion of H+ fig. Reabsorption of HCO3-

3. Excretion of titrable acid- The Na2 HPO4 becomes Na+ and NaHPO4 - . sodium is exchanged

with H+ ions and H+ combines with NaHPO4 – to become Na H2PO4 and gets excreted.

Fig. Excretion of titrable acid and ammonium ions

4. Excretion of NH4 + - Glutamine in DCT becomes glutamate and ammonia. This ammonia is

seceted into the lumen which combines with hydrogen ions to become ammonium ions and

gets excreted.

III. Role of lungs in Acid base balance

When there is fall in pH the respiratory rate is stimulated resulting in hyperventilation.

This would eliminate more CO2 thereby lowering H2CO3 .

In tissues pCO2 is high and pH is low to the formation of acids by the cells like lactate

and production of CO2 by cells. CO2 diffuses into RBC. It combines with water to form carbonic

acid by cabonic anhydrase. And dissociates into H+ and HCO3-. So RBC traps H+ from the tissues.

Some of the HCO3- diffuses out of the cell in exchange for chloride.

In the lungs H+ combines with HCO3- to form H2CO3 which becomes H2O and CO2. This

CO2 is released into the lungs. So lungs reduce the acid load of H2CO3 by excretion of CO2.

In metabolic acidosis lungs hyperventilate to excrete more acid. In Metabolic alkalosis

the reverse happens.

Fig. Reactions in tissues fig.Reaction in lungs

II. Write short notes on: (10 x 5 = 50)

1. Genetic code

Ans.

The letters A, G, T, and C correspond to the nucleotides found in DNA. Within the

protein coding genes these nucleotides are organized into three-letter code words called

codons, and the collection of these codons makes up the genetic code. The code provides a

foundation for explaining the way in which protein defects may cause genetic disease and for

the diagnosis and perhaps the treatment of these disorders. A triplet sequence of nucleotide on

the mRNA is the codon for each aminoacid. There are four different bases they can generate 64

codons.

SALIENT FEATURES:

TRIPLE CODONS: Each codon is consecutive sequence of three bases on the mRNA

e.g. UUU codes for phenyl alanine.

NON OVERLAPPING: The codes are read one after another in a continous manner.

E.g. AUG, GAU, GCA etc.

NONPUNCTUATED: There is no punctuation inbetween codons.

DEGENERATE: 61 codon codes for 20 amino acids so one aminoacid has more than

one codons e.g. serine has 6 codons and glycine has 4 codons this is called

degeneracy of code.

UNAMBIGOUS: Codons are unambiguous that means one codon stands only for one

amino acids.

UNIVERSAL: The codons are same for same aminoacids in all species.

WOBBLING HYPOTHESIS: Reduced stringency between the third base and the

complementary nucleotide in anticodon is wobbling. E.g. GGU, GGC, and GGA codes

for glycine all three pair with anticodon

TERMINATOR CODON: There are three codon do not code for any aminoacids. They

are nonsense codons. The three codons are UAA, UAG, UGA

INITIATOR CODON: AUG acts as initiator codon.

2. Formation of epinephrine

Ans.

Catecholamines:

- tyrosine is first hydroxylated to DOPA by tyrosine hydrolase. It requires tetrahydro

Biopterin.

- DOPA is decarboxylated to form Dopamine by DOPA decarboxylase, a PLP dependent

enzyme

- Dopamine is hydroxylated to Nor epinephrine by dopamine hydrolase

- Nor epinephrine is converted to epinephrine by methyl transferase requiring SAM

Fig. formation of epinephrine

3. Cyt P450

Ans.

One group of cytochrome containing monooxygenases are called cytochrome P450s.

The human genome encodes at least 14 families of these enzymes. Estimates of the number of

distinct cytochrome P450s in human tissues range from approximately 35 to 60. The reaction

catalyzed by a monooxygenase (cytochrome P450) is as follows:

RH + O2 +NADPH +H+ →R-OH +H2O +NADP

Cytochromes P450 are an important superfamily of heme-containing monooxgenases,

and more than 1000 such enzymes are known. Both NADH and NADPH donate reducing

equivalents for the reduction of these cytochromes which in turn are oxidized by substrates in a

series of enzymatic reactions collectively known as the hydroxylase cycle.

