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Biotin (vitamin B7) : Biological functions , clinical indications and
its technological applications
Dr. Rohini C Sane
Historical background of Biotin
• Biotin = vitamin B7 = vitamin H = anti-egg white injury factor(intake of raw
→not boiled egg may cause Biotin deficiency).
• Boas(1927) fed rats with huge quantity of raw eggs → rats developed
dermatitis, nervous manifestations and retardation in growth.
• Vincent du Vigneaud (Noble prize 1955): isolated Biotin.
Structure of Biotin (Vitamin B7 / Vitamin H)
❑Structure of Biotin (Vitamin H) :
❖Sulphur-containing water soluble
heterocyclic monocarboxylic acid
(Vitamin B7) .
❖Imidazole ring fused with
tetrahydrothiophene ring with a
Valeric acid side chain.
• The carboxyl group of biotin forms an
amide linkage with the epsilon(ε)
nitrogen of Lysine residue in the
apoenzyme forming a biotinyl
enzyme.
• Biocytin : a coenzyme form (Active
form)→ (Biotin is covalently bound to
ε- amino group of Lysine (Lysine -
linked by amide bond)in the enzyme.
O
II
C
N H
C H
CH (CH2)4 COOH
H N
H C
H2C
S
ǀ
ǀ
ǀ
ǀ
ǀ
ǀ
CO2 Binding site
Site for binding with Lysine
Biotin = cis-tetrahydro-2-oxo-
1-thienol-(3,4-d)-imidazoline -
4-valeric acid .
Imidazole ring
Tetrahydrothiophene ring
1
2
3
4
5
1
2
3
C10H16 O3N2 S
Molecular weight : 244
Chemical properties of Biotin
1. Existsintwoformswithessentiallyidenticalbiologicalactivities:α-Biotin(egg-yolk)and
β-Biotin(Liver)differingonlyinthenatureofsidechain.
2. Solubility:solubleinwaterandethylalcohol,butinsolubleinetherandchloroform.
3. Stability:heatstable.
4. Destroyedbyoxidizingagentssuchasperoxidesandpermanganates(convertthe
thioethertosulfoxidesandsulfoneswhichdonothavebiotinactivity)andacids/alkalis .
5. Crystallization:whitecrystallinesolidlongneedles,solubleinanaqueoussolution
(pH>7,alkaline)duetoureidoringandionizablecarboxylgroup.
6. Occurrenceinfoodandtissue:freeformandboundforms(Biocytin,oxy-biotinand
Desthiobiotin). ItislinkednoncovalentlyasacomplexwithAvidin(aproteininegg
white).
7. Biocytin(ε-N-Biotinyl-Lysine):BiotinremainboundwithLysineresiduesoftissue
proteinsbyamidebonds.Biocytinisreleasedonhydrolyzingthepeptidebondsbetween
Biotin-boundLysineandpeptidechain.
8. Oxy-biotinandDesthiobiotin:arebiologicallyactiveincertainstrainsofyeastand
bacteria.
Sources of Biotin in human
❖Human beings cannot synthesize Biotin and hence Biotin has to be supplied in diet.
❖Sources of Biotin in human : synthesis by intestinal bacterial flora and dietary
sources .
❖Normal bacterial flora of the intestine provides adequate quantities of
Biotin(biosynthesis of Biotin by E.coli). Moreover, it is distributed ubiquitously(widely)
in animal tissue, fruits and vegetables.
❖Rich food sources of Biotin:
• Milk, cheese , yogurt , yeast , cauliflower , berries , soyabean , tomatoes , sweet
potatoes , spinach , Avocado , lentils , banana , carrots , white mushrooms , Molasses,
wheat germs , oats , sunflower seeds , almonds, walnuts , hazelnuts, peanuts and
grains. (water soluble form in most plants material except cereals and nuts).
• Egg yolk , liver, kidney , pancreas, Sardine , Yellow Tuna, pork , beef , Turkey , chicken ,
lamb and Calf silver. (water insoluble form in animal tissues).
Biotin rich food sources
Co-enzyme-R(Biotin)from Rhizobium
• Co-enzyme R is growth essential for
Rhizobium (nitrogen-fixing
organisms) in the root nodules of
Leguminous plant .
• Co-enzyme R is proved to be Biotin.
• Pimelic acid is possible precursor.
• Desthiobiotin is a probable
intermediate.
Daily dietary requirement of Biotin( RDA)
• Daily dietary requirement of Biotin( RDA) = 200-300 μg
• Estimated Average requirement : Adequate intake(AI)based on urinary excretion of Biotin
and the metabolite 3-hydroxy isovaleric acid.
• Tolerable upper intake level (UL)→ applies to chronic daily use of fortified foods/dietary
supplements → no value established for Biotin .
➢Daily dietary requirement of Biotin(RDA) increases in pregnancy and lactation
(additional 5 μg /kg of body weight) .
➢Intravenous supply of Biotin for adults during TPN = 60 μg /day.
➢Patients receiving hemodialysis or peritoneal dialysis or with biotinidase deficiency require
more .
Age in years Adequate intake(AI)of Biotin (μg/day)
≥ 19 30
14-18(aldolescents) 25
9-13 20
4-8 12
1-3 8
<1 year(Infants) 0.7 μg /kg of body weight
Metabolism of Biotin in the human body
• Sources of Biotin in human : dietary sources(largely protein bound)and free form .It is
biosynthesized biotin by bacterial flora.
• Occurrence /existence in dietary food source : widely distributed in many food sources as
Biocytin (ε- amino-biotinyl lysine)→ Biotin is released after proteolysis.
• Biosynthesis : by intestinal bacteria (human body cannot synthesize Biotin).
• Digestion: proteolysis by gastrointestinal enzyme to produce biotinylate peptides →release of free
Biotin further hydrolysis by intestinal biotinidase.
• Absorption : readily absorbed by intestinal epithelial cells using a biotin carrier (the sodium
dependent multi-vitamin transporter →SMVT). Avidin (present in raw egg white)prevents its
absorption.
• Transport : by circulating blood
• Secretion : in milk
• Uptake of absorbed Biotin : by liver , kidney and muscles (localized in cytosolic and mitochondrial
carboxylases).
• Storage :in Liver and kidneys (limited extent→ 14% of administered dose). Total body content of
Biotin = 1mg
• Excretion : Urinary excretion(10-180 μg/day) > dietary intake (28-100 μg/day) .
Fecal excretion (15-200μg/day) > 3-6 times of dietary intake . Fecal excretion represents unabsorbed
Biotin synthesized by intestinal bacteria.
Absorption and transport of Biotin
Biotin containing food/enzymes
Covalently linked to proteins
Proteolytic enzymes
Biocytin
Pancreatic Biotinidase
Biotin + Lysine
sodium
dependent
multi-vitamin
transporter
→SMVT)
Intestinal Brush border
Biotin
GPR109A
Hydroxycarboxylic acid
(HCA-2) receptor mutation causes biotin –
responsive basal ganglia
disease(encephalopathy )
SLC19A3 Low concentration of
Biotin : active
transport
High concentration
of Biotin : Passive
transport
Basal lateral
membrane
The sodium-dependent multivitamin transporter (SMVT) to facilitate intestinal
absorption of Biotin
• Is located in the intestinal brush border membrane.
• Na+ dependent carrier-mediated process .
• Transports biotin against sodium ion concentration gradient.
• Not specific for vitamin (Biotin) transport.
• Functions in cellular uptake of Biotin ,Pantothenic acid and Lipoic acid with
similar affinities .
• Biotin uptake by intestinal epithelial cells is inhibited by activation of protein
kinase C apparently through phosphorylation of SMVT.
Facilitated transport of Biotin during intestinal absorption
Intestinal Absorption of Biotin at its Low
concentration
Intestinal Absorption of Biotin at its High
concentration
Saturable ,Active and facilitated mechanism
dependent on Na+ .
Passive transport
Facilitated transport inhibited by certain anti-
convulsant drugs and chronic exposure to
ethanol.
Catabolism of Biotin
• The enzyme biocytinase (biotin amidohydrolase) in plasma and erythrocytes
catalyze the hydrolysis biocytin to yield free biotin.
• Free biotin is taken up by tissues (such as liver, muscle and kidney) and
localized in cytosolic and mitochondrial carboxylase.
• Small fraction of Biotin is oxidized to D- and L- sulfoxides ( ureido ring intact
not otherwise degraded).
• Side chain of larger portion of Biotin is degraded via mitochondrial β-oxidation
to yield bis-nor biotin and its degradation products.
• Biotin catabolism in smokers > Biotin catabolism in non-smokers.
Excretion of Biotin
• Source for excretion of Biotin : Half of the absorbed biotin.
• Mean urinary excretion is a reflective of dietary intake (28-100 μg/day for
adult).
• Metabolite Forms for excretion of Biotin:
1. Bis-norbiotin (occurring from β-oxidation of the Valeric acid side chain).
2. Biotin sulfoxide (occurring from oxidation of the sulfur in the heterocyclic
ring).
• Ratio of Biotin : Bis-norbiotin : Biotin sulfoxide = 3:2:1(in circulating plasma
and urine).
• Minor metabolites of biotin for excretion : Bis-norbiotin methyl ketone and
Biotin sulfone.
Laboratory Assessment of Biotin status
❖Bioassay methods
❖Microbiological assay : growth stimulation of yeast cells (Saccharomyces cerevisiae or
Lactobacillus plantarum) is measured . Whole Blood is first digested with papain or acid
hydrolysis to release free biotin . This sample is then added to a Biotin-deficient medium
inoculated with a test organism , such as Lactobacillus plantarum .
❖Measurement of unfound Biotin include Avidin-binding assays : a competitive protein
binding radio-assay with 3H-labelled Biotin or a nonradioactive enzyme-linked sorbent
using Streptavidin as a binding agent .
❖ Measurement of Urinary Biotin and 3-hydroisovaleric acid : by HPLC
Urinary excretion of Biotin and 3-hydroisovaleric acid appear to be better indicator of
biotin status than whole blood concentrations.
❖Urinary Biotin and 3-hydroisovaleric acid : gas chromatography-mass spectrometry.
❖Lymphocyte Propionyl-CoA carboxylase using H14 CO3 - : early indicator and sensitive
indicator of biotin deficiency in patients on prolonged TPN without biotin supplementation
and in children with protein-energy malnutrition.
Reference intervals of Biotin
• Whole blood Biotin levels (physiological) by microbiological method :
200 - 500 pg /ml (0.5- 2.2 nmol/ L with mean 1.31nmol/L)
• Biotin deficiency : Whole blood biotin < 0.5 nmols/L
• Lowered circulating blood levels and urinary excretion are observed in
1. alcoholics
2. Patients with achlorhydria
3. Elderly
4. Athletes
• Biotin content of red cells is similar to that of plasma for a given method.
Co-enzyme and non-coenzyme roles of Biotin
Coenzyme role of Biotin Non-coenzyme role of Biotin
Pyruvate carboxylase Cell proliferation
Acetyl-CoA carboxylase Gene silencing
Propionyl-CoA carboxylase DNA repair
β-methyl crotonyl-CoA Carboxylase Gene expression and cell signaling
Biotin is a coenzyme of carboxylase
reactions . Biotin is a carrier of activated
carbon dioxide (CO2) for the mechanism
of Biotin-dependent carboxylations .
Biotin is the prosthetic of certain enzymes (carboxylases and decarboxylases) that catalyze
CO2 transfer reaction (CO2 fixation reaction/ carboxylation) in human tissue.
Biotin-dependent carboxylases
Biotin
VitaminB7
Vitamin H
Pyruvate Carboxylase
(key enzyme of
Gluconeogenesis, TCA and
Transamination)
Acetyl-CoA Carboxylase
(First committed step in
biosynthesis and
elongation of fatty acids)
Propionyl-CoA
Carboxylase (oxidation of odd chain
fatty acids and synthesis of Succinyl
CoA)
β-methyl crotonyl-CoA
Carboxylase(catabolism of
Leucine)
Biotin is covalently
bound to the ε- amino
groups of Lysine
residues in Biotin –
dependent enzymes.
Functions of Biotin as a prosthetic group of ATP-dependent carboxylases
Enzyme Substrate Product Importance enzyme
Pyruvate Carboxylase Pyruvate Oxaloacetate Gluconeogenesis(synthesisof
Glucosefromnon-carbohydrate
substance),providesoxaloacetate
forTCACycle,Transamination
Acetyl-CoA
Carboxylase
Acetyl-CoA Malonyl-CoA Limiting reaction in Fatty Acid
biosynthesis
Propionyl-CoA
Carboxylase
Propionyl-
CoA
D-Methyl Malonyl-CoA
→Succinyl-CoA
Succinyl-CoA→HemeSynthesis,
Succinyl-CoAoxidizedInTCAcycle
β-methyl crotonyl-
CoA Carboxylase
β-methyl
Crotonyl-CoA
β-Methyl glutaconyl-
CoA
Leucine metabolism (Branched
ChainAminoAcids)
Biotinfunctionsasaprostheticgroupforcarboxylasesandisattachedtotheenzymebyanamidebond
betweenthecarboxylgroupofBiotinandtheterminalε-aminogroupofLysineresidueoftheenzyme,
formingaBiotin-enzyme.
Mechanism of Biotin during carboxylation reactions in the human body
• The peptide biocytin (ε-N-biotinyl lysine) is resistant to hydrolysis by proteolytic
enzymes in intestinal tract but together with biotin is readily absorbed .
