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![• The labelled compound can be prepared by use of two types of
isotopes.
i) Radioactive isotopes.
ii) Stable isotopes.
i) 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.
ii) 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]](https://image.slidesharecdn.com/tracerstechniqueandradioisotopesintheinvestigationofbiogeneticstudies-200918073026/75/Tracers-technique-and-radioisotopes-in-the-investigation-of-biogenetic-studies-13-2048.jpg)
Tracers technique and radioisotopes in the investigation of biogenetic studies
This document discusses tracer techniques and radioisotopes used in biogenetic studies of plants. It provides details on: - Tracer techniques involve using radioactive isotopes as markers incorporated into plant metabolite precursors to study biosynthetic pathways. - Radioisotopes are unstable atoms that decay through emission of particles like alpha, beta, or gamma radiation. Different isotopes have varying half-lives and penetration powers. - Techniques like precursor-product sequence, double labeling, competitive feeding, and sequential analysis use radioisotopes to study the order and pathways of compound formation in plants.
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Tracers technique and radioisotopes in the investigation of biogenetic studies
- 1. Tracer techniques, Radioisotopes used in theinvestigation of Biogenetic studies
- 2. Introduction • Living plantsconsidered as biosynthetic laboratory primary as well as secondary metabolites. • Different biosynthetic pathway:- -Shikmic acid pathway -Mevalonic acid pathway -Amino acids 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 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.
- 3. Radioisotopes • Radioisotopes areradioactive isotopes of an element. • They can also be defined as atoms that contain an unstable combination of neutrons and protons, or excess energy in their nucleus. • The term isotope is formed from the Greek roots isos ("equal") and topos ("place"), meaning "the same place“. • Isotope, one of two or more species of atoms of a chemical element with the same atomic number and position in the periodic table and nearly identical chemical behaviour but with different atomic masses and physical properties. • All isotopes of a given element have the same number of protons but different numbers of neutrons in each atom. • Every chemical element has one or more isotopes. • It was coined by a Scottish doctor and writer Margaret Todd in 1913 Examples: Carbon-12, Carbon-13, and Carbon-14 are three isotopes of the element Carbon with mass numbers 12, 13, and 14, respectively. The atomic number of carbon is 6, which means that every carbon atom has 6 protons, so that the neutron numbers of these isotopes are 6, 7, and 8 respectively.
- 4.
- 5. Radioactive Decay • Anunstable atomic nucleus spontaneously loses energy by emitting ionizing particles and radiation. Radioactive isotope Parent Nuclide Daughter Nuclide • When an unstable nucleus decays, it may give: 1) Alpha or helium Radiation. 2) Beta or Electron Radiation. 3) Gamma Radiation.
- 6. 1) Alpha particledecay • Alpha particles are made up of 2 protons and 2 neutrons. • Are same as helium nucleus. • When a nucleus emits alpha particle, its atomic number decreases by 2 and its atomic mass decreases by 4. • These particles are relatively slow and heavy. • Low penetrating power. • As they have large charge, alpha particles ionize other atoms strongly. • Alpha decay occurs in very heavy elements like Uranium and Radium.
- 7. 2) Beta ParticleDecay • These particles are as same as electrons as they have charge of minus 1. • When a nucleus emits β particle the Atomic number increases by 1 and Atomic mass is unchanged. • They are fast and light. • Have Medium Penetrating Power. • Example of radioisotope emitting β, phosphorus-32. • These particles ionize atoms that they pass, but not as strongly as alpha particles do.
- 8. 3) Gamma rays •Gamma rays are Waves not Particles. • They have no mass and no charge. • Atomic mass and number unchanged. • High Penetrating Power. • Do not directly ionize other atoms. • We don’t find pure gamma source, they are emitted along alpha or beta particles. • Useful gamma source technetium-99, used as tracer in medicine.
- 9. Penetration power ofthe Radiations
- 10. Half-life • The halflife of radioisotope is the time for the radiation level to decrease (decay) to one half of the original value. • Naturally occurring tend to have longer half lives. • Used in nuclear medicine have short half lives.
- 11. Radiation Units 1. Curie(Ci)-measures activity as the number of atoms that decay in one second. 1 Curie (Ci) = 3.7 × 1010 decays/sec. 2. rad (radiations absorbed dose)- Measures the radiation absorbed by the tissues of the body. 1 rad = 0.01 Gy = 0.01 J/kg 3. rem (radiation equivalent mass)- Measures the biological damage caused by different types of radiation. 1 rem = 0.01 Sievert 4. Becquerel (Bq) - 1Bq = 1 Disintegration per sec (dps) 5. Sievert (Sv) = 100 rem 6. Gray (Gy) = 1 J/Kg Tissue
- 12. Tracer technique It canbe defined as techniques which utilizes a labelled compound to find or to trace the different intermediates and various steps in biosynthetic pathways in plants, at a given rate and 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.
- 13. • The labelledcompound can be prepared by use of two types of isotopes. i) Radioactive isotopes. ii) Stable isotopes. i) 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. ii) 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]
- 14. Criteria for tracertechniques • 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.
- 15. Requirement for tracertechnique 1. Preparation of labelled compound. 2. Introduction of labelled compound into a biological system. 3. Separation & determination of labelled compound in various biochemical fractions at later time.
- 16. 1.Preparation of LabelledCompound:- 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:-
- 17. 2. Introduction oflabelled 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. Root feeding: From roots, roots are the main site for biosynthesis.(Rauwolfia-alkaloids, ) Stem feeding: Feeding is done from Stem, mainly for latex containing plants(Rubber, indian hemp, etc) Direct injection: Administrate through injection, for hollow stem plants(Umbeliferious, opium, etc) Infiltration: Suitable for rooted plant. Floating method: Achieved when there is small amount of material is available Spray technique: by spraying method, steroid containing plants
- 18. 3. Separation anddetection of compound:- • Geiger–Muller counter. • Liquid Scintillation counter. • Gas ionization chamber. • Bernstein–Bellentine counter. • Mass spectroscopy. • NMR eletrodemeter. • Autoradiography. • Radio paper chromatography.
- 19. Methods 1. PRECURSOR PRODUCTSEQUENCE: • 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.
- 20. 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 labelled lysine used to determine which hydrogen of lysine molecule was involved in formation of piperidine ring of anabasine in Nicotina glauca.
- 21. 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. 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.
- 22. 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.
- 23. 5. SEQUENTIALANALYSIS: • Theprinciple 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.
- 24. Application of tracertechnique Study of squalene cyclization by use of 14C, 3H labelled mevalonic acid. Interrelationship among 4 – methyl sterols & 4, 4 dimethyl sterols, by use of 14C acetate. Terpenoid biosynthesis by chloroplast isolated in organic solvent, by use of 2- 14C mevalonate. Study the formation of cinnamic acid in pathway of coumarin from labelled coumarin. Origin of carbon & nitrogen atoms of purine ring system by use of 14C or 15N labelled precursor. Study of formation of scopoletin by use of labelled phenylalanine. By use of 45Ca as tracer, - found that the uptake of calcium by plants from the soil. (CaO & CaCO2). 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.
- 25. Advantages High sensitivity. Applicable toall living organism. Wide ranges of isotopes are available. More reliable, easily administration & isolation procedure. Gives accurate result, if proper metabolic time & technique applied.
- 26. Disadvantages Kinetic effect Chemical effect Radiationeffect Radiochemical purity High concentration distorting the result.