In liver microsomes, cytochromes P450 are found together with cytochrome b5 and

have an important role in detoxification. Benzpyrene, aminopyrine, aniline, morphine, and

benzphetamine are hydroxylated, increasing their solubility and aiding their excretion. Many

drugs such as Phenobarbital have the ability to induce the formation of microsomal enzymes

and of cytochromes P450.

The steps of detoxification of compounds is given in the figure.

Fig. Mechanism of detoxification by Cyt P 450

4. Purine salvage pathways

Ans.

- Nucleotides are degraded regularly. Salvage pathway recycles the purines and make

it available for nucleic acid synthesis.

- Adenine is converted to AMP using PRPP by Adenine phosphoribosyl

transferase(APRTase)

- Guanine is converted to GMP using PRPP by Hypoxanthine guanine phosphoribosyl

transferase(HGPRTase)

- The salvage pathway is important for RBC and brain since denovo synthesis of purine

nucleotides are not operative.

- A defect in HGPRTase will lead to Lesch Nyhan syndrome.

Lesch’s Nyhan syndrome

it is a X-linked inborn error of purine metabolism, incidence 1:10,000

deficiency of HGPRTase which acts in salvage pathway

so the salvage pathway is stopped and PRPP accumulates which will go for catabolism to

uric acid

hyperuricemia leads to nephrolithiasis and gout

it is also characterized by self mutilation, mental retardation

5. Dehydration

Ans.

Dehydration is a condition characterized by water depletion in the body. It may be due

to insufficient intake or excessive loss of water.

Causes of dehydration:

It may be due to diarrhea, vomiting, excessive sweating, fluid loss in burns,

adrenocortical dysfunction, kidney diseases, Diabetes Insipidus, etc.,

Features:

The volume of ECF is decreased with rise in electrolyte concentration and osmotic

pressure.

Water moves from ICF to ECF, resulting in cell shrinkage and increased protein

breakdown

ADH secretion increased leading to water retention and low urine volume.

Plasma proteins and blood urea concentrations are increased.

Water depletion may be either depletion of water alone or along with electrolytes.

The clinical features are increased pulse rate, low blood pressure, sunken eyeballs,

decreased skin turgor, lethargy, confusion and coma.

Treatment:

Plenty of water should be taken. Intravenous administration of 5%glucose solution

should be given, if the patient cannot drink orally. If electrolytes are also lost, sufficient

electrolytes must also be given. The cause for dehydration must be identified and treated.

6. Lac operon

Ans.

Operon is a unit of gene expression it includes structural genes, control elements,

regulator/inhibitor gene, promoter and operating areas.

In the bacterial cell the Z gene encodesbeta-galactosidasethe enzyme which hydrolase

lactose to galactose and glucose. The Y gene is responsible for the production of permeases

which transport galactose and lactose into the cell. The A gene codes for the thio

galactoside transacetylase.

The transcription of these gene starts from common promoter (p), located close to Z gene.

The RNA polymers bind to the promoter and transcribe the three structural genes as single

mRNA.

Lacoperon explains the concept of induction, repression, derepression, positive regulator.

When E coli is presented with lactose or some specific lactose analogs the expression of

the activities of β-galactosidase, galactoside permease, and thiogalactoside transacetylase is

increased 100-fold to 1000-fold.

When E coli is exposed to both lactose and glucose as sources of carbon, the organisms

first metabolize the glucose and then temporarily stop growing until the genes of the lac

operon become induced to provide the ability to metabolize lactose as a usable energy source.

Although lactose is present from the beginning of the bacterial growth phase, the cell does not

induce those enzymes necessary for catabolism of lactose until the glucose has been exhausted.

This is due to repression of the lac operon by catabolite of glucose on catabolite gene activator

protein (CAP) in conjunction with cAMP

Constitutive expression: Expression of the normal lacI gene of the lac operon is constitutive; it

is expressed at a constant rate, resulting in formation of the subunits of the lac repressor.