• A biotin carrier , sodium-dependent multivitamin transporter (SMVT) for which
Pantothenic acid and lipoate compete.
• (SMVT) is located in brush borders membrane and transports biotin against a
sodium ion concentration gradient.
• The enzyme biocytinase (Biotin amidohydrolase) is located in plasma and
erythrocytes catalyzes hydrolysis of biocytin to yield free biotin.
• Covalent attachment of Biotin to apoenzyme involves ATP-dependent conversion of
the vitamin to biotinyl-5’-adenylate followed by condensation of the biotinyl moiety
with ε-amino groups of specific Lysyl residues in apoenzyme preformed from
subunits.
• Enzyme responsible for formation of ε- N-biotinyl-l-Lysyl ( biocytinyl) moiety of
proteins is holocarboxylase synthetases (HCS).
Biotin recycling
Holocarboxylase synthetase (HCS) uses ATP to catalyze the covalent bonding of different
apocarboxylases with Biotin to form different biotin-carboxylase complexes called
holocarboxylase . In holocarboxylase –amide bond binds the carboxyl terminal of valeric acid
side chain of Biotin with ε–NH2 group at the end of the side chain of lysine residue of
apocarboxylases.
Mechanism of carboxylation reactions facilitated by Biotin
• In biological system, Biotin functions as the co-enzyme for the enzyme called
carboxylase which catalyze the carbon fixation(carboxylation).
• These enzymes operate via a common mechanism ,which involves
phosphorylation of bicarbonate by ATP to form carbonyl phosphate , followed
by transfer of the carbonyl group to the sterically less hindered nitrogen of the
biotin moiety.
• In this process , Biotin is first gets converted to carboxy-biotin complex by
reaction with ATP and HCO3
- .
• The resulting N(1)-carboxybiotinyl enzyme can then exchange the carboxylate
function with a reactive center in a substrate i.e. CO2 -Biotin complex is the
source of active CO2 which is transferred to the substrate .
• CO2 becomes attached to the biotin coenzyme as above.
Mechanism of carboxylation reactions facilitated by Biotin
Biotin
enzyme
Carbonic
phosphoric
anhydride
Carboxy-
biotin-
enzyme
CO2
ATP
ADP
Pi
Carboxylated substrate
substrate
Biotin acts as
co-enzyme for
carboxylation
reactions. It
captures a
molecule of
CO2 which is
attached to
nitrogen of the
Biotin
molecule.
The energy required for this reaction is provided by ATP . Then the activated carboxyl group is
transferred to the substrate.
Substrate + CO2 + ATP → Product
(Carboxylated substrate) + ADP+ Pi
Biochemical functions of Biotin in carboxylation of Pyruvate to Oxaloacetate
❖ Carboxylation of Pyruvate to Oxaloacetate :
Substrate : Pyruvate
Enzyme : Pyruvate carboxylase
Coenzyme (Carrier of CO2) : Biotin
Energy source : ATP
Product : Oxaloacetate
Mechanism of reaction : Pyruvate carboxylase has Biotin which is bound to the
apoenzyme linked to the ε-amino group of Lysine , forming the active enzyme
(holoenzyme).
Biotin-enzyme reacts with CO2 in presence of ATP to form a carboxy-biotin-
enzyme complex (high energy complex). This high energy complex then hands
over the CO2 to Pyruvate (carboxylation reaction) to produce Oxaloacetate ..
Role of Biotin in conversion of Pyruvate to Oxaloacetic acid by
Pyruvate carboxylase
COOH
CH2
CO
COOH
Pyruvic acid Oxaloacetic acid
CH3
CO
COOH
Pyruvate carboxylase
CO2
Biotin
ATP ADP + Pi
I
I
I
I
I
Acetyl-CoA
Mn2+
❖ Carboxylation of Pyruvate to Oxaloacetate by Pyruvate carboxylase is Biotin-dependent
reaction. Hydrolysis of ATP drives the formation of enzyme-biotin-CO2 intermediate (high
energy complex). This complex subsequently carboxylates Pyruvate to OAA.
❖ Mitochondrial Pyruvate carboxylase(liver and kidney) catalyzes formation of Oxaloacetate,
which together with Acetyl-CoA forms Citrate .
Two important aspects of this reaction:
1. Provides oxaloacetate , that replenishes TCA cycle intermediates that may be depleted,
depending on the synthetic needs of the cell.
2. Is important enzyme in the gluconeogenesis pathway .
Mechanism of carboxylation reaction catalyzed by Biotinylated enzyme
(mitochondrial Pyruvate carboxylase) in formation of Oxaloacetate
Biotin-enzyme + CO2
Biotin-enzyme
ATP
ADP +Pi
S
Lys
NH
(CH 2)4-CO
N NH
H H
O
Enzyme
S
O
O- C-
II
II
- ←Caroxy-biotin-enzyme complex
I
H
O
CH3- C- COO
II -
←Pyruvate
←Oxaloacetate
O
OOC-CH2- C- COO
- II
Pyruvate carboxylase with
covalently attached Biotin
-
Protein portion of enzyme : Acetyl-CoA
carboxylase , Propionyl-CoA carboxylase, Pyruvate
carboxylase, Methyl crotonyl-CoA carboxylase to
catalyze carboxylation of substrate into
corresponding carboxylated product.
Biotin covalently
bound to Lysyl
residue
of a biotin-
dependent enzyme .
Glucose
Glucose -6-phosphate
Glyceraldehyde-3-phosphate
1,3 –Bi phosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Oxaloacetate Pyruvate Acetyl-CoA
Oxaloacetate
Malate Malate Citrate
Fumarate α-Ketoglutarate
Succinyl-CoA
TCA cycle
Glucogenic amino acids
Lactate
Glucogenic amino acids
Glucogenic amino acids
Propionyl CoA
Glucogenic amino acids
Pyruvate carboxylase
ATP , Mg 2+ , Biotin
ATP
ADP + Pi
Glucokinase /Hexokinase
Pi
H2O
Glucose-6-phosphatase
Pi
H2O
Fructose1,6-
biphosphatase
Fructose -6-phosphate
Fructose -1,6-phosphate
GDP +Pi + CO2
GTP
Phosphoenolpyruvate Carboxykinase
ATP
ADP+Pi
Phosphofructokinase
Pathway of Gluconeogenesis(red)
and Glycolysis(blue)
Mitochondrion
Importance of Carboxylation of Pyruvate to Oxaloacetate by Biotin-dependent
Pyruvate carboxylase in Gluconeogenesis and TCA cycle
❖Importance of Carboxylation of
Pyruvate to Oxaloacetate by Biotin-
dependent Pyruvate carboxylase :
• Activated by Acetyl-CoA.
• ATP-dependent.
• Biotin-dependent reaction.
• Replenishes Oxaloacetate which is
an intermediate of TCA cycle
(ensures continuous operation of
Citric acid cycle) in liver and kidney .
• Provides non-carbohydrate
substrates for Gluconeogenesisinliver
andkidneycells .
• An irreversible reaction.
• Pyruvate carboxylase from muscle
cells use OAA produced for
replenishingTCAanddonotsynthesize
glucose.
Pyruvate carboxylase
Biotin
ATP
ADP + Pi
CO2
Acetyl-CoA
+
NADH + H+
NAD
Mitochondrial matrix
Cytosol
Malate
NAD +
NADH + H+
Oxaloacetate Phosphoenol Pyruvate
CO2
Glucose
Gluconeogenesis
*PEPcarboxykinase
+GTP
*
Malate
Oxaloacetate
Malate
dehydrogenase
Pyruvate
Importance of Biotin- dependent carboxylation of Acetyl-CoA to Malonyl-CoA
❖Carboxylation of Acetyl-CoA to Malonyl-CoA(cytosolic reaction) is
• initial (first) and the rate limiting reaction in fatty acid biosynthesis.
• irreversible reaction catalyzed by an enzyme complex , Acetyl-CoA carboxylase, that
requires Biotin as a prosthetic group and utilizes bicarbonate (as a source of CO2) in
presence of ATP.
Acetyl-CoA+ CO2 + ATP Malonyl-CoA +ADP+ Pi
❖Biotin dependent Acetyl-CoA carboxylase is:
▪ an allosteric enzyme activated by Citrate. It is a storage vehicle for biotin.
▪ Inhibited by its end product Palmitoyl-CoA.
➢In addition to high allosteric control , high carbohydrate and low fat diet stimulates
the synthesis of enzyme.
❖Malonyl-CoA is a substrate for fatty acid synthase complex. Fatty acid synthase
subsequently adds 2-carbon units from Malonyl-CoA to growing fatty acid acyl
chain to form Palmitate.
Acetyl-CoA carboxylase
Biotin
Role of Biotin formation of Malonyl-CoA by carboxylation of Acetyl-CoA
O
CH3-C-SCoA
Acetyl-CoA
O
-OOC-CH2-C-SCoA
Malonyl-CoA
Acetyl-CoA carboxylase
CO2
ATP
ADP + Pi
Biotin
II
II
Malonyl-CoA is used for
fatty acid biosynthesis .
Biotin-dependent Acetyl-
CoA carboxylase is
regulatory enzyme in fatty
acid synthesis.
1 Acetyl-CoA
8 Acetyl-CoA Palmitic acid
7 Acetyl-CoA 7 Malonyl-CoA
Site de novo synthesis of fatty
acids : Cytoplasm of liver,
adipose tissue , kidney , brain
and mammary glands
Acetyl-CoA is the starting
material for the biosynthesis
of fatty acids.
The energy for the carbon –
carbon condensations in
fatty acid synthesis is
supplied by the process of
carboxylation and then
decarboxylation of acetyl
groups in cytosol.
Role of Malonyl-CoA in biosynthesis of Palmitic acid
Acetyl-CoA Malonyl-CoA
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
CH3- CH2- CH2- CH2- CH2- CH2- CH2- CH2-CH2- CH2-CH2-CH2-CH2- CH2- CH2- COOH
Cycle I II III IV V VI VII
Carbon atoms 15 and 16 are from Acetyl-CoA and Carbon 1-14 from Malonyl-CoA .
8 Acetyl-CoA are required to make one molecule of Palmitic acid . But in the reaction
mechanism which is explained above only one Acetyl-CoA takes part and other 7 Acetyl-CoA
take part after they are converted into 7 Malonyl-CoA.
Palmitic acid
Role of Biotin in fatty acid biosynthesis
The cytosolic pathway (extra-mitochondrial pathway, De Novo synthesis of fatty acids) is a
major pathway for synthesis of fatty acids. Synthesis of fatty acid from Acetyl-CoA takes place
outside mitochondria . Acetyl-CoA forms Citrate which comes out of the mitochondria can
cleave to give Acetyl-CoA (as such cannot come out of the mitochondria).
Tissue involved in cytosolic fatty
acid biosynthesis: Liver, adipose
tissue, mammary gland ,brain,
kidney
Role of Biotin in fatty acid biosynthesis
Other carboxylases are involved in the
metabolism of odd chain fatty acids and
branched-chain fatty acids.
Carboxylation of Acetyl-CoA to Malonyl-CoA by Biotin-dependent Acetyl-CoA
carboxylase
Energy for the carbon-to-carbon condensation in fatty acid synthesis is supplied by process of
carboxylation and then decarboxylation of acetyl groups in cytosol . The carboxylation of
Acetyl-CoA to Malonyl-CoA is catalyzed by Acetyl-CoA carboxylase which needs CO2 and ATP.
The coenzyme is the vitamin Biotin which is covalently bound to Lysyl residue of carboxylase.
Acetyl-CoA carboxylase (inactive dimer)
Acetyl-CoA carboxylase
(active polymer)
Acetyl-CoA Malonyl-CoA
CO 2
ATP ADP +Pi
Biotin
Allosteric regulation of
Malonyl CoA synthesis by
Acetyl CoA carboxylase. The
carboxyl group is contributed
by dissolved CO 2.
O
CH3-C-S-CoA
O
C-CH2-C-S-CoA
O
O
II II
-
+ H +
2 carbon
compound
3 carbon
compound
→ →
Citrate
+
Long-chain fatty acyl-CoA -
Role of Biotin in Propionyl-CoA metabolism
Propionyl-CoA D-Methyl malonyl-CoA
L-Methyl malonyl-CoA
Biotin
Methyl malonyl-CoA
racemase
Methyl malonyl-CoA
mutase
Deoxy adenosyl cobalamin( vitamin B12)
ATP ADP+ Pi
Heme synthesis TCA
Produced in
metabolism of
Valine ,
Isoleucine,
Threonine
Synthesis of D-Methyl malonyl-CoA : Propionyl CoA is carboxylated forming D-Methyl malonyl-
CoA. The enzyme Propionyl-CoA carboxylase has an absolute requirement for the coenzyme Biotin.
The D-form is isomerized to L-Methyl malonyl-CoA by enzyme Methyl malonyl-CoA racemase.
