BIOC0003
Ratio of fluorescence with A/without A
1/1+(R₀/R)⁶, where R₀ is distance at which half F lost
Kw at 25 degrees
10^-14
[H+] from pH
10^-pH
A260 and OD
1A260 unit is amount of nucleic acid in 1ml that gives 1OD
Pure DNA
260/280 of 1.8
Pure RNA
260/280 of 2.0
Absorbance value of Nucleic Acids
260nm
ssDNA A260
33microg
Oligonucleotides A260
37microg
RNA A260
40microg
dsDNA A260
50microg
To find conc of two components in a mixture from absorbance
A = ([A]xEa x l) + ([B] x Eb x l) for each wavelength, do simultaneous equations
Beer Lambert Law
A = Ecl
Photons in UV/vis/near IR
Cause electronic transitions if enough energy
Chemiluminescence
Chemical reaction excites electrons
Bioluminescence
Chemiluminescence in biological system
GFP structure
Chromophore surrounded by antiparallel beta can which excludes solvent molecules to avoid quenching
Circular dichroism
Difference between absorption of L-CPL and R-CLP
Stokes shift
Difference in intensity of absorbed and emitted light
Prism
Different wavelengths travel at different speeds, longer wavelength (red) refracted less, high efficiency, ~100% transmittance
FRET
Donor absorbs photon, and energy of excited state transferred to Acceptor which then releases a proton, resulting in an increase in A and decrease in D fluorescence
Cons of GFP
Large domain may disturb protein structure
States and fluorescence
Light emitted when S1 state returns to S0 state, light stops being emitted 10⁻⁸s after absorbed
Optical Dichroism
Log Incident/Transmitted
Effect of stray light on absorbance
Log[(I₀+s)/(I+s)]
Emission spectra vs absorption
Longer wavelength, lower energy
Proteins affects on 260/280
Lowers
Units of E
M-1cm-1
Diphenylhexatreiene
Measures physical states of lipid bilayers via anisotropy, detects detergent micelle formation; very hydrophobic, partitions into lipid bilayer with fluorescence indicating bilayer fluidity; lots of conjugated bonds, planar as has resonance structures
Energy of wave
hv = hc/λ
Henderson-Hasselbalch equation
pH = pKa + log [A-]/[HA]
Speed of light
λv, 3e8 m/s
Gibbs, enthalpy and entropy
∆G = ∆H- T∆S
∆G when protons involved
∆G°', remove [H⁺] from Ke, quote pH
∆G from ∆G⁰
∆G⁰ -RTlnQ
Gibbs and equilibrium
∆G⁰=-RTln(Ke)
∆G of cell
−nFE
Countering effect of equipment on emission
Generate correction factor from known standard
Fluourescence
Absorbance of a photon excites an electron, and some of the energy is remitted as light with a longer wavelength
Follow rate of reaction using absorbance spectrometry
Absorbance vs time
Absorbance of multiple species
Additive
Joule
Amount of energy expended when 1 N applied through 1m
∆G⁰ definition
Amount of energy harnessed to do useful work by a reaction starting under standard conditions
pH from [H+]
-log[H+]
Relationship between intensities and absorbance
A= log[I₀/I]
Plot of absorbance vs conc
At high conc, greater difference between ideal and actual behaviour so line stops being straight
Why is the signal measured at 90° to excitation
Avoid detection of unemitted light, possible as fluorescence emitted in all directions
Using an electrode to measure concentration
Barrier between two electrodes only permeable to one ion type, ion causes a pd across barrier related to ion gradient, potential measured between two electrodes changes, calibrate to provide conc readout
F group isolated from/exposed to water
Blue/red shift
Calibration curve of
Conc vs absorbance
If two substances can be interconverted, absorbance at isosbestic point
Constant regardless of conc
Spectrophotometer detector
Converts photon to electron and measures current, can be photomultiplier or photodiode
Effect of equipment on excitation
Correct effect of differential lamp strength vs wavelength by splitting small amount of excitation light and sending it through a secondary standard solution and detector with wavelength independent response
Covalent fluorescence reactive group X
Cysteine reagents, Lyseine reagents
Nernst equation
Eh = Em + R.T./nF x ln[Ox]/[Red] (n is moles of electrons)