Negative regulator The LacI repressor protein molecule, the product of lacI, has a high affinity

for the operator locus. When attached to the operator locus, the LacI repressor molecule

prevents transcription of the operator locus as well as of the distal structural genes, lacZ, lacY,

and lacA. Thus, the LacI repressor molecule is a negative regulator;

Derepression: A lactose analog that is capable of inducing the lac operon while not itself

serving as a substrate for β-galactosidase is an example of a gratuitous inducer. An example is

isopropylthiogalactoside (IPTG). Binding of the inducer to a repressor molecule attached to the

operator locus induces a conformational change in the structure of the repressor and causes it

to dissociate from the DNA. Thus an inducer derepresses the lac operon

Positive regulator: In the presence of glucose—or of glycerol in concentrations sufficient for

growth—the bacteria will lack sufficient cAMP to bind to CAP because the glucose inhibits

adenylyl cyclase, the enzyme that converts ATP to cAMP. Thus, in the presence of glucose or

glycerol, cAMP-saturated CAP is lacking, so that the DNA-dependent RNA polymerase cannot

initiate transcription of the lac operon. Thus, the CAP-cAMP regulator is acting as a positive

regulator because its presence is required for gene expression.

Constitutive expression: When the lacI gene has been mutated so that its product, LacI, is not

capable of binding to operator DNA, the organism will exhibit constitutive expression of the lac

operon

7. Orotic aciduria

Ans.

- The condition results from absence of either or both of the enzymes ORPTase and

OMP decarboxylase

Fig. Orotate metabolism

- It is an autosomal recessive disease

- Growth is retarded and megaloblastic anemia present. Bone marrow cells are fastly

dividing cells which are affected, leading to anemia.

- Orotate crystals are excreted in urine which may cause urinary tract obstruction.

- Treatment is by feeding cytidine or uridine. They are converted into UTP which can

act as feedback inhibitor.

- It also occurs in urea cycle defect like ornithine transcarbamylase deficiency, since

carbamoyl phosphate accumulates and gets diverted to pyrimidine pathway.

8. t-RNA

Ans.

tRNA is a type of RNA. It shows extensive internal base pairing and clover leaf like structure. It

contains unusual bases. They are dihydro uracil, pseudo uridine, hypoxanthine are methylated.

Acceptor arm is at 3´ end: It carries the aminoacids. It has seven base pairs. The end

sequence is CCA-3´. The 3’ end hydroxyl group is bonded with carboxyl end of amino acids.

Anticodon Arm of tRNA: Acceptor arm recognizes the triplet nucleotide codon present in

mRNA.

DHU Arm of tRNA: DHU arm serves as the recognition site for enzymes.

Pseudouridine arm of tRNA: It is involved in binding tRNA to ribosomes.

Fig. Structure of tRNA

Function:

The tRNA molecules serve as adapters for the translation of the information in the

sequence of nucleotides of the mRNA into specific amino acids. There are at least 20 species of

tRNA molecules in every cell, at least one (and often several) corresponding to each of the 20

amino acids required for protein synthesis. The process of recognition and attachment

(charging) proceeds in two steps by one enzyme for each of the 20 amino acids. These enzymes

are termed aminoacyltRNA synthetases.

9. Phenylketonuria

Ans.

Phenyl ketonuria

-it is an autosomal recessive disease with an incidence of 1:1500 births. It is due to

deficiency of phenylalanine hydroxylase. So phenylalanine is not converted into tyrosine and it

accumulates.

- the excess of phenylalanine is converted to phenyl pyruvate, phenyl lactate, and

phenyl acetate and phenyl acetyl glutamine. Phenyl pyruvate, phenyl lactate, phenyl acetate

are excreted in urine.

- the child is mentally retarded and convulsions, tremors agitation, hyperactivity may

present. The child often has hypo pigmentation due to reduced availability of tyr for melanin

production.

- phenyl lactate causes mousy odor of urine.

- blood levels of phenyl alanine are elevated, Guthrie’s test is confirmative. Urine FeCl3

test is positive.

- tapioca based diet which have less phe is the treatment of choice. Gene therapy is

under trial.

10. Water toxicity

Ans.

Overhydration or water intoxication is caused by excessive water retention in the body. The

causes are:

Excessive intake of large volumes of salt free fluids

Renal failure

Psychogenic polydipsia

True polydipsia

o In ADH secreting tumors

o SIADH

Excessive intravenous administration in cases of trauma and post surgery.

Clinical features:

There will be dilution of ECF and ICF with decrease in osmolality. Headache, lethargy

and convulsions may occur. To differentiate pschoenic from true polydipsia, water deprivation

test may be used. Treatment is to stop water intake, diuretics and administration of hypertonic

saline.

