3.4.5 Chemiluminescence (화학발광)
• Chemiluminescence (sometimes "chemoluminescence") is the emission of light
(luminescence), as the result of a chemical reaction.
• Radiative transitions in molecules are 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
• Given reactants A and B, with an excited intermediate ◊, [A] + [B] → [◊] → [Products] + light-따로존재-서로결합
• Molecules such as luciferin and luminol show this property and, when used as substrates by the enzymes firefly luciferase and peroxidase, respectively, emit light which may be measured or detected.
Chemiluminescence - luminometer
• Chemiluminescence is measured in a luminometer.-자체적인 화학적발광
• The reaction leading to light emission is carried out in a cuvette.
• The wavelength and intensity of emitted light is detected.
Chemiluminescence - ATP quantitation
The level of ATP (or of any biomolecule which may be coupled to ATP production) may be quantified using luciferin and luciferase.
This assay is extremely sensitive allowing detection at the pM/fM level.
•
Bioluminescent Reactions Catalyzed by Firefly Luciferase.
Firefly luciferase, using ATP, catalyzes the two-step oxidation of luciferin to oxyluciferin, which yields light at 560 nm.
In this reaction, ATP is hydrolyzed to AMP and pyrophosphate.
Chemiluminescence
Luminol is activated with an oxidant. In the presence of a catalyst such as an iron compound (hemoglobin), luminol generates very unstable intermediate, resulting in blue light.
•
•
These reactions are highly efficient and may be readily incorporated into sensitive assay systems or in staining of protein or nucleic acid blots.
Since peroxidase-conjugated antibodies are extensively used in immunodetection systems, luminol in particular has wide applications in this general area.
안티젠의 양이많다-웨스턴블락
Chemiluminescence – Western
Chemiluminescent detection of immobilized proteins (Western Blot) or immobilized nucleic acids (Southern and Northern Blot) conjugated with horseradish peroxidase (HRP) directly or indirectly.
Principle of protein detection procedure:
Principle of nucleic acid detection procedure:
Figure 3.22. Use of chemiluminescence in specific staining of protein and nucleic acid blots.
(a) Peroxidase may be covalently attached to an antibody as shown. After transfer of protein to a suitable membrane, specific proteins may be visualized by staining with luminol. Light emitted by chemiluminescence may be detected on photographic film.
3.5 Spectroscopic techniques using plane- polarized light – Circular dichroism (CD)
Plane-polarized light
Lights by linear vs circular polarization
•
Linearly polarized light:
Electric vector direction constant
but magnitude varies.
Circularly polarized light:
Electric vector direction varies but
magnitude constant
Lights by linear vs circular polarization
Circularly Polarized Light
Two forms: left and right handed Used for circular dichroism (CD)
Linear-circular-방향성 wavelength마다 달라짐
Circular Dichroism (CD)
• The technique circular dichroism (CD) measures the difference between어떠한물질 달라지냐 -방향성
the absorption of left and right handed circularly-polarized light.
• These interact equally with non-chiral chromophores, but differently when the chromophore has a right- or left-handed form. The result is a small difference in the extinction coefficients for left- and right-polarized light, which varies with wavelength.-protein 방향성
• The CD signal is a very small difference between two large originals.
• ∆ε is the difference in the extinction coefficients, typically < 10 M-1cm-1
Circular Dichroism (CD)
• After passing through the sample, the L & R beams have different amplitudes and the combination of the two unequal beams gives elliptically polarized light.
• CD measures the ellipticity of the transmitted light.
• ∆ε, ∆A, or ellipticity is plotted against wavelength in a CD spectrum.
CD equipment-signal특성
• Light is first plane-polarized by polaroid.
• L- or R-polarization can be selected using an electro-optic modulator (photoelastic modulator) in which a piezoelectric crystal of quartz, or other optically active material, is subjected to an alternating electric filed which distorts the crystal in such a way as to transmit left or right circularly polarized light alternately.
• This passes through the sample and into the detector and analysis circuitry, which are electrically synchronized with modulator so that light transmitted in L- or R-polarization may be measured separately. => ∆ε, ∆A, or ellipticity
CD equipment-큐벳-absorbance
CD signals for different protein secondary structures
• The phenomenon of circular dichroism is very sensitive to the secondary structure of proteins. Secondary structure elements such as alpha helix and beta sheet impose additional chirality on the polypeptide chain.
• This gives rise to CD effects in the far-UV (190-240 nm), arising mainly from the peptide backbone groups, that are characteristic of the overall secondary structure of the protein.
Circular dichroism spectra typical of membrane proteins composed of different secondary structural types: predominantly antiparallel alpha-helical bundle (red: a sodium channel pore); predominantly beta-barrel (blue: BTUB outer membrane cobalamin transporter), mixed helical, beta sheet and unordered structure (green: WZA translocon for capsular polysaccharides
Applications of CD to prediction of secondary structure ration
• Myoglobin example: qt = xaqa + xbqb + xcqc
• Fits best with
xa =80%, xb=0%, xc=20%
• Agrees well with structure 78% helix, 22% coil
Applications of CD to protein stability
• Protein loses secondary structure when denatured. Thus, CD can be employed to measure protein stability.
CD signals for different DNA forms
다른형태-방향성
솔트농도 피에이치 dna protein
Chap 3. Spectroscopy
Part 5 – Surface plasmon resonance (SPR) and biolayer interferometry (BLI)
3.10. Surface plasmon resonance (SPR) Principle of SPR
• Surface plasmon resonance (SPR) is an optical technique which depends on changes in refractive index near metal surfaces.
• When two surfaces, one a metal and the other a dielectric material, are exposed to a beam of plane-polarized light of wavelength, λ, a longitudinal charge density wave (a surface plasmon) is propagated along the interface between them.
• This only happens when one of the surfaces is a metal and works best with silver, gold, copper and aluminum.
• This is because metals contain free oscillating electrons called plasmons.
Principle of SPR
• At a certain incident angle, known by a resonance angle, light is absorbed by the electron on the metal film of the sensor chip, causing them to resonate. The result is a intensity loss in reflected beams (appears to be dark band).
• The surface plasmon is sensitive to changes in any change of this boundary (environment
< 300 nm), such as the adsorption of molecules to the surface, and therefore has potential as a sensing probe.
• Linear relationship is found between an angle shift and the mass of bound molecules on the surface.
Principle of SPR
• Intensityofreflectedlightdepletes.
• Anglechangeswithassociationordissociationofmoleculesonthesurface.
• Thechangeinangleisconvertedtoasignaldirectlyproportionaltomass
bound at surface
