Lecture 7
Bio 451 Molecular Biology Techniques
An atom is made up of proton and neutrons that form the nucleus of the atom, and electrons that revolve around the nucleus. The protons have a positive charge, the neutrons have no charge and the electrons have a negative charge. The atomic number is determined by the number of protons which is equal to the number of electrons. The atomic number determines the identity of the element. The atomic mass is determined by the sum of the protons and neutrons. Although all atoms of the same element have the same number of protons, they do not always have the same the same number of neutrons. Each atom of the same element with different number of neutrons is called an isotope of an element. A radioisotope is an unstable isotope. Therefore, the radioisotope decays. That is, the nucleus disintegrates releasing energy and particles from the nucleus (in the case of a and b emitters).
Commonly used radioisotopes
|
Radioisotope |
Half life |
|
3H |
12.26 years |
|
14C |
5,730 years |
|
22Na |
2.62 years |
|
32P |
14.28 days |
|
33P |
25.4 days |
|
35S |
87.9 days |
|
42K |
12.36 hrs |
|
45Ca |
163 days |
|
59Fe |
45.6 days |
|
125I |
60.2 days |
|
131I |
8.04 days |
|
203Hg |
49.9 days |
The use of radioisotopes to label specific molecules in a defined way has greatly furthered the discovery and dissection of biochemical pathways. Although most of these protocols involve the use of only microcurie (µCi) amount of radioactivity, some particularly those describing the metabolic labeling of protein or nucleic acids within cells, require amounts on the order of tens of millicuries (mCi).
Each element is characterized by its atomic number, defined as the number of orbital electrons or the number of protons in the nucleus of that atom.
Isotopes of a given element exist because some atoms of each element while having the same number of protons, have a different number of neutrons and therefore a different nuclear weight. The number of electrons outside the nucleus remains the same for all isotopes of a given element, so all isotopes of a given element are equivalent with respect to their chemical reactivity.
· Radioactive decay occurs when subatomic particles are released from the nucleus of an atom of a heavy isotope. This often results in the conversion of an atom of one isotope to an isotope of a different element, because the original isotope’s atomic number changes after decay. The subatomic particles released from naturally occurring radioisotopes are of three basic types: a and b particles and g rays.
· An a particle is essentially the nucleus of a helium atom, or two protons plus two neutrons. It is a relatively large, heavy particle that moves slowly and usually only across short distances before it encounters some other atom with which it interacts. These particles are released from isotopes with large nuclei (atomic number greater than 82; e.g., plutoniun or uraniun); such isotopes are not commonly used in biological research.
· b particles are light, high speed charged particles. Negatively charged b particles are essentially electrons of nuclear origin that are released when a neutron i converted to a proton. Release of a b particle thus changes the atomic number and elemental status of the isotope.
· g rays refer to the electromagnetic radiation and is not deflected by electric and magnetic fields. Unlike b particle release, the release of g radiation by itself produces an isotopic change rather than an elemental one; however, the resultant nuclei are unstable and often decay further, releasing b particles.
· The energy of all a particles and g rays is fixed because they are of specific composition or wavelength. The energy of b particles, however, varies depending on the atom they originate from. There are high-energy b particles released during the decay of 32P and low-energy b particles released when tritium (3H) decays.
· a, b and g emissions all have the potential, upon encountering an atom, to knock out its electrons, thereby creating ions. Thus, these three types of emissions are called ionizing radiation. The formation of such ions may result in the perturbation of biological processes: therein lies the danger associated with radioactivity.
1 Becquerel (Bq)= 1 disintegration per second
The more commonly encountered unit is the Curie (Ci):
1 Ci=3.7X107 Bq
1 Ci=2.22X1012 disintegrations per minute (dpm)
1 mCi=2.22X109 dpm
1µCi=2.22X106 dpm
counts per minute (cpm) is what appears in the b counters or g counters:
cpm=dpmX (counting efficiency of machine)
When designing any experiment using radioactivity, every effort should be made to limit the time spent directly handling the vials or tubes containing the radioactive material.
Distance helps to determine dose. When possible, experiments involving radioactivity should be performed in an area separate from the rest of the lab.
The energy of the particles released during the decay of an isotope determines what, if any, type of shielding is appropriate. b particles released during the decay of 14C and 35S possess roughly ten times the energy of those released when 3H decays. All three b particles are of relatively low energy, do not travel very far in air, and cannot penetrate solid surfaces. No barriers are necessary for shielding against this type of b radiation. The major health threat from these isotopes occurs through their accidental ingestion, inhalation, or injection. b particles released during the decay of 32P have 10-fold higher energy than those released from 14C and pose significant threat to workers. (one reported hazard is the potential for induction of cataracts in the unshielded eye).
Low density materials such as water, glass, and plastic are suitable shields against the 32P b radiation because of the Bremsstrahlung radiation.
When mCi amounts of 32P are used at one time it is necessary to also block the Bremsstrahlung radiation by adding layer of high-density material such as 4-6mm lead to the outside of Plexiglas shield.
g rays released during the decay of 125I have much higher penetrance than the b particles from 32P decay; this radiation must be stopped by very-high density material, such as lead.