Gram/small calorie
Energy needed to increase 1g of water by 1°C
Non covalent fluorescent probes
Ethidium bromide,diphenylhexatriene
Beers Law Fluorescence
F ~ E l c Qy
Covalent fluorescent probes
Fl group (F) attached to chemically reactive group (X) which reacts with protein or is directly attached to biomolecule
Buffer in blood
H2CO3/HCO3-
pKa from titration
Half of end point, pH when [A-] = [HA]
Inner filter effect
Higher conc of fluorescent molecules decreases proportion of light interacting with molecule of interest; linear relationship between F and C tails off at high c
Properties of fluorescent compounds
Highly conjugated, condensed system of fused rings, electron donating groups, rigid, planar
Standard hydrogen electrode
H⁺ + e⁻ ⇌ 1/2H₂ (g)
Effect of stray light at high absorbency
Increases as less light transmitted
Action spectra
Indirectly monitor a process, eg photosynthesis depends on light absorption
Emission spectra
Intensity of em vs wavelength of em; gives colours of light, measure using emission monochromator
Excitation spectra
Intensity of em vs wavelength of ex, vary excitation monochromator, gives coloursof light able to cause excitation
Scintillation
Interaction with radioactive particle excites electron
Ethidium bromide
Intercalates between DNA bases; large, largely planar, many conj bonds
Pros of GFP
Non toxic, can be used on whole organism
Eh
Overall cell potential
Colours of haemoglobin
Ox- brown, red- purple, O2 bound red- red
Lower E
Oxidised
Vibrational splitting
Photons absorbed excite electrons to higher states, which then trickle down to emit photon at lower energy and longer wavelength; all excited vibrational levels return to ground state prior to emission
Solvent effects
Polar solvent rearranges self to stabilise and lower energy of system, electron transitions occur too quickly for this to occur immediately but overall solvent interactions lower the energy of the states
Fluorescence Activated Cell Sorting
Primary antibody binds to cell epitope,second fluorescent antibody binds to constant region of first, flow cytometer sorts via electrostatic deflection
Dispersion element types
Prism or diffraction grating
Uses of covalent fluorescent probes
Probe local environment of individual amino acid residue, monitor conformational changes, determine membrane protein topography, detect molecule location in cell, determine abundance of CSM, FACS
Fluorescence and conc of fluorophore
Proportional with exception of stray light
Collisional quenching
Random non interactive collisions deactivate excited state, this type requires near contact
Quantum yield Qy
Ratio of emitted to absorbed photons, range from 0-1, number of events triggered per photon absorbed
Electrode potential of cell
Red-ox
Cathode
Reduction
Static quenching
Reporter and quencher form intramolecular dimer, non flu ground state complex forms
Monochromator
Selects wavelength of light
Source of fluorescence in GFP
Ser 65- Tyr 66- Gly 67 specially bonded on alpha helix
Isosbestic point
Wavelength at which two species have the same E
Spectrophotometers
Source, entrance slit, dispersion device, monochromator, sample, detector
Circular Dichroism Set up
Source, monochromator, linear polariser, modulator, sample, detector
Em
Standard cell potential
Quenching types
Static and Dynamic/collisional
Diffraction grating
Surface with closely spread grooves, splits and diffracts light, groove period must be on order of desired wavelength, constant dispersion
Phosphorescence
T1 returns to S0 state, as T1 is long lived, emission can occur after source turned off
Intrinsic fluorescence
Trp, Tyr, somewhat Phe
Features of a good quencher
Unpaired electrons, heavy atoms (affect electron spin spin state, increase S1 to T1 transition), electron transfer event
Reasons for differences in emission vs absorption
Vibrational splitting, solvent effects
Spectrofluorometer
Xe lam, excitation monochromator, sample, 90° to emission monochromator, detector
Kw equation
[H+][OH-]