III. Short answer questions ( 10 x 2 = 20 )

1. Xeroderma pigmentosum

Ans.

Xeroderma pigmentosum: it is an autosomal recessive condition. There is defect in Nucleotide

excision repair mechanism. The patient is highly sensitive to UV rays. Sunlight causes blisters in

the skin. Death occurs in second decade due to skin cancer.

2. Hbs

Ans.

In the 6th position of B chain of hemoglobin, when glutamate is replaced by Valine, HbS

results. The hemoglobin can carry oxygen, but when deoxygenated, the Hb sticks with other Hb

molecules leading to the formation of sickle shaped plugs which can obstruct vasculature. This

also leads to reduced life span of RBC leading to hemolytic anemia.

Types:

- Sickle cell disease- homozygous-severe manifestations

- Sickle cell trait- heterozygous- symptoms only in precipitating conditions.

Diagnosis:

Hemoglobin electrophoresis reveals S band which is diagnostic.

3. Functions of PTH

Ans.

PTH is secreted by four parathyroid glands in the thyroid tissue. Decreased serum calcium

leads to release of PTH from parathyroids. PTH activates adenylyl cyclase in target cells and

increases intracellular calcium concentration. A protein kinase is activated which activates

enzyme systems. PTH acts on

1. PTH and bones- PTH causes demineralization in bones. It activates pyrophosphatase in

osteoclasts leading to bone resorption and solubilising calcium. Calcium is released into

the blood stream and increases blood calcium level. This leads to loss of bone matrix.

2. PTH and kidneys: PTH causes decreased renal excretion of calcium and increased

excretion of phosphates and increased reabsorption of calcium leading to increased

blood calcium level.

3. PTH and intestines: PTH stimulates increased production of VIT D3 which acts on

intestine to absorb more calcium leading to increased calcium level in blood.

4. Mention two second messenger

Ans.

calcium and cAMP

5. Symport

Ans.

Symport is a type of active transport requiring energy. Here simultaneously two

different compounds are moved across the membrane in same direction. Eg. Sodium glucose

transporter(SGLT). One molecule of ATP is utilized for this reaction. The substances are moved

against concentration gradient. The opposite of symport is antiport.

6. Oxytocin

Ans.

Oxytocin is an hormone synthesized by posterior pituitary. It is an peptide molecule.

The stimulant for oxytocin secretion from the pituitary is suckling of the nipple of the

mother by newborn baby.

Oxytocin causes the pregnant uterus to contract and induce labor. It causes contraction

of myoepithelial cells of the breast leading to ejection of milk from breast. Oxytocin inhibits

synthesis of steroids by the ovary.

7. Addison’s disease

Ans.

Reduced production of cortisol from the adrenals leads to Addison’s disease. It may be

primary due to adrenal dysfunction or secondary due to pituitary or hypothalamic dysfunction.

There will be hypoglycemia, loss of weight, anorexia, muscle wasting, impaired cardiac

function, low blood pressure, decreased Na+ and increased K+ level in serum. Patients are more

prone for stress.

8. Functions of glucagon

Ans.

Glucagon increases blood glucose by following mechanisms:

- Inhibiting glycolysis- inhibits glucokinase, phosphofructokinase, pyruvate kinase

- Stimulating gluconeogenesis- by stimulating PEPCK, pyruvate carboxylase, Fructose

1,6 bisphosphatase, Glucose 6 phosphatase

- Stimulates glycogenolysis-by activating phosphorylase

- Inhibits glycogenesis- by inactivating glycogen synthase

9. GABA

Ans.

Glutamate on decarboxylation produces GABA(gamma amino butyric acid). It is

produced in brain. GABA is an inhibitory neurotransmitter because it opens chloride channels in

post synaptic membranes in CNS.

Both formation and catabolism of GABA requires PLP. So in Pyridoxine deficiency GABA

will be deficient leading to convulsions. Sodium valproate inhibits GABA oxidase and is used for

epilepsy treatment.

10. Hartnup’s disease

Ans.

- It is an autosomal recessive disease.

- Absorption of aromatic aminoacids by the intestine and the renal tubules are

defective. So these aminoacids are excreted in large amounts in urine.

- Pellagra like symptoms appear due to deficiency of Niacin, which is produced from

Tryptophan

- Obermeyer test will be positive.

- Supplementation of Niacin and high protein diet improves the symptoms.