CO2
Propionyl-CoA carboxylase
Succinyl-CoA
Gluconeogenesis (only example of glucogenic
precursor from fatty acid oxidation)
β-oxidation of odd chain fatty acids
Mn2+
Gluconeogenesis (only
example of glucogenic
precursor from fatty
acid oxidation)
Metabolism of Propionyl-CoA to Succinyl-CoA
β-Oxidation
odd chain fatty acid
CH3
CH2
CO-S-CoA
Propionyl-CoA
Propionyl-CoA carboxylase
CH3
H C COO
-
CO-S-CoA
ATP
AMP +PPi
CO2
D-Methyl malonyl-CoA
CH3
- OOC C H
CO S–CoA
Methyl malonyl-CoA racemase /
epimerase
L-Methyl malonyl-CoA
COO
-
CH2
CH2
CO S CoA
Methyl malonyl-CoA mutase
Deoxy adenosyl
cobalamin( vitamin B12)
Succinyl-CoA
Heme synthesis
TCA
I
I
I
I
I
I
I
I
I
ǀ
ǀ
ǀ
ǀ
ǀ
ǀ
ǀ
Produced in metabolism of Valine ,
Isoleucine, Threonine
Propionyl-CoA carboxylase has an absolute
requirement for the coenzyme Biotin
Biotin
Importance of Biotin dependent metabolism of Leucine
❖In the metabolism of Leucine following reaction is dependent on Biotin.
β-Methyl crotonyl-CoA β-MethylGlutaconyl-CoA
❖Importance of Hydroxymethyl glutaryl-CoA (HMG) which is produced during
catabolism of Leucine:
• Precursor for of Cholesterol biosynthesis.
• Precursor for ketone body formation.
Biotin
β-methyl crotonyl-CoA Carboxylase
Hydroxymethyl glutaryl-CoA (HMG)
Biotin-dependent reactions in catabolism of branched chain amino acids
Valine Isoleucine* Leucine*
α-keto isovalerate α-keto-β-methyl valerate α-keto isocaproate
Iso butyryl-CoA 2-methylbutyryl-CoA Iso valeryl-CoA
Methacrylyl-CoA Tiglyl-CoA β-Methyl crotonyl-CoA
HMG-CoA
Propionyl-CoA
Succinyl-CoA
Transamination
Oxidative
decarboxylation
α-keto acid dehydrogenase
TPP TPP TPP
CO2 CO2 CO2
CO2 , ATP , Biotin
β-methyl crotonyl-CoA Carboxylase
Acetoacetate
Lyase
Acetyl-CoA
CO2 , ATP , Biotin
Methyl malonyl-CoA
Deoxy adenosyl cobalamin( vitamin B12)
α-keto acid dehydrogenase
Maple syrup
urine disease
Valine and Isoleucine that generate
Propionyl-CoA ,which is converted to
Succinyl-CoA by Biotin and Vitamin B12
requiring reactions.
* Ketogenic
Glucogenic
Biotin Dependent Enzymes from amino acid metabolism
❑Biotin Dependent Enzymes is : Threonine Deaminase
Role of Biotin in catabolism of Threonine(Threonine Deaminase activity)
• Threonine dehydratase produces
α-ketobutyrate .
• Subsequently , α-ketobutyrate is
oxidatively decarboxylated by
Threonine Deaminase to yield
Propionyl-CoA .
• Propionyl-CoA is then carboxylated
to Methyl malonyl-CoA .
• Methyl malonyl-CoA is isomerized to
Succinyl-CoA.
• Succinyl-CoA enters the Kreb’s
cycle(TCA) and give rise to Pyruvate.
It is a precursor for heme synthesis .
• Threonine is glucogenic amino acid.
Threonine Deaminase activity
Threonine
α-ketobutyrate
Propionyl-CoA
Methyl malonyl-CoA
Threonine dehydratase PLP
NH4
+
Threonine Deaminase Oxidative decarboxylation
Biotin
Glucose
Pyruvate Heme
Methyl malonyl-CoA
mutase
Succinyl-CoA
Deoxyadenosyl
cobalamin(vitaminB12)
TCA
Propionyl-CoAcarboxylase
Role of Biotin in Methionine metabolism
Biotin independent carboxylation reactions
1. Carbamoyl phosphate synthetase , which is stepping stone for urea and
pyrimidine synthesis .
2. Malic enzyme , converting Pyruvate to Malate during gluconeogenesis .
3. Carboxylation of Glutamate to form γ-carboxyglutamate (Gla) in activation of
blood clotting factors.
Role of Biotin in purine biosynthesis
• Biotin is required for fixation of CO2 in carbon 6 of the purine nucleus during
the de novo purine biosynthesis.
Biotin→
Incorporation of CO2 in
Purine synthesis do not
require Biotin .
Role of Biotin in biosynthesis of Carbamoyl phosphate of Urea cycle
Formation of Carbamoyl phosphate in urea cycle do not require Biotin.
Vitamin K cycle in carboxylation reaction
• Vitamin K is required in the hepatic
synthesis of prothrombin and blood clotting
factors II, VII, IX and X . These proteins are
synthesized as inactive precursor molecules
.
• Formation of the clotting factors requires
the vitamin K-dependent carboxylation of
Glutamic acid residue to γ-
carboxyglutamate (Gla) .
• This forms a mature clotting factor that
contains γ-carboxyglutamate (Gla) and
capable of subsequent activation.
• The reaction requires O2 , CO2 and
Hydroquinone form of vitamin K .
• The formation of (Gla) is sensitive to
inhibition by Dicumarol (an anticoagulant
occurring in naturally in spoiled sweet
clover)and by Warfarin (synthetic analog of
vitamin K) .
Biotin-independent Carboxylation of Glutamate to form γ-
carboxyglutamate (Gla) in activation of blood clotting factors
H
N CH C
CH2 O
CH2
COO-
H
N CH C
CH2 O
CH
II
I
I
I
I
-
COO- COO-
I
- - II
I
I
γ-carboxyglutamate (Gla) residue
Precursors of clotting factors II , VII ,IX, X
Mature of clotting factors II , VII ,IX, X
Glutamyl residue
Polypeptide
of clotting
factor
CO 2
O 2
Hydroquinone(active Vitamin K)
⃝
⃝ +
Warfarin,
Dicumarol
-
vvvvvvvv-
-vvvvvvvv
-vvvvvvvv vvvvvvvv-
-
Liver : site of synthesis
and activation of clotting
factors II , VII ,IX, X
Role of Biotin in gene silencing
Biotinylation of proteins play role in epigenic regulation of gene function
Histone biotinylation and DNA repair
Regulation of gene expression by Biotin
Biotin
Biotinyl-AMP
soluble Guanylate cyclase
Guanosine triphosphate c-GMP
Protein kinase G
Phosphorylated proteins
Transcriptional activation of gene
Holocarboxylase synthetase
Proteins
⃝
⃝
⃝
+
+
+
Altered gene expression
during Biotin deficiency and
new enzymatic activities of
the enzyme biotinidase is
confirming suggestions of a
role for Biotin in the
regulation of gene expression.
Role of Biotin in gene expression
Biotin cycle and Holocarboxylase synthetase (HCS)-dependent transcriptional
regulation
Biotin and cell cycle regulation
Deficiency of Biotin
• The addition of raw egg white to the diet as a source of protein induces
symptoms of biotin deficiency.
• Raw egg contains a glycoprotein Avidin ,which tightly binds Biotin and
prevents its absorption from the intestine.
• With normal diet ,however it has been estimated that 20 eggs / day would be
required to induce deficiency syndrome of Biotin.
• Thus ,inclusion of an occasional raw egg in the diet does not lead to Biotin
deficiency.
• Although, eating of raw eggs is generally not recommended due to possibility
of salmonella infection.
• Multiple carboxylase deficiency: results from a defect in ability to link Biotin
to carboxylases or to remove it from carboxylases during their degradation.
Treatment is Biotin supplementation.
Intake of perfect boiled eggs → No danger of Biotin
deficiency
Causes of Deficiency of Biotin
❖Deficiency of Biotin is uncommon and does not occur naturally . Since it is widely
distributed in food and also supplied by the intestinal bacteria.
❖Causes of deficiency manifestations include:
1. Prolonged use of intestinal-sterilizing antibiotics e.g. Sulphonamides
supplementation, broad spectrum oral antibiotics → causes destruction of
intestinal flora→ biosynthesis of Biotin is affected/decreased .
2. Prolonged / High consumption of raw (uncooked) egg whites which contain
Avidin (a glycoprotein). Avidin has high affinity for Biotin → binds with the
imidazole ring of Biotin → blocks its absorption of Biotin by intestinal epithelial
cells . (intake of 20 more eggs/day causes Biotin deficiency) . Therefore, Avidin
from egg white serves as an antagonist/ anti-vitamin.
3. Genetic Deficiency of Holo-carboxylase synthetase (HCS is required for
attachment of Biotin to apoenzyme) or genetic deficiency of Biotinidase.
4. Leiner’s Disease : In breast fed infants with persistent diarrhea.
5. Patients who are on long term Total parental nutrition (TPN) with inadequate
Biotin supplementation .
Symptoms in deficiency manifestations of Biotin
1. Nausea ,vomiting
2. Anorexia (Loss of appetite) → weakness
3. Anemia(pallor)
4. Seborrheic /dry scaly dermatitis
5. Hair : loss (Alopecia) , graying and thinning
6. Spectacle eyed –due to circum-ocular alopecia
7. Brittle nails
8. Glossitis
9. Muscle pain
10. Depression/ Hallucination/sleepiness/ insomnia
11. neurological manifestations
Injection of Biotin 100-300mg
will bring about rapid cure of
these symptoms .
Symptoms develop after
5-7 weeks.
Leiner’sDisease(Acquireddeficiencyof Biotin)
• Leiner’s Disease (Acquired deficiency of Biotin): Erythroderma
desquamative or exfoliative dermatitis in young infants.
• Occurrence of Leiner’s disease : in breast-fed infants frequently in
association with persistent diarrhea.
• Cause of deficiency of biotin in Leiner’s Disease : low Biotin content
of human milk and poor absorption of biotin due to diarrhea.
• Management of Leiner’s Disease : administration of Biotin.
Congenital deficiencyof Biotin
❖Congenital deficiency of Biotin :
• A rare genetic deficiency of holocarboxylase(holoenzyme synthetase →
reflected in inadequate conversion of apocarboxylases to holocarboxylases ) .
• Genetic enzyme defect in Propionyl-CoA carboxylase(reflected in a
distinguishing acidemia).
• Affected child cannot utilize biotin in metabolic role.
• Clinical manifestation of Congenital deficiency of Biotin :
1. Dry-scaly dermatitis
2. Greying of hair
3. Alopecia (loss of hair)
4. Incoordination of movement
5. Urine : high content lactate, β-hydroxy propionate and β-methyl crotonate
(due to the failure of corresponding enzyme activities).
Fetal birth defects in biotin deficiency
Pregnancy (more utilization of Biotin)
Deficiency of Biotin
ReducedactivityBiotindependentenzymes(Acetyl-CoAcarboxylaseandPropionyl-CoAcarboxylase)
Alteration of lipid metabolism
Alteration in metabolism of Polyunsaturated fatty acids (PUFA) and Prostaglandins
Deranged(defective) muscle development
Fetal birth defects like cleft palate , Micrognathia and Micromyelia
Molecular basis of fetal birth defects in Biotin deficiency
Pathophysiology of Biotinidase deficiency
❖Biotinidase deficiency ( OMIM number :253260):
• Enzyme /transport defect : Biotinidase deficiency (<10% of normal serum activity)
(disorder of organic acid Metabolism) .
• Incidence in USA → > 1: 75000 population , carrier frequency for heterozygous for
biotinidase gene mutation → 1:120 in general population.
• Symptoms observed :
1. Alopecia
2. Periorificial skin rash
3. Conjunctivitis
4. Developmental delay
5. Hypotonia , seizures
• Modality of acute episodes : ketoacidosis in early life
• Major biochemical markers in newborns : Biotinidase (serum) , 3-hydroxy isovaleric
acid (urine)
• Prenatal diagnosis : enzyme assay in amniocytes
• Sudden unexpected death : Anecdotal reports
Formation of 3-hydroxyisovaleric acid under conditions of Biotin deficiency
β-Methylcrotonyl-CoA carboxylase
Biotin
β-Methylcrotonyl-CoA
Leucine
Biotin deficiency
3-hydroxyisovaleric acid →
excreted in urine
β-Methylglutaconyl-CoA
H3C SCoA
CH3 O
H3C OH OH
CH3 O
HO SCoA
O CH3 O
Biotinidase deficiency
Management of Biotinidase deficiency
• Treatable genetic condition with supplementation of Biotin (not dietary) .
• Life long supplementation of Biotin(5-20mg/day) in preventing or relieving
most symptoms .
• Seizures resolves within hours to days .
• Cutaneous symptoms resolve within weeks.
• Resolution of Neurological defects variable.
Multiple carboxylase deficiency (Juvenile/late form)
Active
Inactive
Multiple carboxylase deficiency : results from a defect in ability to link Biotin to
carboxylases or to remove it from carboxylases during their degradation. Treatment is
Biotin supplementation.
Dermatitis in Biotin deficiency :1
Seborrheic dermatitis
Biotin responsive dermatoses
Dermatitis red rash around genital area
Spectacle eyed appearance due to circum –
ocular alopecia .
Dermatitis red rash around eyes ,nose and
mouth.
Dermatitis in Biotin deficiency : 2
Alopecia(hair loss)
H. Pylori
infection
Deficiency manifestations of Biotin: Alopecia and dry brittle nails
Management of Biotin deficiency
Toxicity of Biotin
• No adverse effects of biotin in doses up to 300 times normal dietary intake
(as in patients with biotinidase deficiency).
