test 1 Flashcards
fluorescence polarization anisotropy
stable receptor-ligand complex has a large molecular mass
the complex tumbles and rotates very slowly in solution
when this complex is excited with polarized laser light, most of the fluorescing molecules emit light with a very similar polarization
high throughput screening (HTS) definition
competitive binding assay measured by fluorescence polarization anisotropy
high throughput screening (HTS)
if the compound is capable of binding more strongly to the receptor, the labelled ligand will be forced out
fluorescently labelled ligand is diffusing and rotating freely in the solution
its rotational diffusion is faster
most photons are now emitted with an arbitrary, very different polarization
by measuring this different polarization, the binding constant of each chemical compound can be determined very accurately
Absorption spectroscopy
determination of concentration of certain biomolecules
determining DNA purity
measuring oxygen saturation in blood
measuring cooperativity of hemoglobin
Intrinsic fluorescence spectroscopy
identification of the presence of fluorescing or fluorescently labelled biomolecules
Determination of metabolic activity from NADPH-fluorescence
fluorescence labelling
identification of labelled biomolecules in complex biological environments
FPA (fluorescence polarization anisotropy)
determining binding processes or other processes that can affect the rotational diffusion of labelled species
Receptor-ligand binding
molecular mass estimate
enzyme kinetics
enzyme inhibition
Forster resonance energy transfer (FRET)
determining binding processes, conformational transitions, and biomolecular distances
Receptor-ligand binding
conformation changes in DNA
determining distances during protein unfolding
membrane fusion
Fluorescence kinetics
sensing changes in the environmental polarity of biomolecules or other processes that can affect the excited state lifetime of fluorescence markers
detecting FRET or FPA via fluorescence kinetics
Fluorescence recovery after photobleaching
membrane diffusion
Biochemiluminescence
monitoring ATP concentrations
monitoring protein expression
Circular dichroism (CD), optical rotation dispersion
determination of amount of secondary structure elements in proteins
Light scattering
determination of molecular mass of biomolecules
aggregation and shape of biological objects
vibrational spectroscopy (infrared / Raman spectroscopy)
determination of secondary structure elements
label-free identification of chemical composition
Nuclear magnetic resonance (NMR)
structure determination under physiological conditions
determination of structural flexibility of biomolecules
label-free observation of biomolecular processes
comparison of NMR structure of insulin and severin with X-ray structure
flexibility of severin structure under physiological conditions
Electron paramagnetic resonance (EPR)
observation of redox reactions in photosynthesis or haemoglobin
determination of rotational diffusion using spin labels
determination of membrane structure and dynamics
Mass spectrometry
identifying biomolecules from mixtures
determining biomolecular structures from fragments
peptide sequencing
Fluorescence microscopy
imaging of labelled proteins in a whole cell context
real-time imaging of biological processes
Light
a wave of oscillating electric and magnetic fields propagating through space
Light field components
The electric and magnetic field components of electromagnetic radiation are oscillating in phase perpendicular to the propagation direction and with respect to each other
The smallest possible unit of light is…
photons
Photons
smallest possible unit of light and have particle-light and wave-light character
equation for energy including frequency or wavelength
E = hv = hc / wavelength, E = hr, v = 1 / wavelength
h variable
Planck’s constant
The energy of a photon is….
linear proportional to the frequency v and wave number
The wavelength of a photon is…
inversely proportional to the energy
UV is important for…
the characterization of proteins and DNA
The visible region of light is important for…
most fluorescence techniques
Infrared region is important for…
the identification and investigation of biomolecules based on their characteristic molecular vibrations
Radio-waves and microwaves are used for…
nuclear and magnetic resonance techniques
If a molecular possesses a suitable electronic structure…
it can absorb a photon of a specific wavelength, resulting in an electronic structure rearrangement
During the absorption of a photon…
the electronic structure of the molecules is usually transferred from an electronic ground state into an energetically higher electronic excited state
Only photons having __________ corresponding to _________ can be absorbed by the molecules
energies
the energy difference between these electronic states
electrons display…
wave-light properties
In phase
the two lines make a big squiggly line
out of phase
the two lines make a straight line
Molecular orbitals
the possible wave functions for single electrons in the presence of several nuclei
An orbital can only be occupies by…
a maximum of two electrons with opposite spins
If two atomic s-orbitals with opposite signs (out of phase) are combined, they form…
an antibonding molecular sigma orbital
Electrons in antibonding have…
higher energies
antibonding orbitals can lead to…
bond breakage
the energy of the newly formed molecular orbitals…
increases with the number of nodal planes
electrons from the original p-orbitals are filled into molecular orbitals, starting with…
the lowest energy orbitals and obeying the rule that only a maximum of two electrons are allowed per molecular orbital
HOMO
highest occupied molecular orbital
LUMO
lowest unoccupied molecular orbital
The electronic ground state corresponds to the situation…
that all orbitals up to the HOMO electrons are occupied by two electrons
The first excited state corresponds to the situation…
that one electron has been promoted from the HOMO to the LUMO
The electronic ground state and the first possible electronically excited state of molecules are often referred to as…
S0 and S1 states
The absorption band observed for the longest wavelength (lowest photon energy), corresponds to…
the absorption of photons having energies corresponding to transitions from the electronic ground state to the first possible electronically excited state (S0 - S1)
absorption spectrum
describes the frequency or wavelength dependence of the probability that a molecule absorbs photons of the corresponding photon energy
The absorption band observed for the longest wavelength corresponds to…
the absorption of photons having energies corresponding to transitions from the electronic ground state to the first possible electronically excited state
The absorption band observed for the lowest photon energy corresponds to…
the absorption of photons having energies corresponding to transitions from the electronic ground state to the first possible electronically excited state
The bands observed at shorter wavelengths correspond to
transitions in which vibrations of the nuclei in the molecules are excited in addition to the pure electronic excitation
The bands observed at higher photon energies corresponds to
transitions in which vibrations of the nuclei in the molecules are excited in addition to the pure electronic excitation
Vibrational ground state is defined as
v = 0
Frank-Condon principle
In the first molecule, the equilibrium positions of the atoms in the excited state are very similar to those in the S0 state
No vibrations are induced by the electronic transition, and the molecule is preferentially excited into the vibrational ground state of the S1 state
In the absorption spectrum, the band corresponding to this transition dominates
Vibrational relaxation
quick release of the entire vibrational excess energy in the S1 state to the surrounding
What indicates rapid vibrational relaxation?
