test 2 Flashcards
the fluorescence intensity is proportional to ______
the concentration of the utilized fluorescent conjugate at a certain time and/or location
an absolute determination of fluorescence intensities depends on…
sample and instrument characteristics
fluorescence photobleaching
the presence of other light-absorbing molecules
the measurement of fluorescence intensity and spectrum is often used for…
qualitative not quantitative
for quantitative information…
ratiometric measurement methods are often necessary
ratiometric methods (definition)
based on the measurement of at least two parameters which are influenced by possible artefacts in the same or at least very similar way
comparison of these two parameters then allows us to determine the pure information of interest independent from the artefacts
ratiometric fluorescence dyes (definition)
designed so that the parameter of interest can be deduced directly from the measured intensity of these two bands
fluorescence polarization assays are based on…
simultaneous measurement and comparison of two photophysical parameters of fluorescence, the horizontal and vertical component of the fluorescence polarization
fluorescence polarization measurements allow the determination of…
the time constant of the rotational diffusion of fluorescently labelled biomolecules or complexes
since the rotational diffusion depends very much on the size of these objects, it can be used for…
quantitative determination of receptor-ligand binding, proteolysis, protein-DNA interactions, and membrane fluidity when the corresponding components are fluorescently labelled
the only fluorescence dyes that are excited are those…
that are aligned with their transition dipole moment for absorption are the same as or similar to the polarization vector of the exciting light
photoselection (definition)
the excitation of only this subpopulation of fluorescence dyes
for large particles, which is bigger: I(ii) or I (i)?
I(ii)
for small particles, I(ii) and I(i) are what?
approximately equal
I(ii) and I(i) can be used for…
calculating the parameters of fluorescence polarization and fluorescence anisotropy
I(ii) and I(i) both increase with…
increasing rotational diffusion time and molecular size of the labelled species
P = (equation)
P = (I(ii) - I(i)) / (I(ii) + I(i))
r = (equation)
r = (I(ii) - I(i)) / (I(ii) + 2I(i))
r and P increase with…
binding
r and P decrease with…
dissociation
I(ii) bound _____ I(ii) unbound
>
I(i) unbound ______ I(i) bound
>
mixtures of different fluorescently labelled species…
exhibit anisotropies that can be linearly calculated from the fractions of the individual fluorescently labelled components
r prime
average anisotropy measured for the mixture
fi
the fraction of the individually fluorescently labelled component i in relation to all components labelled with the same fluorescence dye
ri
the corresponding fluorescence anisotropy that would be measured if only the individual component i were present
r equals …
f(1)r(1) + f(2)r(2
f(1), f(2),… are…
fractions of the free and bound ligand
r(1) and r(2) are…
anisotropies of the free and bound ligand
fret is the transfer of…
excitation energy from an electronically excited molecule, called a donor (D), to an acceptor (A) molecule in the electronic ground state
if donor and acceptor have a large separation…
little energy transfer occurs after selective donor excitation
fluorescence of the donor molecule will dominate
after selective excitation of the donor…
vibrational relaxation occurs in the donor
donor-acceptor transitions can be estimated from…
the spectral overlap, J, of the donor fluorescence with the acceptor absorption
fret efficiency is calculated by…
R(0) ^6 / (R(0)^6 + r^6)
R(0)
forster distance
forster distance
defines the donor-acceptor distance, r, for which a transfer efficiency of 50% is observed
the transfer efficiency measured in a fret experiment allows us to estimate…
the distance between donor and acceptor fluorescence dyes under different biological conditions
fluorescence kinetics of a fluorescence dye depends on…
environmental conditions such as solvent polarity
the rate of recovery of fluorescence is an indicator of…
the mobility of the fluorescing molecules adjacent to the photobleached volume and relates to their diffusion coefficient
chiroptical methods are based on…
the use of circular-polarized light and the differences obtained in experiments using left circular polarized or right circular polarized light
circular dichroism (CD)
based on the differences in the absorption or extinction coefficient of biomolecules for left and right circular polarized light
the observed difference (CD) is quantified by…
ellipticity
O(lambda) =
(2.303[ODL(lambda) - ODR(lambda)] * 180) / 4 pi
molar ellipticity equation
(100 * O(lambda)) / CL
alpha helix CD graph
high above 0 to low below 0 to 0
beta sheet CD graph
slightly above 0 to slightly below 0 to slightly above 0
CD equation that’s with the graph (idk)
O(lambda) = f * each O of each protein shape
CD graph b form DNA
kind of negative to very negative to kind of positive to 0
CD graph z form DNA
kind of positive to very positive to very negative to 0
optical rotatory dispersion (ORD) is based on
differences in the refractive index, n, of a sample for left and right circular polarized light
wavelength-dependent optical rotation equation
(180 l [n1 - nr])/ wavelength
circular dichromism is based on differences in…
absorptions measured with right or left handed circular polarized light
optical rotatory dispersion is based on differences in…
the refractive index with right or left handed circular polarized light
circular birefringence
n1 - nr
in order for a molecule to exhibit circular birefringence and circular dichroism, it must be…
chiral
why is the measurement of light scattered at biomolecules important?
used to determine particle size, shape, aggregations, and diffusional processes
the electric field of light interacts with…
electron clouds to induce transitions
elastic light scattering
if the scattered light has the same frequency and energy has incident photons but different propagation vectors
infrared spectral range (IR)
caused by the interaction between the IR electric field vector and the molecular dipole transition moments related to the molecular vibrations
major disadvantage for IR
high absorption of IR radiation by water
raman spectroscopy
based on inelastic scattering of photons at the biomolecules (photons change their frequency)
in raman spectroscopy, the frequency change is caused by…
a loss or gain in the photon energy corresponding to transitions between vibrational levels of the molecules at which the scattering occurred
hv = hv1 (raman)
elastic scattering
hv doesn’t equal hv1
inelastic scattering
alpha helix vibrational spectra
1650
beta sheet vibrational spectra
1630
nmr spectroscopy is used for the investigation of…
structural, mechanistic, and dynamical properties of biomolecules under physiological conditions
alpha spin
the magnetic moment is oriented with the external magnetic field
beta spin
the magnetic moment is oriented against the external magnetic field
nmr spectroscopy is based on the measurement of
the energy difference between these two states by absorption or emission of electromagnetic radiation on the radiowave scale
