dr gihan gawish 1. a chromophore is part (or moiety) of a molecule responsible for its color. when...
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1Dr Gihan Gawish
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A chromophore is part (or moiety) of a molecule responsible for its color.
When a molecule absorbs certain wavelengths of visible light and transmits or reflects others, the molecule has a color.
Chromophores almost always arise in one of two forms: conjugated pi systems and metal complexes
Dr Gihan Gawish
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In the conjugated system , the energy levels that the electrons jump between are extended pi orbitals created by a series of alternating single and double bonds, often in aromatic systems.
Common examples include retinal (used in the eye to detect light), various food colorings, fabric dyes (azo compounds), lycopene, β-carotene, and anthocyanins.
Dr Gihan Gawish
Azo compoundsAzo compounds
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A chemically conjugated system is a system of atoms covalently bonded with alternating single and multiple (e.g. double) bonds (e.g., C=C-C=C-C) in a molecule of an organic compound.
This system results in a general delocalization of the electrons across all of the adjacent parallel aligned p-orbitals of the atoms
Dr Gihan Gawish
1. Conjugated system
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Conjugated systems have unique properties that give rise to strong colors.
Many pigments make use of conjugated electron systems, such as beta-carotene's long conjugated hydrocarbon chain resulting in a strong orange color.
When an electron in the system absorbs a photon of light of the right wavelength, it can be promoted to a higher energy level.
Dr Gihan Gawish
1. Conjugated system
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Conjugated systems of fewer than eight conjugated
double bonds absorb only in the ultraviolet region and
are colorless to the human eye.
Dr Gihan Gawish
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Chemical structure of beta-carotene. The eleven conjugated double bonds that form the chromophore of the molecule are highlighted in red
Dr Gihan Gawish
1-1. β-carotene
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It is important to note that merely possessing alternating double and single bonds is not enough for a system to be strongly conjugated.
Some cyclic hydrocarbons (such as cyclooctatetraene) do indeed possess alternating single and double bonds.
Although the molecule may appear planar if one looks only at its chemical structure, it is in fact not, and typically adopts a "tub" conformation.
Dr Gihan Gawish
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Because the p-orbitals of the molecule do not align themselves well in this non-planar molecule, the electrons are not as easily shared between the carbon atoms.
Dr Gihan Gawish
cyclooctatetraenecyclooctatetraene
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Vitamin D is a prohormone, meaning that it has no hormone activity itself, but is converted to the active hormone 1,25-D through a tightly regulated synthesis mechanism.
Production of vitamin D in nature always appears to require the presence of some UV light; even vitamin D in foodstuffs is ultimately derived from organisms, which are not able to synthesize it except through the action of sunlight at some point in the synthetic chain.
Dr Gihan Gawish
1-2. Vitamin D
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Vitamin D3 is synthesized from 7-dehydrocholesterol, a
derivative of cholesterol, which is then photolyzed by ultraviolet light in 6-electron conrotatory electro cyclic reaction. The product is pre-vitamin D3.
Dr Gihan Gawish
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The metal complex chromophores arise from the splitting of d-orbitals by binding of a transition metal to ligands.
Examples of such chromophores can be seen in chlorophyll (used by plants for photosynthesis), hemoglobin, hemocyanin
Dr Gihan Gawish
2. The metal complex
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Chlorophyll is a chlorine pigment, which is structurally similar to and produced through the same metabolic pathway as other porphyrin pigments such as heme.
At the center of the chlorine ring is a magnesium ion.
The chlorine ring can have several different side chains, usually including a long phytol chain.
There are a few different forms that occur naturally, but the most widely distributed form is chlorophyll a.
Dr Gihan Gawish
2-1. Chlorophyll
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ChlorophyllChlorophyll
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Measurement of the absorption of light is complicated by the solvent used to extract it from plant material, which affects the values obtained,
In diethyl ether, chlorophyll a has approximate absorbance maxima of 430 nm and 662 nm, while chlorophyll b has approximate maxima of 453 nm and 642 nm
The absorption peaks of Chlorophyll a are at 665 nm and 465 nm. Chlorophyll a fluoresces at 673 nm.
Dr Gihan Gawish
Spectrophotometry of Chlorophyll
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A heme group consists of an iron (Fe) ion (charged atom) held in a heterocyclic ring, known as a porphyrin.
Dr Gihan Gawish
2-2. Hemoglobin
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A common motif in biochemistry is chromophores consisting of four
pyrrole ringsThese come in two types: the pyrroles form an open chain, no metal:
phytochrome, phycobilin, bilirubin
the pyrroles form a ring (porphyrin), with a metal in the center: heme, chlorophyll
Dr Gihan Gawish
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PorphyrinPorphyrin
PyrrolePyrrole
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Intrinsic Bases form part of
the structure of biomolecules.