green wavy arrows
The final result of fast vibrational relaxation is…
that the molecule is now in the lowest vibrational state of the electronic excited state
The probability of fluorescence is proportional to the…
transition dipole moment
The fluorescence spectrum has a …
mirror image like shape in comparison to the absorption spectrum
0-0 transitions
the absorption and fluorescence bands with the largest and smallest wavelengths define the 0-0 transitions
In the fluorescence spectrum, the _______ dominates
the vibrational band
Absorption and fluorescence v values
S0, v = 0, S1, v = 0
Stokes shift
the general observation that emitted photons have longer wavelengths than the absorbed photons
If the energy gap between the s1 state and the s0 state is large
fluorescence dominates and the de-excitation of the moleces
If the energy gap becomes smaller
nonradiative de-excitation of the s1 state often becomes dominant
the smaller the energy gap between s1 and s0
the higher the chance that there is a good overlap between the vibrational ground state wave function of the s1 state with wave functions of highly excited vibrations in the electronic ground state
Internal conversion
the molecule can switch from the electronic excited state into the electronic ground state without the emission of a photon
Kasha’s rule
fluorescence occurs from the lowest electronically excited state in its lowest vibrational state
Pauli rule
no electron in a molecule can have the exact same properties as another electron in the molecule
An orbital can be occupied by two electrons only if…
they have different spins
Triplet states
electronic states with two unpaired electrons of the same spin
Triplet states are _________ compared to respective singlet states
lower in energy
Intersystem crossing
the crossing from singlet to triplet
Intersystem crossing is…
favored when a good overlap exists between vibrational wave functions of the excited S1 state, with vibrational wave functions of the T1 state
Phosphorescence
radiative relaxation from the triplet state
Phosphorescence and intersystem crossing require a…
spin flip
0-0 transitions are the ______ when on a graph
right between the absorption last peak and the fluorescence first peak (where the mirror image begins)
Similar to fluorescence, the phosphorescence spectrum often appears to be a ….
mirror image of the ground-state absorption because the vibrational modes are often very similar in all electronic states
Jablonski diagram
only energy levels of the electronic states are depicted as horizontal lines
all radiative processes are depicted using straight arrows (absorption or emission of a photon)
nonradiative processes are portrayed using wavy arrows
rate constant is proportional to
the probability per time unit that the corresponding process occurs
The fluorescence quantum efficiency is
the number of photons emitted by a molecule as fluorescence divided by the number of photons that were previously absorbed by the moleucle
Fluorescence quantum efficiency equation
number of photons emitted as fluorescence / number of absorbed photons
quantum efficiency of any process is defined as
the number of quanta undergoing the process divided by the number of absorbed photons or quanta originally present in the initial state from which this process occurs
quantum efficiency is a direct measure of
the probability of certain processes in a molecule
the quantum efficiency of a process can be calculated by
dividing the rate constant of the process of interest by the sum of the rate constants of all processes simultaneously depopulating the initial state
to calculate the phosphorescence quantum yield
you have to multiply the quantum yield for s1 - t1 intersystem crossing by the probability for radiative emission from the triplet state
the sum of all radiative and nonradiative processes…
must be one
time constant
inverse of its corresponding rate constant
the absorption of a compound for a certain wavelength can be evaluated by..
a comparison between the light intensity measures after the sample cell and the light intensity measured after the reference cell
wavelength-dependent transmission equation
T (wavelength) = light intensity after sample cell / light intensity after reference cell
the reduction of the light intensity is…
proportional to the concentration of the absorbing compound and the length of the sample cell
Beer-Lambert Law
log (light intensity reference cell / light intensity sample cell) = the molar absorption * concentration * total optical path length of the light through the sample cell
The absorption of a compound at a given compound is defined as
A = ECL
the molar absorption coefficient is proportional to
the corresponding electronic transition dipole moment
Transition-dipole moment describes
the magnitude of absorption
The Franck-Condon integral descibres
which wavelength will result in absorption maxima
the fluorescence intensity is proportional to
the concentration of the compound
proteins and peptides show ___________
unspecific absorptions at wavelengths below 240nm
which amino acids have distinctive absorbance and fluorescence properties
tryptophan, tyrosine, and phenylalanine
Redshifts
shifts of absorption or fluorescence bands towards longer wavelengths (blueshifts are the opposite)
DNA and RNA both have _______ that show ______
conjugates pi electron systems
significant absorption around 260nm
pure DNA value is
1.8
pure RNA value is
2.0
when you want to run a blank sample on the spectrophotometer, you should use
whatever your sample is dissolved into
easy way to remember Beer’s law
A = ECL
haem is a cofactor for
protein hemogloblin, which is responsible for the transportaion of oxygen in red blood cells
the absorption change between _____________ is used for _______
hemoglobin and oxyhemoglobin
determine the oxygen saturation in a patient’s blood
flavins
an important class of cofactors that dominate the optical signals from cells