Summary of Biotin
Name of
water
soluble
vitamin
Co-
enzyme
form
RDA
(μg/day)
Main reaction
using
co-enzyme
Direct and indirect
assay
Deficiency disease
Biotin Biocytin 200-300 Carboxylation
of
Pyruvate,
Acetyl CoA ,
Propionyl CoA,
β-methyl
crotonyl CoA
Microbiological,
Competitive
protein binding
(CPB),carboxylases,
avidin binding
No specific disease.
Consumption of
large amount of
raw egg whites
(which contains
protein Avidin that
binds Biotin) can
induce a biotin
deficiency.
Technical applications of Biotin Antagonist
❖Avidin asBiotinantagonist:
• aglycoproteinpresentineggwhite.Whenfedtoanimalsinlargeamount,canproduce
Biotindeficiency(namedasanti-egg-white-injury-factor).
• hasgreataffinityforBiotin(presentineggyolkandboundnon-covalentlywithAvidin).
• Heatlabile(boilingofeggwilldenatureAvidinandneutralizeitsinhibitoryactivity).
• Onemoleculecombineswith4moleculesofBiotin.
❖AffinityofAvidinisgreaterthanmostoftheusualantigen-antibodyreactions Therefore,
Avidin-BiotinsystemiscommonlyutilizedfordetectionofpathogensbyELISA.
❖DNAisgenerallylabeledbyradioactivenucleotides.BiotinlabellingofDNAisbecoming
morepopular.Biotinisaddedtonucleotides,whichwillbeincorporatedintothenewly
synthesizingDNA.ThefixedbiotincanbeidentifiedbyreactionwithAvidin.
❖StreptavidinpurifiedfromStreptomycesavidinii,canbind4moleculesofBiotin.
❖Biotinantagonists:Desthiobiotin,Biotinsulphonicacid.
Signal amplification by Biotin-Avidin Complex in ELISA
• Biotin and other affinity labels do not generate detectable signals on
their own.
• They can initiate signal amplification mediated by high-affinity binding
antibodies or in case of biotin , with Avidin or streptavidin.
• Biotin will tightly bind with Avidin .
• Instead of enzyme directly fixed over antibody ,Biotin is labelled on the
first antibody. The avidin-conjugated enzyme is added and color
reaction is done as before using horse radish peroxidase or alkaline
phosphatase.
• The advantage here is that for each Biotin fixed ,4 Avidin molecules
therefore so 4 enzyme molecules are fixed .
• The intensity of color the assay is thus increased many times .
Signal amplification by Biotin-Avidin Complex in ELISA
Biotin can be used to introduce amplification into an immunoassay . The
binding constant of Biotin-Avidin complex is extremely high (10 15 L/mol ) ;
capitalizing on this system allows immunoassay systems to be devised even
more sensitive than the simple antibody systems.
Signal amplification by Biotin –Avidin Complex in ELISA
Biotin-Avidin System : is Used in Elisa reaction for detection of pathogens in Elisa test(great
affinity of avidin amplifies the signal) .
It uses Biotin-labeled antibody . Biotin can be attached to the antibody without loss of
immunoreactivity by the antibody . When Avidin-conjugated label is added , a complex of
Ag: Ab: Ab-Biotin: Avidin label is formed . Further amplification is achieved by a Biotin : Avidin
linkage because the binding ratio of Biotin : Avidin is 4:1.
Biotinylated probes
• NeedofBiotinylatedprobes:thedisposalofradioactiveprobesisbecoming
increasinglyexpensivethereforenonradioactiveprobeshavebeendeveloped.
❖ApplicationsofBiotinylatedprobes:Fluorescentprobescanallowdetection
andlocalizationofDNA/mRNA/miRNAsequencesincellortissuepreparations,
aprocesscalledinsituhybridization.
• Themostsuccessfulprobes: basedonvitaminBiotin(Biotinylatedprobes).
❖PropertiesofBiotinrequisiteforpreparationofBiotinylatedprobes:Biotin
• canbechemicallycoupledtothenucleotidesusedtosynthesizetheprobe.
• bindstenaciouslytoAvidin.
❖ PropertiesofAvidinsuitableforitsuseininsituhybridizationofDNAor
mRNA/miRNAsequences byBiotinylatedprobes:Avidin
• isareadilyavailableproteincontainedinchickeneggwhites.
• canbeattachedtoafluorescentdyedetectableopticallywithgreatsensitivity.
❖DNAfragment(displayedbygelelectrophoresis)that hybridizeswith
Biotinylatedprobescanbemadevisiblebyimmersingthegelinasolutionof
fluorescentdye-coupledwithAvidin(afterwashingexcessAvidin).
Non-isotopic probes using Biotin
• The first practical example of non-isotopic probe labelling used a
biotin label analog of dUTP.
• Despite of altered steric configuration ,this nucleotide is incorporated
by DNA polymerase and terminal transferase.
• Alternately ,oligonucleotide probes can be label during synthesis
with biotin for subsequent attachment to indicator molecules.
• Labels at either the 5’ or 3’ end of the molecule are preferred
(because central modifications may interfere with hybridization).
• If label is biotinylated enzyme ,then large number of enzyme
molecules in the complete complex provide a large increase of
enzyme activity coupled with small amount of antigen to be
determined and the antigen assay is correspondingly more sensitive.
In situ hybridization using Biotinylated probes for detection of target DNA using
gel electrophoresis
• Technique used to detect DNA or m-RNA/
miRNA by in situ hybridization using
Biotinylated probes : Gel electrophoresis
.
• Procedure : DNA fragment that
hybridizes with the Biotinylated probe in
gel and can be made visible by immersing
gel in a solution of fluorescent dye-
coupled Avidin.
• Results : After washing away the excess
Avidin , the DNA fragment that binds the
probe is fluorescent and is displayed.
• Application : DNA/mRNA/ miRNA can be
viewed in the context of the tissue
morphology.
When a probe is with fluorescein ,it
can be seen under UV microscope ,
then it is called FISH( fluorescent in situ
hybridization) .
Biotinylated hybridization probes for detection of target DNA or m-RNA
Alkaline phosphatase and Horse- radish
peroxidase can act on luminescent
substrate to emit light.
Principle and technique of in situ hybridization using biotinylated probes
• Avidin or Streptavidin are linked to enzymes
(e.g. Horse-radish peroxidase or Alkaline
phosphatase) connecting single target to a
single enzyme.
• Enzyme activity is monitored according to
enzyme substrate used (colorimetric,
fluorescent or chemiluminescent).
• Affinity labels can be used to capture and
localize targets to an area of solid support.
• Biotinylated probes can affixed to a
streptavidin-coated surface.
• After incubation with target nucleic acid , a
second probe is added ,which is either
directly labeled with fluorescence or
conjugated with affinity label to an enzyme.
• Multiple separation and washing steps to
decrease the background OR non specific
localization of reagents (results in
amplification of undesired signals along
with desired signals) .
Analytical detection of limitsof immunoassays can be increased using enzyme labels
❖If the label is an biotinylated enzyme , then large numbers of enzyme
molecules in complete complex provide a large increase of enzyme activity
coupled with small amount of antigen being determined and the antigen
assays is correspondingly more sensitive.
❖Enzymes predominate in Elisa :
1. Alkaline phosphatase
2. Horseradish peroxidase
3. Glucose -6-phosphate dehydrogenase
4. β-galactosidase
❖Alkaline phosphatase and Horse-raddish
peroxidase can act on luminescent
substrate to emit light .
In situ hybridization
• Allows to examine the tissue first by microscope.
• Is a modified version of DNA –DNA hybridization.
• If a metaphase spread chromosome preparation is probed with a gene , location of the
gene on a specific chromosome can be identified e.g. Philadelphia chromosome
abnormality.
• Principle may be applied to histology slide also. In tissue preparation ,DNA is denatured ,the
specific probe is tagged with fluorescent labels ,incubated , washed and seen under
fluorescent microscope.
• The process is known as in situ hybridization(FISH).
• Histology section, single cells may be treated with specific antibodies tagged with
biotinylated fluorescent tag and seen under microscope .Histology section may be treated
with antibody linked with peroxidase and color developed.
Philadelphia
In situ hybridization
cells with specific antibody showing surface
immunofluorescence.
Human breast cancer cells with specific
antibody showing Immuno-peroxidase
technique.
RNA FISH
Fluorescent labels
DNA-DNA hybridization
Double stranded DNA
DNA denatured and
strands separated by
heat or alkali.
Add biotinylated probes
Parent DNA and probe
hybridized if
complementary
sequences available.
Fluorescent in situ (FISH) is a molecular technique that uses
fluorescent probes that bind to only those parts of
chromosomes with complementary sequences.
Biotinylated DNA probe
Application biotinylated
DNA probe in southern
blotting technique
Genomic DNA
DNA fragments
DNA cut by Restriction endonuclease
AgaroseGelelectrophoresis
Agarose Gel
Denature by mild alkali NaOH
Blot transfer on Nitrocellulose (or nylon
membrane) followed by fixation of DNA
on the membrane by baking at 80 ⁰C.
Biotinylated DNA probes
_ _
_ _
- -
- -
_ _
_ _
- -
- -
Long DNA fragments
Small DNA fragments
Southern blotting refers
to the transfer of DNA
from Agarose gel to
nitrocellulose or nylon
for DNA hybridization.
detection of complementary
nucleotide sequence in the host DNA
Band visualization by Autoradiography
Applications of Southern blotting technique
• An invaluable method in gene analysis( i.e. to detect a specific
segment of DNA in the whole genome).
• Important for conformation of DNA cloning.
• Forensically applied to detect minute quantities of DNA (to identify
parenthood , thieves and rapists etc.)
• Highly useful for the determination of restriction fragment length
polymorphism (RFLP) associated with pathological conditions.
• Mutant genes such as HbS , Cystic fibrosis, Phenylketonuria ,DMD as
well as presence of viral DNA (Hepatitis B and C) can be identified by
this method.
Principle and technique of in situ hybridization using biotinylated probes
Northern blotting is a procedure by which RNA molecules are
transferred from agarose gel to diazo benzyloxymethyl(DBM)
paper or nylon membrane by capillary action for hybridization.
Denaturation of RNA by
formaldehyde .
RNA immobilized on the
membrane is hybridized
to single-stranded c-DNA
probes . Determination
of the size of a transcript.
Applications of Northern blotting technique
❖Applications of Northern
blotting:
• is theoretically a good
technique for determining the
number of genes (through
specific mRNA).
• Determinationofhybridization
patternsinmRNAsamples
(RNA-DNAhybridization).
• Analysis of gene expression in a
tissue.
• Determination of the size of a
transcript.
RNA extract
Agarose Gel electrophoresis
rRNA brands
Diazobenzyloxymethyl paper(DMB)
Biotinylated c-DNA probes
Hybridization of Biotinylated c-DNA
probes with rRNA
Results of Multiplex miRNA northern blots via hybridization
Near infrared fluorescent
Northern blot
Advances in oligonucleotide synthesis and fluorescence detection systems have made
fluorescently labeled biotinylated affinity probes , the preferred reporter for nucleic acid
analysis.
Application of biotinylated probes in western blotting:1
❖Western blotting is a technique by which a protein is transferred from a
polyacrylamide gel to nitrocellulose or nylon membrane after electrophoresis.
• The proteins are isolated from the tissue and electrophoresis is done.
• The separated proteins are the transferred on to a nitrocellulose membrane.
• After fixation, it is probed with radioactive antibody and autoradiographed.
• Alternately ,the specific antibody is poured over, washed and a second
antibody carrying biotinylated horse-radish peroxidase is added .
• Hydrogen peroxide and a chromogen are layered .
❖Application of western blotting: very useful to identify the specific protein in
tissue , thereby showing the expression of a particular gene.
Application of biotinylated probes in western blotting :2
Blot transfer techniques
Southern (for DNA) Northern blot(for RNA) Western blot(for proteins)
DNA* C-DNA* Antibody*
DNA/RNA fragments or proteins
placed in the well and then
electrophoresed.
Transfer to nitrocellulose or
DMB or nylon membrane .
Biotinylated probes added.
Autoradiograph/
colorimetric, fluorescent or
chemiluminescent analysis
_ _ _ _
_ _
_ _
- -
- -
_ _
_ _
_ _
- -
- -
_ _
_ _
_ _
_ _
- -
- -
- -
Examples of diagnostic applications of ISH and FISH
1. Assessment of gene rearrangement in leukemia.
2. Diagnosis of B-cell lymphoma by demonstration of reduced light-chain
mRNA.
3. Determination of amplification of HER2 in breast cancer.
4. Diagnosis of various types of lymphomas.
5. Chromogenic in situ hybridization (CISH) for diagnosis of melanoma and
lymphomas using mRNA probes for kappa and lambda chains.
Acute promyelocytic leukemia with cryptic insertion with
RARA into PML Diagnosis of B-cell lymphoma
HER2 receptors and HER2 positive breast cancer Melanoma cells
Use of Biotin-labeled probes in Elisa and Polymerase chain reaction(PCR)
DuringPCRamplification,thePCRproductstypicallyislabeledwithnucleotidesthateitherradioactiveor
fluorescentorhaveattachedaffinitylabels(e.g.biotinylatedprobes) .Ifdesired,singlestrandedprobescanbe
obtainedbyusingBiotinlabeledprimerfollowedbysolidphaseseparationwithstreptavidin.