Such as aromatic amino acids and nucleotide of proteins and DNA
Extrinsic It is chemically to
attach artificial groups with strong absorption spectra to proteins (Reporter groups).
Dr Gihan Gawish
Chromophore
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They should have a single site of attachment to the target macromolecule.
They should not affect the normal structure and function of macromolecules.
Dr Gihan Gawish
Reporter groups
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Some chromogenic enzyme substrates
These substrates have negligible absorbance at λ max but, when converted into product by the enzymes, a strong absorbance is detected at this wavelength
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Physical Basis of Fluorescence and Related PhenomenaPhysical Basis of Fluorescence and Related Phenomena
FluorescenceFluorescence PhosphorescencePhosphorescence ChemiluminescenceChemiluminescence
Some of the light energy initially absorbed is lost in transitions between vibrational energy levels, the light energy emitted is always of longer wavelength than that absorbedFluors (chromophores) have a characteristic fluorescence or emission spectrum as absorbance spectrum
Some of the light energy initially absorbed is lost in transitions between vibrational energy levels, the light energy emitted is always of longer wavelength than that absorbedFluors (chromophores) have a characteristic fluorescence or emission spectrum as absorbance spectrum
According to Pauli exclusion principle the atom can have three distinct energy levels(-1&0&+1), it exists in a triplet stateWhen the molecule returne to the ground state , a radiative triplet-single transittion occurs (phosphorescence ), it is a much slower process than fluroscence
According to Pauli exclusion principle the atom can have three distinct energy levels(-1&0&+1), it exists in a triplet stateWhen the molecule returne to the ground state , a radiative triplet-single transittion occurs (phosphorescence ), it is a much slower process than fluroscence
It occurs in molecules which can be promoted to an excited state as a result of a chemical reaction and which then return to the ground state with the emission of light Such as luciferin and luminol.
It occurs in molecules which can be promoted to an excited state as a result of a chemical reaction and which then return to the ground state with the emission of light Such as luciferin and luminol.
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Chemical structures of some common fluors and chemiluminescent compounds
Chemical structures of some common fluors and chemiluminescent compounds
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Use of chemiliminescence in specific staining of protein and nucleic acid blots. Peroxidase may be covalently attached to an antibody or an oligonucleotide as shown. After transfer of (a) protein or (b) nucleic acids to a suitable membrane , specific proteins or nucleic acids may be visualized by staining with luminol. Light emitted by chemiluminescence may be detected on photographic films.
Use of chemiliminescence in specific staining of protein and nucleic acid blots. Peroxidase may be covalently attached to an antibody or an oligonucleotide as shown. After transfer of (a) protein or (b) nucleic acids to a suitable membrane , specific proteins or nucleic acids may be visualized by staining with luminol. Light emitted by chemiluminescence may be detected on photographic films.
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FISHFluorescence In Situ Hybridization
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Immunofluorescence microscopy Immunofluorescence microscopy
Direct immunostainingDirect immunostaining
Indirect immunostainingIndirect immunostaining
Secondary antibodies are raised to the constant parts of immunoglobins from a particular class and species. Note the amplication of signal possible with indirect immunostaining
Secondary antibodies are raised to the constant parts of immunoglobins from a particular class and species. Note the amplication of signal possible with indirect immunostaining
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•In situ hybridization with fluorescently labelled oligonucleotide. A fluore such as fluorescein is incorporated into an oligonucleotide of defined sequence.
•The labelled oligonucleotide recognises a complementary sequence in the target nucleic acid (DNA or RNA) and hybridizes to it
•Fluorescence allows visualization of this hybrid in a microscope.
•This technique allows us to determine the location of specific nucleic acids in cell and tissue samples
•In situ hybridization with fluorescently labelled oligonucleotide. A fluore such as fluorescein is incorporated into an oligonucleotide of defined sequence.
•The labelled oligonucleotide recognises a complementary sequence in the target nucleic acid (DNA or RNA) and hybridizes to it
•Fluorescence allows visualization of this hybrid in a microscope.
•This technique allows us to determine the location of specific nucleic acids in cell and tissue samples
FISH technique
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Example: Using FISH to detect chromosome aberrations in children with Acute Lymphoblastic Leukemia
Dr Gihan Gawish
childhood acute lymphoblastic leukemia case shows red signal which is ABL on chromosome 9 and green signal which is the breakpoint cluster region (BCR) on chromosome 22 for children with Philadelphia negative acute lymphoblastic leukemia (Ph‾ ALL).
childhood acute lymphoblastic leukemia case shows red signal which is ABL on chromosome 9, green signal which is the breakpoint cluster region (BCR) on chromosome 22 and pale orange signal which is the fusion (BCR/ABL) for children with Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL).