Dr Gihan Gawish 1

 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 complexesconjugated pi systemsmetal complexes  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 complexesconjugated pi systemsmetal complexes Dr Gihan Gawish 2

chromophorechromophore 3

 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.  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 compounds 4

 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 5

 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 6

 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 7

Chemical structure of beta-carotene. The eleven conjugated double bonds that form the chromophore of the molecule are highlighted in red Dr Gihan Gawish 8

 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.cyclooctatetraene  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 9

 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 cyclooctatetraene 10

 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 11

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 12

 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 13

 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 14

Dr Gihan Gawish ChlorophyllChlorophyll 15

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 16

 A heme group consists of an iron (Fe) ion (charged atom) held in a heterocyclic ring, known as a porphyrin. Dr Gihan Gawish 17

A common motif in biochemistry is chromophores consisting of four pyrrole rings These 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 A common motif in biochemistry is chromophores consisting of four pyrrole rings These 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 18

Dr Gihan Gawish Porphyrin PyrrolePyrrole 19

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 20

 They should have a single site of attachment to the target macromolecule.  They should not affect the normal structure and function of macromolecules.  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 21

Dr Gihan Gawish 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 22

Dr Gihan Gawish Physical Basis of Fluorescence and Related Phenomena Fluorescence Phosphorescence Chemiluminescence 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 absorbed Fluors (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 absorbed Fluors (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 state When 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 state When 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. 23

Dr Gihan Gawish 24 Chemical structures of some common fluors and chemiluminesc ent compounds

Dr Gihan Gawish 25 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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Dr Gihan Gawish 27 Immunofluorescence microscopy Direct immunostaining 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

Dr Gihan Gawish 28 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 Gawish30 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).