Signal amplification for detection of PSA using Biotin-Avidin system
Role of Biotin azide in purification of DNA –protein complex using iPOND methaodology
The iPOND methodology enables purification of proteins bound directly or indirectly to the nascent DNA at replication forks.
The method relies on labeling short fragments of nascent DNA with EdU, a nucleoside analog of thymidine. EdU contains an
alkyne functional group that permits copper-catalyzed cycloaddition to a biotin azide to yield a stable covalent linkage This
facilitates a single-step purification of DNA-protein complexes based on the high affinity biotin-streptavidin interaction.
PMCID: PMC3671908
NIHMSID: NIHMS362
809
PMID: 22383038
Role of Biotin in isolation of proteins on Nascent DNA in normoxia,
hypoxia and reoxygenation conditions
Biotin (vitamin b7)  biological functions, clinical indications and its technological applications

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Biotin (vitamin b7) biological functions, clinical indications and its technological applications

  • 1. Biotin (vitamin B7) : Biological functions , clinical indications and its technological applications Dr. Rohini C Sane
  • 2. Historical background of Biotin • Biotin = vitamin B7 = vitamin H = anti-egg white injury factor(intake of raw →not boiled egg may cause Biotin deficiency). • Boas(1927) fed rats with huge quantity of raw eggs → rats developed dermatitis, nervous manifestations and retardation in growth. • Vincent du Vigneaud (Noble prize 1955): isolated Biotin.
  • 3. Structure of Biotin (Vitamin B7 / Vitamin H) ❑Structure of Biotin (Vitamin H) : ❖Sulphur-containing water soluble heterocyclic monocarboxylic acid (Vitamin B7) . ❖Imidazole ring fused with tetrahydrothiophene ring with a Valeric acid side chain. • The carboxyl group of biotin forms an amide linkage with the epsilon(ε) nitrogen of Lysine residue in the apoenzyme forming a biotinyl enzyme. • Biocytin : a coenzyme form (Active form)→ (Biotin is covalently bound to ε- amino group of Lysine (Lysine - linked by amide bond)in the enzyme. O II C N H C H CH (CH2)4 COOH H N H C H2C S ǀ ǀ ǀ ǀ ǀ ǀ CO2 Binding site Site for binding with Lysine Biotin = cis-tetrahydro-2-oxo- 1-thienol-(3,4-d)-imidazoline - 4-valeric acid . Imidazole ring Tetrahydrothiophene ring 1 2 3 4 5 1 2 3 C10H16 O3N2 S Molecular weight : 244
  • 4. Chemical properties of Biotin 1. Existsintwoformswithessentiallyidenticalbiologicalactivities:α-Biotin(egg-yolk)and β-Biotin(Liver)differingonlyinthenatureofsidechain. 2. Solubility:solubleinwaterandethylalcohol,butinsolubleinetherandchloroform. 3. Stability:heatstable. 4. Destroyedbyoxidizingagentssuchasperoxidesandpermanganates(convertthe thioethertosulfoxidesandsulfoneswhichdonothavebiotinactivity)andacids/alkalis . 5. Crystallization:whitecrystallinesolidlongneedles,solubleinanaqueoussolution (pH>7,alkaline)duetoureidoringandionizablecarboxylgroup. 6. Occurrenceinfoodandtissue:freeformandboundforms(Biocytin,oxy-biotinand Desthiobiotin). ItislinkednoncovalentlyasacomplexwithAvidin(aproteininegg white). 7. Biocytin(ε-N-Biotinyl-Lysine):BiotinremainboundwithLysineresiduesoftissue proteinsbyamidebonds.Biocytinisreleasedonhydrolyzingthepeptidebondsbetween Biotin-boundLysineandpeptidechain. 8. Oxy-biotinandDesthiobiotin:arebiologicallyactiveincertainstrainsofyeastand bacteria.
  • 5. Sources of Biotin in human ❖Human beings cannot synthesize Biotin and hence Biotin has to be supplied in diet. ❖Sources of Biotin in human : synthesis by intestinal bacterial flora and dietary sources . ❖Normal bacterial flora of the intestine provides adequate quantities of Biotin(biosynthesis of Biotin by E.coli). Moreover, it is distributed ubiquitously(widely) in animal tissue, fruits and vegetables. ❖Rich food sources of Biotin: • Milk, cheese , yogurt , yeast , cauliflower , berries , soyabean , tomatoes , sweet potatoes , spinach , Avocado , lentils , banana , carrots , white mushrooms , Molasses, wheat germs , oats , sunflower seeds , almonds, walnuts , hazelnuts, peanuts and grains. (water soluble form in most plants material except cereals and nuts). • Egg yolk , liver, kidney , pancreas, Sardine , Yellow Tuna, pork , beef , Turkey , chicken , lamb and Calf silver. (water insoluble form in animal tissues).
  • 7. Co-enzyme-R(Biotin)from Rhizobium • Co-enzyme R is growth essential for Rhizobium (nitrogen-fixing organisms) in the root nodules of Leguminous plant . • Co-enzyme R is proved to be Biotin. • Pimelic acid is possible precursor. • Desthiobiotin is a probable intermediate.
  • 8. Daily dietary requirement of Biotin( RDA) • Daily dietary requirement of Biotin( RDA) = 200-300 μg • Estimated Average requirement : Adequate intake(AI)based on urinary excretion of Biotin and the metabolite 3-hydroxy isovaleric acid. • Tolerable upper intake level (UL)→ applies to chronic daily use of fortified foods/dietary supplements → no value established for Biotin . ➢Daily dietary requirement of Biotin(RDA) increases in pregnancy and lactation (additional 5 μg /kg of body weight) . ➢Intravenous supply of Biotin for adults during TPN = 60 μg /day. ➢Patients receiving hemodialysis or peritoneal dialysis or with biotinidase deficiency require more . Age in years Adequate intake(AI)of Biotin (μg/day) ≥ 19 30 14-18(aldolescents) 25 9-13 20 4-8 12 1-3 8 <1 year(Infants) 0.7 μg /kg of body weight
  • 9. Metabolism of Biotin in the human body • Sources of Biotin in human : dietary sources(largely protein bound)and free form .It is biosynthesized biotin by bacterial flora. • Occurrence /existence in dietary food source : widely distributed in many food sources as Biocytin (ε- amino-biotinyl lysine)→ Biotin is released after proteolysis. • Biosynthesis : by intestinal bacteria (human body cannot synthesize Biotin). • Digestion: proteolysis by gastrointestinal enzyme to produce biotinylate peptides →release of free Biotin further hydrolysis by intestinal biotinidase. • Absorption : readily absorbed by intestinal epithelial cells using a biotin carrier (the sodium dependent multi-vitamin transporter →SMVT). Avidin (present in raw egg white)prevents its absorption. • Transport : by circulating blood • Secretion : in milk • Uptake of absorbed Biotin : by liver , kidney and muscles (localized in cytosolic and mitochondrial carboxylases). • Storage :in Liver and kidneys (limited extent→ 14% of administered dose). Total body content of Biotin = 1mg • Excretion : Urinary excretion(10-180 μg/day) > dietary intake (28-100 μg/day) . Fecal excretion (15-200μg/day) > 3-6 times of dietary intake . Fecal excretion represents unabsorbed Biotin synthesized by intestinal bacteria.
  • 10. Absorption and transport of Biotin Biotin containing food/enzymes Covalently linked to proteins Proteolytic enzymes Biocytin Pancreatic Biotinidase Biotin + Lysine sodium dependent multi-vitamin transporter →SMVT) Intestinal Brush border Biotin GPR109A Hydroxycarboxylic acid (HCA-2) receptor mutation causes biotin – responsive basal ganglia disease(encephalopathy ) SLC19A3 Low concentration of Biotin : active transport High concentration of Biotin : Passive transport Basal lateral membrane
  • 11. The sodium-dependent multivitamin transporter (SMVT) to facilitate intestinal absorption of Biotin • Is located in the intestinal brush border membrane. • Na+ dependent carrier-mediated process . • Transports biotin against sodium ion concentration gradient. • Not specific for vitamin (Biotin) transport. • Functions in cellular uptake of Biotin ,Pantothenic acid and Lipoic acid with similar affinities . • Biotin uptake by intestinal epithelial cells is inhibited by activation of protein kinase C apparently through phosphorylation of SMVT.
  • 12. Facilitated transport of Biotin during intestinal absorption Intestinal Absorption of Biotin at its Low concentration Intestinal Absorption of Biotin at its High concentration Saturable ,Active and facilitated mechanism dependent on Na+ . Passive transport Facilitated transport inhibited by certain anti- convulsant drugs and chronic exposure to ethanol.
  • 13. Catabolism of Biotin • The enzyme biocytinase (biotin amidohydrolase) in plasma and erythrocytes catalyze the hydrolysis biocytin to yield free biotin. • Free biotin is taken up by tissues (such as liver, muscle and kidney) and localized in cytosolic and mitochondrial carboxylase. • Small fraction of Biotin is oxidized to D- and L- sulfoxides ( ureido ring intact not otherwise degraded). • Side chain of larger portion of Biotin is degraded via mitochondrial β-oxidation to yield bis-nor biotin and its degradation products. • Biotin catabolism in smokers > Biotin catabolism in non-smokers.
  • 14. Excretion of Biotin • Source for excretion of Biotin : Half of the absorbed biotin. • Mean urinary excretion is a reflective of dietary intake (28-100 μg/day for adult). • Metabolite Forms for excretion of Biotin: 1. Bis-norbiotin (occurring from β-oxidation of the Valeric acid side chain). 2. Biotin sulfoxide (occurring from oxidation of the sulfur in the heterocyclic ring). • Ratio of Biotin : Bis-norbiotin : Biotin sulfoxide = 3:2:1(in circulating plasma and urine). • Minor metabolites of biotin for excretion : Bis-norbiotin methyl ketone and Biotin sulfone.
  • 15. Laboratory Assessment of Biotin status ❖Bioassay methods ❖Microbiological assay : growth stimulation of yeast cells (Saccharomyces cerevisiae or Lactobacillus plantarum) is measured . Whole Blood is first digested with papain or acid hydrolysis to release free biotin . This sample is then added to a Biotin-deficient medium inoculated with a test organism , such as Lactobacillus plantarum . ❖Measurement of unfound Biotin include Avidin-binding assays : a competitive protein binding radio-assay with 3H-labelled Biotin or a nonradioactive enzyme-linked sorbent using Streptavidin as a binding agent . ❖ Measurement of Urinary Biotin and 3-hydroisovaleric acid : by HPLC Urinary excretion of Biotin and 3-hydroisovaleric acid appear to be better indicator of biotin status than whole blood concentrations. ❖Urinary Biotin and 3-hydroisovaleric acid : gas chromatography-mass spectrometry. ❖Lymphocyte Propionyl-CoA carboxylase using H14 CO3 - : early indicator and sensitive indicator of biotin deficiency in patients on prolonged TPN without biotin supplementation and in children with protein-energy malnutrition.
  • 16. Reference intervals of Biotin • Whole blood Biotin levels (physiological) by microbiological method : 200 - 500 pg /ml (0.5- 2.2 nmol/ L with mean 1.31nmol/L) • Biotin deficiency : Whole blood biotin < 0.5 nmols/L • Lowered circulating blood levels and urinary excretion are observed in 1. alcoholics 2. Patients with achlorhydria 3. Elderly 4. Athletes • Biotin content of red cells is similar to that of plasma for a given method.
  • 17. Co-enzyme and non-coenzyme roles of Biotin Coenzyme role of Biotin Non-coenzyme role of Biotin Pyruvate carboxylase Cell proliferation Acetyl-CoA carboxylase Gene silencing Propionyl-CoA carboxylase DNA repair β-methyl crotonyl-CoA Carboxylase Gene expression and cell signaling Biotin is a coenzyme of carboxylase reactions . Biotin is a carrier of activated carbon dioxide (CO2) for the mechanism of Biotin-dependent carboxylations . Biotin is the prosthetic of certain enzymes (carboxylases and decarboxylases) that catalyze CO2 transfer reaction (CO2 fixation reaction/ carboxylation) in human tissue.
  • 18. Biotin-dependent carboxylases Biotin VitaminB7 Vitamin H Pyruvate Carboxylase (key enzyme of Gluconeogenesis, TCA and Transamination) Acetyl-CoA Carboxylase (First committed step in biosynthesis and elongation of fatty acids) Propionyl-CoA Carboxylase (oxidation of odd chain fatty acids and synthesis of Succinyl CoA) β-methyl crotonyl-CoA Carboxylase(catabolism of Leucine) Biotin is covalently bound to the ε- amino groups of Lysine residues in Biotin – dependent enzymes.
  • 19. Functions of Biotin as a prosthetic group of ATP-dependent carboxylases Enzyme Substrate Product Importance enzyme Pyruvate Carboxylase Pyruvate Oxaloacetate Gluconeogenesis(synthesisof Glucosefromnon-carbohydrate substance),providesoxaloacetate forTCACycle,Transamination Acetyl-CoA Carboxylase Acetyl-CoA Malonyl-CoA Limiting reaction in Fatty Acid biosynthesis Propionyl-CoA Carboxylase Propionyl- CoA D-Methyl Malonyl-CoA →Succinyl-CoA Succinyl-CoA→HemeSynthesis, Succinyl-CoAoxidizedInTCAcycle β-methyl crotonyl- CoA Carboxylase β-methyl Crotonyl-CoA β-Methyl glutaconyl- CoA Leucine metabolism (Branched ChainAminoAcids) Biotinfunctionsasaprostheticgroupforcarboxylasesandisattachedtotheenzymebyanamidebond betweenthecarboxylgroupofBiotinandtheterminalε-aminogroupofLysineresidueoftheenzyme, formingaBiotin-enzyme.
