New Microsoft PowerPoint Presentation
New Microsoft PowerPoint Presentation
New Microsoft PowerPoint Presentation
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:
CH3MgBr + 14CO2 CH3 14COOHMgBr + H2O
CH314COOH + Mg(OH)Br
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.
h) Radio paper chromatography.
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.
Application:
• Stopping of hordenine production in barley seedling after 15 – 20
daysof 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 biochemical incorporation of precursor arises
double & triple labelling. In this method specifically labelled precursor
and their subsequent degradation of recover product are more
employed.
Application: -
• This method is extensively applied to study the biogenesis of plant
secondary metabolite.
• Used for study of morphine alkaloid.
E.g. Leete, use Doubly labeled lysine used to determine which
hydrogen of lysine molecule was involved in formation of piperidine
ring of anabasine in Nicotina glauca.
3. COMPETITIVE FEEDING: - If incorporation is obtained it is necessary to
consider whether this infact, the normal route of synthesis in plant not the
subsidiary pathway. Competitive feeding can distinguish whether B & B’ is normal
intermediate in the formation of C from A.
OR
Application:
• This method is used for elucidation of biogenesis of propane alkaloids.
• Biosynthesis of hemlock alkaloids (conline, conhydrine etc) e.g. biosynthesis of
alkaloids of Conium maculactum (hemlock) using 14C labelled compounds.
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.
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