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tracer techniques

The tracer technique is a method used to investigate biosynthetic pathways in plants by utilizing labeled compounds, primarily radioactive and stable isotopes, to trace intermediates and steps in metabolic processes. This technique allows for high sensitivity and accuracy in detecting the location and quantity of compounds, although it has limitations such as kinetic and chemical effects. Various methods within the tracer technique include precursor product sequence, double and multiple labeling, and sequential analysis, which have applications in studying plant metabolism and compound formation.

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Megha Kamble
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15 views

tracer techniques

The tracer technique is a method used to investigate biosynthetic pathways in plants by utilizing labeled compounds, primarily radioactive and stable isotopes, to trace intermediates and steps in metabolic processes. This technique allows for high sensitivity and accuracy in detecting the location and quantity of compounds, although it has limitations such as kinetic and chemical effects. Various methods within the tracer technique include precursor product sequence, double and multiple labeling, and sequential analysis, which have applications in studying plant metabolism and compound formation.

Uploaded by

Megha Kamble
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Tracer technique

• INTRODUCTION -iving plants considered as biosynthetic


laboratory primary as well as secondary metabolite.
• Different biosynthetic pathway: - » Shikmic acid pathway »
Mevalonic acid pathway » Acetate pathway

• Various intermediate and steps are involved in biosynthetic


pathway in plants can be investigated by means of following
techniques: - » Tracer technique » Use of isolated organ »
Grafting methods » Use of mutant strain

• Definition: - It can be defined as technique which utilizes a


labelled compound to find out or to trace the different
intermediates and various steps in biosynthetic pathways in
plants, at a given rate & time. OR
• In this technique different isotope, mainly the radioactive
isotopes which are incorporated into presumed precursor of
plant metabolites and are used as marker in biogenic
experiments.

• The labelled compound can be prepared by use of two


types of isotopes. » Radioactive isotopes. » Stable isotopes.
• Radioactive isotopes: - [e.g. 1H, 14C, 24Na, 42K, 35S, 35P,
131I decay with emission of radiation] – For biological
investigation – carbon & hydrogen. – For metabolic studies – S,
P, and alkali and alkaline earth metals are used. – For studies
on protein, alkaloids, and amino acid – labelled nitrogen atom
give more specific information. – 3H compound is
commercially available.
• Stable isotopes: - [e.g. 2H, 13C, 15N, 18O] – Used for labelling
compounds as possible intermediates in biosynthetic
pathways. – Usual method of detection are: – MASS
spectroscopy [15N, 18O] – NMR spectroscopy [2H, 13C]

• SIGNIFICANCE OF TRACER TECHNIQUE • Tracing of


Biosynthetic Pathway: - e.g. By incorporation of radioactive
isotope of 14C into phenylalanine, the biosynthetic
cyanogenetic glycoside prunasin, can be detected.
• Location & Quantity of compound containing tracer: - 14C
labelled glucose is used for determination of glucose in
biological system Different tracers for different studies: - For
studies on nitrogen and amino acid. (Labelled nitrogen give
specific information than carbon) Convenient and suitable
technique
• CRITERIA FOR TRACER TECHIQUE -The starting concentration
of tracer must be sufficient withstand resistance with dilution
in course of metabolism.
• Proper Labelling: - for proper labelling physical & chemical
nature of compound must be known.
• Labelled compound should involve in the synthesis reaction.
• Labelled should not damage the system to which it is used.

ADVANTAGES • High sensitivity. • Applicable to all living


organism. • Wide ranges of isotopes are available. • More reliable,
easily administration & isolation procedure. • Gives accurate result,
if proper metabolic time & technique applied. LIMITATION • Kinetic
effect • Chemical effect • Radiation effect • Radiochemical purity •
High concentration distorting the result.
•
• REQUIREMENT FOR TRACER TECHNIQUE – Preparation of
labelled compound. – Introduction of labelled compound into a
biological system. – Separation & determination of labelled
compound in various biochemical fractions at later time. I.
Preparation of Labelled Compound: - The labelled compound
produce by growing chlorella in atmosphere of 14CO2 . All
carbon compounds 14C labelled. The 3H (tritium) labelled
compound are commercially available. Tritium labelling is
effected by catalytic exchange in aqueous media by
hydrogenation of unsaturated compound with tritium gas.
Tritium is pure β – emitter of low intensity & its radiation
energy is lower than 14C. By the use of organic synthesis: - 14
CH3MgBr + 14CO2 CH3 COOHMgBr + H2O 14

• II. Introduction of labelled compound: - PRECAUTION: - The


precursor should react at necessary site of synthesis in plant.
Plant at the experiment time should synthesize the compound
under investigation •The dose given is for short period. 1. Root
feeding 2. Stem feeding 3. Direct injection 4. Infiltration 5.
Floating method 6. Spray technique III. Separation and
detection of compound: - a) Geiger – Muller counter. b) Liquid
Scintillation counter. c) Gas ionization chamber. d) Bernstein –
Bellentine counter. e) Mass spectroscopy. f) NMR
eletrodemeter. g) Autoradiography.
•
• METHODS IN TRACER TECHNIQUE 1. PRECURSOR PRODUCT
SEQUENCE: - In this technique, the presumed precursor of the
constituent under investigation on a labelled form is fed into
the plant and after a suitable time the constituent is isolated,
purified and radioactivity is determined.
• Disadvantage: - The radioactivity of isolated compound alone
is not usually sufficient evidence that the particular compound
fed is direct precursor, because substance may enter the
general metabolic pathway and from there may become
randomly distributed through a whole range of product.
• Application: - •Stopping of hordenine production in barley
seedling after 15 – 20 days of germination. •Restricted
synthesis of hyoscine, distinct from hyoscyamine in Datura
stramonium. •This method is applied to the biogenesis of
morphine & ergot alkaloids
•
•

2. DOUBLE & MULTIPLE LABELLING: - This method give the evidence for nature of

•
•

3. COMPETITIVE FEEDING: - If incorporation is obtained it is necessary to consider wh

•
• 4. ISOTOPE INCORPORATION: - This method provides
information about the position of bond cleavage & their
formation during reaction. E.g. Glucose – 1- phosphatase
cleavage as catalyzed by alkaline phosphatase this reaction
occur with cleavage of either C – O bond or P – O bond. CH2OH
CH2OH O O 18 OH OH + H2 O OH + H2PO 4 OPO 3H OH OH
OH OH
•
• 5. SEQUENTIAL ANALYSIS: - The principle of this method of
investigation is to grow plant in atmosphere of 14CO2 & then
analyze the plant at given time interval to obtain the sequence
in which various correlated compound become labelled.
Application: - 14CO2 & sequential analysis has been very
successfully used in elucidation of carbon in photosynthesis.
Determination of sequential formation of opium hemlock and
tobacco alkaloids. Exposure as less as 5 min. 14CO2, is used in
detecting biosynthetic sequence as – Piperitone --------- (-)
Menthone ---------- (-) Menthol in Mentha piperita.
•
• APPLICATION OF TRACER TECHNIQUE 1. Study of
squalene cyclization by use of 14C, 3H labelled mevalonic acid.
2. Interrelationship among 4 – methyl sterols & 4, 4 dimethyl
sterols, by use of 14C acetate. 3. Terpenoid biosynthesis by
chloroplast isolated in organic solvent, by use of 2- 14C
mevalonate. 4. Study the formation of cinnamic acid in
pathway of coumarin from labelled coumarin. 5. Origin of
carbon & nitrogen atoms of purine ring system by use of 14C
or 15N labelled precursor. 6. Study of formation of scopoletin
by use of labelled phenylalanine. 7. By use of 45Ca as tracer, -
found that the uptake of calcium by plants from the soil. (CaO
& CaCO2). 8. By adding ammonium phosphate labelled with
32P of known specific activity the uptake of phosphorus is
followed by measuring the radioactivity as label reaches first
in lower part of plant, than the upper part i.e. branches, leaves
etc.

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