  • 20. Mechanism of Biotin during carboxylation reactions in the human body • The peptide biocytin (ε-N-biotinyl lysine) is resistant to hydrolysis by proteolytic enzymes in intestinal tract but together with biotin is readily absorbed . • A biotin carrier , sodium-dependent multivitamin transporter (SMVT) for which Pantothenic acid and lipoate compete. • (SMVT) is located in brush borders membrane and transports biotin against a sodium ion concentration gradient. • The enzyme biocytinase (Biotin amidohydrolase) is located in plasma and erythrocytes catalyzes hydrolysis of biocytin to yield free biotin. • Covalent attachment of Biotin to apoenzyme involves ATP-dependent conversion of the vitamin to biotinyl-5’-adenylate followed by condensation of the biotinyl moiety with ε-amino groups of specific Lysyl residues in apoenzyme preformed from subunits. • Enzyme responsible for formation of ε- N-biotinyl-l-Lysyl ( biocytinyl) moiety of proteins is holocarboxylase synthetases (HCS).
  • 21. Biotin recycling Holocarboxylase synthetase (HCS) uses ATP to catalyze the covalent bonding of different apocarboxylases with Biotin to form different biotin-carboxylase complexes called holocarboxylase . In holocarboxylase –amide bond binds the carboxyl terminal of valeric acid side chain of Biotin with ε–NH2 group at the end of the side chain of lysine residue of apocarboxylases.
  • 22. Mechanism of carboxylation reactions facilitated by Biotin • In biological system, Biotin functions as the co-enzyme for the enzyme called carboxylase which catalyze the carbon fixation(carboxylation). • These enzymes operate via a common mechanism ,which involves phosphorylation of bicarbonate by ATP to form carbonyl phosphate , followed by transfer of the carbonyl group to the sterically less hindered nitrogen of the biotin moiety. • In this process , Biotin is first gets converted to carboxy-biotin complex by reaction with ATP and HCO3 - . • The resulting N(1)-carboxybiotinyl enzyme can then exchange the carboxylate function with a reactive center in a substrate i.e. CO2 -Biotin complex is the source of active CO2 which is transferred to the substrate . • CO2 becomes attached to the biotin coenzyme as above.
  • 23. Mechanism of carboxylation reactions facilitated by Biotin Biotin enzyme Carbonic phosphoric anhydride Carboxy- biotin- enzyme CO2 ATP ADP Pi Carboxylated substrate substrate Biotin acts as co-enzyme for carboxylation reactions. It captures a molecule of CO2 which is attached to nitrogen of the Biotin molecule. The energy required for this reaction is provided by ATP . Then the activated carboxyl group is transferred to the substrate. Substrate + CO2 + ATP → Product (Carboxylated substrate) + ADP+ Pi
  • 24. Biochemical functions of Biotin in carboxylation of Pyruvate to Oxaloacetate ❖ Carboxylation of Pyruvate to Oxaloacetate : Substrate : Pyruvate Enzyme : Pyruvate carboxylase Coenzyme (Carrier of CO2) : Biotin Energy source : ATP Product : Oxaloacetate Mechanism of reaction : Pyruvate carboxylase has Biotin which is bound to the apoenzyme linked to the ε-amino group of Lysine , forming the active enzyme (holoenzyme). Biotin-enzyme reacts with CO2 in presence of ATP to form a carboxy-biotin- enzyme complex (high energy complex). This high energy complex then hands over the CO2 to Pyruvate (carboxylation reaction) to produce Oxaloacetate ..
  • 25. Role of Biotin in conversion of Pyruvate to Oxaloacetic acid by Pyruvate carboxylase COOH CH2 CO COOH Pyruvic acid Oxaloacetic acid CH3 CO COOH Pyruvate carboxylase CO2 Biotin ATP ADP + Pi I I I I I Acetyl-CoA Mn2+ ❖ Carboxylation of Pyruvate to Oxaloacetate by Pyruvate carboxylase is Biotin-dependent reaction. Hydrolysis of ATP drives the formation of enzyme-biotin-CO2 intermediate (high energy complex). This complex subsequently carboxylates Pyruvate to OAA. ❖ Mitochondrial Pyruvate carboxylase(liver and kidney) catalyzes formation of Oxaloacetate, which together with Acetyl-CoA forms Citrate . Two important aspects of this reaction: 1. Provides oxaloacetate , that replenishes TCA cycle intermediates that may be depleted, depending on the synthetic needs of the cell. 2. Is important enzyme in the gluconeogenesis pathway .
  • 26. Mechanism of carboxylation reaction catalyzed by Biotinylated enzyme (mitochondrial Pyruvate carboxylase) in formation of Oxaloacetate Biotin-enzyme + CO2 Biotin-enzyme ATP ADP +Pi S Lys NH (CH 2)4-CO N NH H H O Enzyme S O O- C- II II - ←Caroxy-biotin-enzyme complex I H O CH3- C- COO II - ←Pyruvate ←Oxaloacetate O OOC-CH2- C- COO - II Pyruvate carboxylase with covalently attached Biotin - Protein portion of enzyme : Acetyl-CoA carboxylase , Propionyl-CoA carboxylase, Pyruvate carboxylase, Methyl crotonyl-CoA carboxylase to catalyze carboxylation of substrate into corresponding carboxylated product. Biotin covalently bound to Lysyl residue of a biotin- dependent enzyme .
  • 27. Glucose Glucose -6-phosphate Glyceraldehyde-3-phosphate 1,3 –Bi phosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate Oxaloacetate Pyruvate Acetyl-CoA Oxaloacetate Malate Malate Citrate Fumarate α-Ketoglutarate Succinyl-CoA TCA cycle Glucogenic amino acids Lactate Glucogenic amino acids Glucogenic amino acids Propionyl CoA Glucogenic amino acids Pyruvate carboxylase ATP , Mg 2+ , Biotin ATP ADP + Pi Glucokinase /Hexokinase Pi H2O Glucose-6-phosphatase Pi H2O Fructose1,6- biphosphatase Fructose -6-phosphate Fructose -1,6-phosphate GDP +Pi + CO2 GTP Phosphoenolpyruvate Carboxykinase ATP ADP+Pi Phosphofructokinase Pathway of Gluconeogenesis(red) and Glycolysis(blue) Mitochondrion
  • 28. Importance of Carboxylation of Pyruvate to Oxaloacetate by Biotin-dependent Pyruvate carboxylase in Gluconeogenesis and TCA cycle ❖Importance of Carboxylation of Pyruvate to Oxaloacetate by Biotin- dependent Pyruvate carboxylase : • Activated by Acetyl-CoA. • ATP-dependent. • Biotin-dependent reaction. • Replenishes Oxaloacetate which is an intermediate of TCA cycle (ensures continuous operation of Citric acid cycle) in liver and kidney . • Provides non-carbohydrate substrates for Gluconeogenesisinliver andkidneycells . • An irreversible reaction. • Pyruvate carboxylase from muscle cells use OAA produced for replenishingTCAanddonotsynthesize glucose. Pyruvate carboxylase Biotin ATP ADP + Pi CO2 Acetyl-CoA + NADH + H+ NAD Mitochondrial matrix Cytosol Malate NAD + NADH + H+ Oxaloacetate Phosphoenol Pyruvate CO2 Glucose Gluconeogenesis *PEPcarboxykinase +GTP * Malate Oxaloacetate Malate dehydrogenase Pyruvate
  • 29. Importance of Biotin- dependent carboxylation of Acetyl-CoA to Malonyl-CoA ❖Carboxylation of Acetyl-CoA to Malonyl-CoA(cytosolic reaction) is • initial (first) and the rate limiting reaction in fatty acid biosynthesis. • irreversible reaction catalyzed by an enzyme complex , Acetyl-CoA carboxylase, that requires Biotin as a prosthetic group and utilizes bicarbonate (as a source of CO2) in presence of ATP. Acetyl-CoA+ CO2 + ATP Malonyl-CoA +ADP+ Pi ❖Biotin dependent Acetyl-CoA carboxylase is: ▪ an allosteric enzyme activated by Citrate. It is a storage vehicle for biotin. ▪ Inhibited by its end product Palmitoyl-CoA. ➢In addition to high allosteric control , high carbohydrate and low fat diet stimulates the synthesis of enzyme. ❖Malonyl-CoA is a substrate for fatty acid synthase complex. Fatty acid synthase subsequently adds 2-carbon units from Malonyl-CoA to growing fatty acid acyl chain to form Palmitate. Acetyl-CoA carboxylase Biotin
  • 30. Role of Biotin formation of Malonyl-CoA by carboxylation of Acetyl-CoA O CH3-C-SCoA Acetyl-CoA O -OOC-CH2-C-SCoA Malonyl-CoA Acetyl-CoA carboxylase CO2 ATP ADP + Pi Biotin II II Malonyl-CoA is used for fatty acid biosynthesis . Biotin-dependent Acetyl- CoA carboxylase is regulatory enzyme in fatty acid synthesis. 1 Acetyl-CoA 8 Acetyl-CoA Palmitic acid 7 Acetyl-CoA 7 Malonyl-CoA Site de novo synthesis of fatty acids : Cytoplasm of liver, adipose tissue , kidney , brain and mammary glands Acetyl-CoA is the starting material for the biosynthesis of fatty acids. The energy for the carbon – carbon condensations in fatty acid synthesis is supplied by the process of carboxylation and then decarboxylation of acetyl groups in cytosol.
  • 31. Role of Malonyl-CoA in biosynthesis of Palmitic acid Acetyl-CoA Malonyl-CoA 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 CH3- CH2- CH2- CH2- CH2- CH2- CH2- CH2-CH2- CH2-CH2-CH2-CH2- CH2- CH2- COOH Cycle I II III IV V VI VII Carbon atoms 15 and 16 are from Acetyl-CoA and Carbon 1-14 from Malonyl-CoA . 8 Acetyl-CoA are required to make one molecule of Palmitic acid . But in the reaction mechanism which is explained above only one Acetyl-CoA takes part and other 7 Acetyl-CoA take part after they are converted into 7 Malonyl-CoA. Palmitic acid
  • 32. Role of Biotin in fatty acid biosynthesis The cytosolic pathway (extra-mitochondrial pathway, De Novo synthesis of fatty acids) is a major pathway for synthesis of fatty acids. Synthesis of fatty acid from Acetyl-CoA takes place outside mitochondria . Acetyl-CoA forms Citrate which comes out of the mitochondria can cleave to give Acetyl-CoA (as such cannot come out of the mitochondria). Tissue involved in cytosolic fatty acid biosynthesis: Liver, adipose tissue, mammary gland ,brain, kidney
  • 33. Role of Biotin in fatty acid biosynthesis Other carboxylases are involved in the metabolism of odd chain fatty acids and branched-chain fatty acids.
  • 34. Carboxylation of Acetyl-CoA to Malonyl-CoA by Biotin-dependent Acetyl-CoA carboxylase Energy for the carbon-to-carbon condensation in fatty acid synthesis is supplied by process of carboxylation and then decarboxylation of acetyl groups in cytosol . The carboxylation of Acetyl-CoA to Malonyl-CoA is catalyzed by Acetyl-CoA carboxylase which needs CO2 and ATP. The coenzyme is the vitamin Biotin which is covalently bound to Lysyl residue of carboxylase. Acetyl-CoA carboxylase (inactive dimer) Acetyl-CoA carboxylase (active polymer) Acetyl-CoA Malonyl-CoA CO 2 ATP ADP +Pi Biotin Allosteric regulation of Malonyl CoA synthesis by Acetyl CoA carboxylase. The carboxyl group is contributed by dissolved CO 2. O CH3-C-S-CoA O C-CH2-C-S-CoA O O II II - + H + 2 carbon compound 3 carbon compound → → Citrate + Long-chain fatty acyl-CoA -
  • 35. Role of Biotin in Propionyl-CoA metabolism Propionyl-CoA D-Methyl malonyl-CoA L-Methyl malonyl-CoA Biotin Methyl malonyl-CoA racemase Methyl malonyl-CoA mutase Deoxy adenosyl cobalamin( vitamin B12) ATP ADP+ Pi Heme synthesis TCA Produced in metabolism of Valine , Isoleucine, Threonine Synthesis of D-Methyl malonyl-CoA : Propionyl CoA is carboxylated forming D-Methyl malonyl- CoA. The enzyme Propionyl-CoA carboxylase has an absolute requirement for the coenzyme Biotin. The D-form is isomerized to L-Methyl malonyl-CoA by enzyme Methyl malonyl-CoA racemase. CO2 Propionyl-CoA carboxylase Succinyl-CoA Gluconeogenesis (only example of glucogenic precursor from fatty acid oxidation) β-oxidation of odd chain fatty acids Mn2+
  • 36. Gluconeogenesis (only example of glucogenic precursor from fatty acid oxidation) Metabolism of Propionyl-CoA to Succinyl-CoA β-Oxidation odd chain fatty acid CH3 CH2 CO-S-CoA Propionyl-CoA Propionyl-CoA carboxylase CH3 H C COO - CO-S-CoA ATP AMP +PPi CO2 D-Methyl malonyl-CoA CH3 - OOC C H CO S–CoA Methyl malonyl-CoA racemase / epimerase L-Methyl malonyl-CoA COO - CH2 CH2 CO S CoA Methyl malonyl-CoA mutase Deoxy adenosyl cobalamin( vitamin B12) Succinyl-CoA Heme synthesis TCA I I I I I I I I I ǀ ǀ ǀ ǀ ǀ ǀ ǀ Produced in metabolism of Valine , Isoleucine, Threonine Propionyl-CoA carboxylase has an absolute requirement for the coenzyme Biotin Biotin
  • 37. Importance of Biotin dependent metabolism of Leucine ❖In the metabolism of Leucine following reaction is dependent on Biotin. β-Methyl crotonyl-CoA β-MethylGlutaconyl-CoA ❖Importance of Hydroxymethyl glutaryl-CoA (HMG) which is produced during catabolism of Leucine: • Precursor for of Cholesterol biosynthesis. • Precursor for ketone body formation. Biotin β-methyl crotonyl-CoA Carboxylase Hydroxymethyl glutaryl-CoA (HMG)
  • 38. Biotin-dependent reactions in catabolism of branched chain amino acids Valine Isoleucine* Leucine* α-keto isovalerate α-keto-β-methyl valerate α-keto isocaproate Iso butyryl-CoA 2-methylbutyryl-CoA Iso valeryl-CoA Methacrylyl-CoA Tiglyl-CoA β-Methyl crotonyl-CoA HMG-CoA Propionyl-CoA Succinyl-CoA Transamination Oxidative decarboxylation α-keto acid dehydrogenase TPP TPP TPP CO2 CO2 CO2 CO2 , ATP , Biotin β-methyl crotonyl-CoA Carboxylase Acetoacetate Lyase Acetyl-CoA CO2 , ATP , Biotin Methyl malonyl-CoA Deoxy adenosyl cobalamin( vitamin B12) α-keto acid dehydrogenase Maple syrup urine disease Valine and Isoleucine that generate Propionyl-CoA ,which is converted to Succinyl-CoA by Biotin and Vitamin B12 requiring reactions. * Ketogenic Glucogenic
  • 39. Biotin Dependent Enzymes from amino acid metabolism ❑Biotin Dependent Enzymes is : Threonine Deaminase
  • 40. Role of Biotin in catabolism of Threonine(Threonine Deaminase activity) • Threonine dehydratase produces α-ketobutyrate . • Subsequently , α-ketobutyrate is oxidatively decarboxylated by Threonine Deaminase to yield Propionyl-CoA . • Propionyl-CoA is then carboxylated to Methyl malonyl-CoA . • Methyl malonyl-CoA is isomerized to Succinyl-CoA. • Succinyl-CoA enters the Kreb’s cycle(TCA) and give rise to Pyruvate. It is a precursor for heme synthesis . • Threonine is glucogenic amino acid. Threonine Deaminase activity Threonine α-ketobutyrate Propionyl-CoA Methyl malonyl-CoA Threonine dehydratase PLP NH4 + Threonine Deaminase Oxidative decarboxylation Biotin Glucose Pyruvate Heme Methyl malonyl-CoA mutase Succinyl-CoA Deoxyadenosyl cobalamin(vitaminB12) TCA Propionyl-CoAcarboxylase
  • 41. Role of Biotin in Methionine metabolism
  • 42. Biotin independent carboxylation reactions 1. Carbamoyl phosphate synthetase , which is stepping stone for urea and pyrimidine synthesis . 2. Malic enzyme , converting Pyruvate to Malate during gluconeogenesis . 3. Carboxylation of Glutamate to form γ-carboxyglutamate (Gla) in activation of blood clotting factors.
  • 43. Role of Biotin in purine biosynthesis • Biotin is required for fixation of CO2 in carbon 6 of the purine nucleus during the de novo purine biosynthesis. Biotin→ Incorporation of CO2 in Purine synthesis do not require Biotin .
  • 44. Role of Biotin in biosynthesis of Carbamoyl phosphate of Urea cycle Formation of Carbamoyl phosphate in urea cycle do not require Biotin.
  • 45. Vitamin K cycle in carboxylation reaction • Vitamin K is required in the hepatic synthesis of prothrombin and blood clotting factors II, VII, IX and X . These proteins are synthesized as inactive precursor molecules . • Formation of the clotting factors requires the vitamin K-dependent carboxylation of Glutamic acid residue to γ- carboxyglutamate (Gla) . • This forms a mature clotting factor that contains γ-carboxyglutamate (Gla) and capable of subsequent activation. • The reaction requires O2 , CO2 and Hydroquinone form of vitamin K . • The formation of (Gla) is sensitive to inhibition by Dicumarol (an anticoagulant occurring in naturally in spoiled sweet clover)and by Warfarin (synthetic analog of vitamin K) .
  • 46. Biotin-independent Carboxylation of Glutamate to form γ- carboxyglutamate (Gla) in activation of blood clotting factors H N CH C CH2 O CH2 COO- H N CH C CH2 O CH II I I I I - COO- COO- I - - II I I γ-carboxyglutamate (Gla) residue Precursors of clotting factors II , VII ,IX, X Mature of clotting factors II , VII ,IX, X Glutamyl residue Polypeptide of clotting factor CO 2 O 2 Hydroquinone(active Vitamin K) ⃝ ⃝ + Warfarin, Dicumarol - vvvvvvvv- -vvvvvvvv -vvvvvvvv vvvvvvvv- - Liver : site of synthesis and activation of clotting factors II , VII ,IX, X
  • 47. Role of Biotin in gene silencing
  • 48. Biotinylation of proteins play role in epigenic regulation of gene function
  • 50. Regulation of gene expression by Biotin Biotin Biotinyl-AMP soluble Guanylate cyclase Guanosine triphosphate c-GMP Protein kinase G Phosphorylated proteins Transcriptional activation of gene Holocarboxylase synthetase Proteins ⃝ ⃝ ⃝ + + + Altered gene expression during Biotin deficiency and new enzymatic activities of the enzyme biotinidase is confirming suggestions of a role for Biotin in the regulation of gene expression.
  • 51. Role of Biotin in gene expression
  • 52. Biotin cycle and Holocarboxylase synthetase (HCS)-dependent transcriptional regulation
  • 53. Biotin and cell cycle regulation
  • 54. Deficiency of Biotin • The addition of raw egg white to the diet as a source of protein induces symptoms of biotin deficiency. • Raw egg contains a glycoprotein Avidin ,which tightly binds Biotin and prevents its absorption from the intestine. • With normal diet ,however it has been estimated that 20 eggs / day would be required to induce deficiency syndrome of Biotin. • Thus ,inclusion of an occasional raw egg in the diet does not lead to Biotin deficiency. • Although, eating of raw eggs is generally not recommended due to possibility of salmonella infection. • Multiple carboxylase deficiency: results from a defect in ability to link Biotin to carboxylases or to remove it from carboxylases during their degradation. Treatment is Biotin supplementation. Intake of perfect boiled eggs → No danger of Biotin deficiency
  • 55. Causes of Deficiency of Biotin ❖Deficiency of Biotin is uncommon and does not occur naturally . Since it is widely distributed in food and also supplied by the intestinal bacteria. ❖Causes of deficiency manifestations include: 1. Prolonged use of intestinal-sterilizing antibiotics e.g. Sulphonamides supplementation, broad spectrum oral antibiotics → causes destruction of intestinal flora→ biosynthesis of Biotin is affected/decreased . 2. Prolonged / High consumption of raw (uncooked) egg whites which contain Avidin (a glycoprotein). Avidin has high affinity for Biotin → binds with the imidazole ring of Biotin → blocks its absorption of Biotin by intestinal epithelial cells . (intake of 20 more eggs/day causes Biotin deficiency) . Therefore, Avidin from egg white serves as an antagonist/ anti-vitamin. 3. Genetic Deficiency of Holo-carboxylase synthetase (HCS is required for attachment of Biotin to apoenzyme) or genetic deficiency of Biotinidase. 4. Leiner’s Disease : In breast fed infants with persistent diarrhea. 5. Patients who are on long term Total parental nutrition (TPN) with inadequate Biotin supplementation .
  • 56. Symptoms in deficiency manifestations of Biotin 1. Nausea ,vomiting 2. Anorexia (Loss of appetite) → weakness 3. Anemia(pallor) 4. Seborrheic /dry scaly dermatitis 5. Hair : loss (Alopecia) , graying and thinning 6. Spectacle eyed –due to circum-ocular alopecia 7. Brittle nails 8. Glossitis 9. Muscle pain 10. Depression/ Hallucination/sleepiness/ insomnia 11. neurological manifestations Injection of Biotin 100-300mg will bring about rapid cure of these symptoms . Symptoms develop after 5-7 weeks.
  • 57. Leiner’sDisease(Acquireddeficiencyof Biotin) • Leiner’s Disease (Acquired deficiency of Biotin): Erythroderma desquamative or exfoliative dermatitis in young infants. • Occurrence of Leiner’s disease : in breast-fed infants frequently in association with persistent diarrhea. • Cause of deficiency of biotin in Leiner’s Disease : low Biotin content of human milk and poor absorption of biotin due to diarrhea. • Management of Leiner’s Disease : administration of Biotin.
  • 58. Congenital deficiencyof Biotin ❖Congenital deficiency of Biotin : • A rare genetic deficiency of holocarboxylase(holoenzyme synthetase → reflected in inadequate conversion of apocarboxylases to holocarboxylases ) . • Genetic enzyme defect in Propionyl-CoA carboxylase(reflected in a distinguishing acidemia). • Affected child cannot utilize biotin in metabolic role. • Clinical manifestation of Congenital deficiency of Biotin : 1. Dry-scaly dermatitis 2. Greying of hair 3. Alopecia (loss of hair) 4. Incoordination of movement 5. Urine : high content lactate, β-hydroxy propionate and β-methyl crotonate (due to the failure of corresponding enzyme activities).
  • 59. Fetal birth defects in biotin deficiency
  • 60. Pregnancy (more utilization of Biotin) Deficiency of Biotin ReducedactivityBiotindependentenzymes(Acetyl-CoAcarboxylaseandPropionyl-CoAcarboxylase) Alteration of lipid metabolism Alteration in metabolism of Polyunsaturated fatty acids (PUFA) and Prostaglandins Deranged(defective) muscle development Fetal birth defects like cleft palate , Micrognathia and Micromyelia Molecular basis of fetal birth defects in Biotin deficiency
  • 61. Pathophysiology of Biotinidase deficiency ❖Biotinidase deficiency ( OMIM number :253260): • Enzyme /transport defect : Biotinidase deficiency (<10% of normal serum activity) (disorder of organic acid Metabolism) . • Incidence in USA → > 1: 75000 population , carrier frequency for heterozygous for biotinidase gene mutation → 1:120 in general population. • Symptoms observed : 1. Alopecia 2. Periorificial skin rash 3. Conjunctivitis 4. Developmental delay 5. Hypotonia , seizures • Modality of acute episodes : ketoacidosis in early life • Major biochemical markers in newborns : Biotinidase (serum) , 3-hydroxy isovaleric acid (urine) • Prenatal diagnosis : enzyme assay in amniocytes • Sudden unexpected death : Anecdotal reports
  • 62. Formation of 3-hydroxyisovaleric acid under conditions of Biotin deficiency β-Methylcrotonyl-CoA carboxylase Biotin β-Methylcrotonyl-CoA Leucine Biotin deficiency 3-hydroxyisovaleric acid → excreted in urine β-Methylglutaconyl-CoA H3C SCoA CH3 O H3C OH OH CH3 O HO SCoA O CH3 O Biotinidase deficiency
  • 63. Management of Biotinidase deficiency • Treatable genetic condition with supplementation of Biotin (not dietary) . • Life long supplementation of Biotin(5-20mg/day) in preventing or relieving most symptoms . • Seizures resolves within hours to days . • Cutaneous symptoms resolve within weeks. • Resolution of Neurological defects variable.
  • 64. Multiple carboxylase deficiency (Juvenile/late form) Active Inactive Multiple carboxylase deficiency : results from a defect in ability to link Biotin to carboxylases or to remove it from carboxylases during their degradation. Treatment is Biotin supplementation.
  • 65. Dermatitis in Biotin deficiency :1 Seborrheic dermatitis Biotin responsive dermatoses Dermatitis red rash around genital area
  • 66. Spectacle eyed appearance due to circum – ocular alopecia . Dermatitis red rash around eyes ,nose and mouth. Dermatitis in Biotin deficiency : 2
  • 67. Alopecia(hair loss) H. Pylori infection Deficiency manifestations of Biotin: Alopecia and dry brittle nails
  • 68. Management of Biotin deficiency
  • 69. Toxicity of Biotin • No adverse effects of biotin in doses up to 300 times normal dietary intake (as in patients with biotinidase deficiency).
  • 70. Summary of Biotin Name of water soluble vitamin Co- enzyme form RDA (μg/day) Main reaction using co-enzyme Direct and indirect assay Deficiency disease Biotin Biocytin 200-300 Carboxylation of Pyruvate, Acetyl CoA , Propionyl CoA, β-methyl crotonyl CoA Microbiological, Competitive protein binding (CPB),carboxylases, avidin binding No specific disease. Consumption of large amount of raw egg whites (which contains protein Avidin that binds Biotin) can induce a biotin deficiency.
  • 71. Technical applications of Biotin Antagonist ❖Avidin asBiotinantagonist: • aglycoproteinpresentineggwhite.Whenfedtoanimalsinlargeamount,canproduce Biotindeficiency(namedasanti-egg-white-injury-factor). • hasgreataffinityforBiotin(presentineggyolkandboundnon-covalentlywithAvidin). • Heatlabile(boilingofeggwilldenatureAvidinandneutralizeitsinhibitoryactivity). • Onemoleculecombineswith4moleculesofBiotin. ❖AffinityofAvidinisgreaterthanmostoftheusualantigen-antibodyreactions Therefore, Avidin-BiotinsystemiscommonlyutilizedfordetectionofpathogensbyELISA. ❖DNAisgenerallylabeledbyradioactivenucleotides.BiotinlabellingofDNAisbecoming morepopular.Biotinisaddedtonucleotides,whichwillbeincorporatedintothenewly synthesizingDNA.ThefixedbiotincanbeidentifiedbyreactionwithAvidin. ❖StreptavidinpurifiedfromStreptomycesavidinii,canbind4moleculesofBiotin. ❖Biotinantagonists:Desthiobiotin,Biotinsulphonicacid.
  • 72. Signal amplification by Biotin-Avidin Complex in ELISA • Biotin and other affinity labels do not generate detectable signals on their own. • They can initiate signal amplification mediated by high-affinity binding antibodies or in case of biotin , with Avidin or streptavidin. • Biotin will tightly bind with Avidin . • Instead of enzyme directly fixed over antibody ,Biotin is labelled on the first antibody. The avidin-conjugated enzyme is added and color reaction is done as before using horse radish peroxidase or alkaline phosphatase. • The advantage here is that for each Biotin fixed ,4 Avidin molecules therefore so 4 enzyme molecules are fixed . • The intensity of color the assay is thus increased many times .
  • 73. Signal amplification by Biotin-Avidin Complex in ELISA Biotin can be used to introduce amplification into an immunoassay . The binding constant of Biotin-Avidin complex is extremely high (10 15 L/mol ) ; capitalizing on this system allows immunoassay systems to be devised even more sensitive than the simple antibody systems.
  • 74. Signal amplification by Biotin –Avidin Complex in ELISA Biotin-Avidin System : is Used in Elisa reaction for detection of pathogens in Elisa test(great affinity of avidin amplifies the signal) . It uses Biotin-labeled antibody . Biotin can be attached to the antibody without loss of immunoreactivity by the antibody . When Avidin-conjugated label is added , a complex of Ag: Ab: Ab-Biotin: Avidin label is formed . Further amplification is achieved by a Biotin : Avidin linkage because the binding ratio of Biotin : Avidin is 4:1.
  • 75. Biotinylated probes • NeedofBiotinylatedprobes:thedisposalofradioactiveprobesisbecoming increasinglyexpensivethereforenonradioactiveprobeshavebeendeveloped. ❖ApplicationsofBiotinylatedprobes:Fluorescentprobescanallowdetection andlocalizationofDNA/mRNA/miRNAsequencesincellortissuepreparations, aprocesscalledinsituhybridization. • Themostsuccessfulprobes: basedonvitaminBiotin(Biotinylatedprobes). ❖PropertiesofBiotinrequisiteforpreparationofBiotinylatedprobes:Biotin • canbechemicallycoupledtothenucleotidesusedtosynthesizetheprobe. • bindstenaciouslytoAvidin. ❖ PropertiesofAvidinsuitableforitsuseininsituhybridizationofDNAor mRNA/miRNAsequences byBiotinylatedprobes:Avidin • isareadilyavailableproteincontainedinchickeneggwhites. • canbeattachedtoafluorescentdyedetectableopticallywithgreatsensitivity. ❖DNAfragment(displayedbygelelectrophoresis)that hybridizeswith Biotinylatedprobescanbemadevisiblebyimmersingthegelinasolutionof fluorescentdye-coupledwithAvidin(afterwashingexcessAvidin).
  • 76. Non-isotopic probes using Biotin • The first practical example of non-isotopic probe labelling used a biotin label analog of dUTP. • Despite of altered steric configuration ,this nucleotide is incorporated by DNA polymerase and terminal transferase. • Alternately ,oligonucleotide probes can be label during synthesis with biotin for subsequent attachment to indicator molecules. • Labels at either the 5’ or 3’ end of the molecule are preferred (because central modifications may interfere with hybridization). • If label is biotinylated enzyme ,then large number of enzyme molecules in the complete complex provide a large increase of enzyme activity coupled with small amount of antigen to be determined and the antigen assay is correspondingly more sensitive.
  • 77. In situ hybridization using Biotinylated probes for detection of target DNA using gel electrophoresis • Technique used to detect DNA or m-RNA/ miRNA by in situ hybridization using Biotinylated probes : Gel electrophoresis . • Procedure : DNA fragment that hybridizes with the Biotinylated probe in gel and can be made visible by immersing gel in a solution of fluorescent dye- coupled Avidin. • Results : After washing away the excess Avidin , the DNA fragment that binds the probe is fluorescent and is displayed. • Application : DNA/mRNA/ miRNA can be viewed in the context of the tissue morphology. When a probe is with fluorescein ,it can be seen under UV microscope , then it is called FISH( fluorescent in situ hybridization) .
  • 78. Biotinylated hybridization probes for detection of target DNA or m-RNA Alkaline phosphatase and Horse- radish peroxidase can act on luminescent substrate to emit light.
  • 79. Principle and technique of in situ hybridization using biotinylated probes • Avidin or Streptavidin are linked to enzymes (e.g. Horse-radish peroxidase or Alkaline phosphatase) connecting single target to a single enzyme. • Enzyme activity is monitored according to enzyme substrate used (colorimetric, fluorescent or chemiluminescent). • Affinity labels can be used to capture and localize targets to an area of solid support. • Biotinylated probes can affixed to a streptavidin-coated surface. • After incubation with target nucleic acid , a second probe is added ,which is either directly labeled with fluorescence or conjugated with affinity label to an enzyme. • Multiple separation and washing steps to decrease the background OR non specific localization of reagents (results in amplification of undesired signals along with desired signals) .
  • 80. Analytical detection of limitsof immunoassays can be increased using enzyme labels ❖If the label is an biotinylated enzyme , then large numbers of enzyme molecules in complete complex provide a large increase of enzyme activity coupled with small amount of antigen being determined and the antigen assays is correspondingly more sensitive. ❖Enzymes predominate in Elisa : 1. Alkaline phosphatase 2. Horseradish peroxidase 3. Glucose -6-phosphate dehydrogenase 4. β-galactosidase ❖Alkaline phosphatase and Horse-raddish peroxidase can act on luminescent substrate to emit light .
  • 81. In situ hybridization • Allows to examine the tissue first by microscope. • Is a modified version of DNA –DNA hybridization. • If a metaphase spread chromosome preparation is probed with a gene , location of the gene on a specific chromosome can be identified e.g. Philadelphia chromosome abnormality. • Principle may be applied to histology slide also. In tissue preparation ,DNA is denatured ,the specific probe is tagged with fluorescent labels ,incubated , washed and seen under fluorescent microscope. • The process is known as in situ hybridization(FISH). • Histology section, single cells may be treated with specific antibodies tagged with biotinylated fluorescent tag and seen under microscope .Histology section may be treated with antibody linked with peroxidase and color developed. Philadelphia
  • 82. In situ hybridization cells with specific antibody showing surface immunofluorescence. Human breast cancer cells with specific antibody showing Immuno-peroxidase technique.
  • 84. DNA-DNA hybridization Double stranded DNA DNA denatured and strands separated by heat or alkali. Add biotinylated probes Parent DNA and probe hybridized if complementary sequences available. Fluorescent in situ (FISH) is a molecular technique that uses fluorescent probes that bind to only those parts of chromosomes with complementary sequences.
  • 86. Application biotinylated DNA probe in southern blotting technique Genomic DNA DNA fragments DNA cut by Restriction endonuclease AgaroseGelelectrophoresis Agarose Gel Denature by mild alkali NaOH Blot transfer on Nitrocellulose (or nylon membrane) followed by fixation of DNA on the membrane by baking at 80 ⁰C. Biotinylated DNA probes _ _ _ _ - - - - _ _ _ _ - - - - Long DNA fragments Small DNA fragments Southern blotting refers to the transfer of DNA from Agarose gel to nitrocellulose or nylon for DNA hybridization. detection of complementary nucleotide sequence in the host DNA Band visualization by Autoradiography
  • 87. Applications of Southern blotting technique • An invaluable method in gene analysis( i.e. to detect a specific segment of DNA in the whole genome). • Important for conformation of DNA cloning. • Forensically applied to detect minute quantities of DNA (to identify parenthood , thieves and rapists etc.) • Highly useful for the determination of restriction fragment length polymorphism (RFLP) associated with pathological conditions. • Mutant genes such as HbS , Cystic fibrosis, Phenylketonuria ,DMD as well as presence of viral DNA (Hepatitis B and C) can be identified by this method.
  • 88. Principle and technique of in situ hybridization using biotinylated probes Northern blotting is a procedure by which RNA molecules are transferred from agarose gel to diazo benzyloxymethyl(DBM) paper or nylon membrane by capillary action for hybridization. Denaturation of RNA by formaldehyde . RNA immobilized on the membrane is hybridized to single-stranded c-DNA probes . Determination of the size of a transcript.
  • 89. Applications of Northern blotting technique ❖Applications of Northern blotting: • is theoretically a good technique for determining the number of genes (through specific mRNA). • Determinationofhybridization patternsinmRNAsamples (RNA-DNAhybridization). • Analysis of gene expression in a tissue. • Determination of the size of a transcript. RNA extract Agarose Gel electrophoresis rRNA brands Diazobenzyloxymethyl paper(DMB) Biotinylated c-DNA probes Hybridization of Biotinylated c-DNA probes with rRNA
  • 90. Results of Multiplex miRNA northern blots via hybridization Near infrared fluorescent Northern blot Advances in oligonucleotide synthesis and fluorescence detection systems have made fluorescently labeled biotinylated affinity probes , the preferred reporter for nucleic acid analysis.
  • 91. Application of biotinylated probes in western blotting:1 ❖Western blotting is a technique by which a protein is transferred from a polyacrylamide gel to nitrocellulose or nylon membrane after electrophoresis. • The proteins are isolated from the tissue and electrophoresis is done. • The separated proteins are the transferred on to a nitrocellulose membrane. • After fixation, it is probed with radioactive antibody and autoradiographed. • Alternately ,the specific antibody is poured over, washed and a second antibody carrying biotinylated horse-radish peroxidase is added . • Hydrogen peroxide and a chromogen are layered . ❖Application of western blotting: very useful to identify the specific protein in tissue , thereby showing the expression of a particular gene.
  • 92. Application of biotinylated probes in western blotting :2
  • 93. Blot transfer techniques Southern (for DNA) Northern blot(for RNA) Western blot(for proteins) DNA* C-DNA* Antibody* DNA/RNA fragments or proteins placed in the well and then electrophoresed. Transfer to nitrocellulose or DMB or nylon membrane . Biotinylated probes added. Autoradiograph/ colorimetric, fluorescent or chemiluminescent analysis _ _ _ _ _ _ _ _ - - - - _ _ _ _ _ _ - - - - _ _ _ _ _ _ _ _ - - - - - -
  • 94. Examples of diagnostic applications of ISH and FISH 1. Assessment of gene rearrangement in leukemia. 2. Diagnosis of B-cell lymphoma by demonstration of reduced light-chain mRNA. 3. Determination of amplification of HER2 in breast cancer. 4. Diagnosis of various types of lymphomas. 5. Chromogenic in situ hybridization (CISH) for diagnosis of melanoma and lymphomas using mRNA probes for kappa and lambda chains.
  • 95. Acute promyelocytic leukemia with cryptic insertion with RARA into PML Diagnosis of B-cell lymphoma HER2 receptors and HER2 positive breast cancer Melanoma cells
  • 96. Use of Biotin-labeled probes in Elisa and Polymerase chain reaction(PCR) DuringPCRamplification,thePCRproductstypicallyislabeledwithnucleotidesthateitherradioactiveor fluorescentorhaveattachedaffinitylabels(e.g.biotinylatedprobes) .Ifdesired,singlestrandedprobescanbe obtainedbyusingBiotinlabeledprimerfollowedbysolidphaseseparationwithstreptavidin.
  • 97. Signal amplification for detection of PSA using Biotin-Avidin system
  • 98. Role of Biotin azide in purification of DNA –protein complex using iPOND methaodology The iPOND methodology enables purification of proteins bound directly or indirectly to the nascent DNA at replication forks. The method relies on labeling short fragments of nascent DNA with EdU, a nucleoside analog of thymidine. EdU contains an alkyne functional group that permits copper-catalyzed cycloaddition to a biotin azide to yield a stable covalent linkage This facilitates a single-step purification of DNA-protein complexes based on the high affinity biotin-streptavidin interaction. PMCID: PMC3671908 NIHMSID: NIHMS362 809 PMID: 22383038
  • 99. Role of Biotin in isolation of proteins on Nascent DNA in normoxia, hypoxia and reoxygenation conditions