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Biophysical Chem > test 2 > Flashcards

test 2 Flashcards

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1
Q

the fluorescence intensity is proportional to ______

A

the concentration of the utilized fluorescent conjugate at a certain time and/or location

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2
Q

an absolute determination of fluorescence intensities depends on…

A

sample and instrument characteristics

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3
Q

fluorescence photobleaching

A

the presence of other light-absorbing molecules

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4
Q

the measurement of fluorescence intensity and spectrum is often used for…

A

qualitative not quantitative

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5
Q

for quantitative information…

A

ratiometric measurement methods are often necessary

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6
Q

ratiometric methods (definition)

A

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

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7
Q

ratiometric fluorescence dyes (definition)

A

designed so that the parameter of interest can be deduced directly from the measured intensity of these two bands

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8
Q

fluorescence polarization assays are based on…

A

simultaneous measurement and comparison of two photophysical parameters of fluorescence, the horizontal and vertical component of the fluorescence polarization

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9
Q

fluorescence polarization measurements allow the determination of…

A

the time constant of the rotational diffusion of fluorescently labelled biomolecules or complexes

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10
Q

since the rotational diffusion depends very much on the size of these objects, it can be used for…

A

quantitative determination of receptor-ligand binding, proteolysis, protein-DNA interactions, and membrane fluidity when the corresponding components are fluorescently labelled

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11
Q

the only fluorescence dyes that are excited are those…

A

that are aligned with their transition dipole moment for absorption are the same as or similar to the polarization vector of the exciting light

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12
Q

photoselection (definition)

A

the excitation of only this subpopulation of fluorescence dyes

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13
Q

for large particles, which is bigger: I(ii) or I (i)?

A

I(ii)

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14
Q

for small particles, I(ii) and I(i) are what?

A

approximately equal

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15
Q

I(ii) and I(i) can be used for…

A

calculating the parameters of fluorescence polarization and fluorescence anisotropy

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16
Q

I(ii) and I(i) both increase with…

A

increasing rotational diffusion time and molecular size of the labelled species

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17
Q

P = (equation)

A

P = (I(ii) - I(i)) / (I(ii) + I(i))

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18
Q

r = (equation)

A

r = (I(ii) - I(i)) / (I(ii) + 2I(i))

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19
Q

r and P increase with…

A

binding

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20
Q

r and P decrease with…

A

dissociation

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21
Q

I(ii) bound _____ I(ii) unbound

A

>

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22
Q

I(i) unbound ______ I(i) bound

A

>

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23
Q

mixtures of different fluorescently labelled species…

A

exhibit anisotropies that can be linearly calculated from the fractions of the individual fluorescently labelled components

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24
Q

r prime

A

average anisotropy measured for the mixture

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25
Q

fi

A

the fraction of the individually fluorescently labelled component i in relation to all components labelled with the same fluorescence dye

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26
Q

ri

A

the corresponding fluorescence anisotropy that would be measured if only the individual component i were present

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27
Q

r equals …

A

f(1)r(1) + f(2)r(2

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28
Q

f(1), f(2),… are…

A

fractions of the free and bound ligand

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29
Q

r(1) and r(2) are…

A

anisotropies of the free and bound ligand

30
Q

fret is the transfer of…

A

excitation energy from an electronically excited molecule, called a donor (D), to an acceptor (A) molecule in the electronic ground state

31
Q

if donor and acceptor have a large separation…

A

little energy transfer occurs after selective donor excitation

fluorescence of the donor molecule will dominate

32
Q

after selective excitation of the donor…

A

vibrational relaxation occurs in the donor

33
Q

donor-acceptor transitions can be estimated from…

A

the spectral overlap, J, of the donor fluorescence with the acceptor absorption

34
Q

fret efficiency is calculated by…

A

R(0) ^6 / (R(0)^6 + r^6)

35
Q

R(0)

A

forster distance

36
Q

forster distance

A

defines the donor-acceptor distance, r, for which a transfer efficiency of 50% is observed

37
Q

the transfer efficiency measured in a fret experiment allows us to estimate…

A

the distance between donor and acceptor fluorescence dyes under different biological conditions

38
Q

fluorescence kinetics of a fluorescence dye depends on…

A

environmental conditions such as solvent polarity

39
Q

the rate of recovery of fluorescence is an indicator of…

A

the mobility of the fluorescing molecules adjacent to the photobleached volume and relates to their diffusion coefficient

40
Q

chiroptical methods are based on…

A

the use of circular-polarized light and the differences obtained in experiments using left circular polarized or right circular polarized light

41
Q

circular dichroism (CD)

A

based on the differences in the absorption or extinction coefficient of biomolecules for left and right circular polarized light

42
Q

the observed difference (CD) is quantified by…

A

ellipticity

43
Q

O(lambda) =

A

(2.303[ODL(lambda) - ODR(lambda)] * 180) / 4 pi

44
Q

molar ellipticity equation

A

(100 * O(lambda)) / CL

45
Q

alpha helix CD graph

A

high above 0 to low below 0 to 0

46
Q

beta sheet CD graph

A

slightly above 0 to slightly below 0 to slightly above 0

47
Q

CD equation that’s with the graph (idk)

A

O(lambda) = f * each O of each protein shape

48
Q

CD graph b form DNA

A

kind of negative to very negative to kind of positive to 0

49
Q

CD graph z form DNA

A

kind of positive to very positive to very negative to 0

50
Q

optical rotatory dispersion (ORD) is based on

A

differences in the refractive index, n, of a sample for left and right circular polarized light

51
Q

wavelength-dependent optical rotation equation

A

(180 l [n1 - nr])/ wavelength

52
Q

circular dichromism is based on differences in…

A

absorptions measured with right or left handed circular polarized light

53
Q

optical rotatory dispersion is based on differences in…

A

the refractive index with right or left handed circular polarized light

54
Q

circular birefringence

A

n1 - nr

55
Q

in order for a molecule to exhibit circular birefringence and circular dichroism, it must be…

A

chiral

56
Q

why is the measurement of light scattered at biomolecules important?

A

used to determine particle size, shape, aggregations, and diffusional processes

57
Q

the electric field of light interacts with…

A

electron clouds to induce transitions

58
Q

elastic light scattering

A

if the scattered light has the same frequency and energy has incident photons but different propagation vectors

59
Q

infrared spectral range (IR)

A

caused by the interaction between the IR electric field vector and the molecular dipole transition moments related to the molecular vibrations

60
Q

major disadvantage for IR

A

high absorption of IR radiation by water

61
Q

raman spectroscopy

A

based on inelastic scattering of photons at the biomolecules (photons change their frequency)

62
Q

in raman spectroscopy, the frequency change is caused by…

A

a loss or gain in the photon energy corresponding to transitions between vibrational levels of the molecules at which the scattering occurred

63
Q

hv = hv1 (raman)

A

elastic scattering

64
Q

hv doesn’t equal hv1

A

inelastic scattering

65
Q

alpha helix vibrational spectra

A

1650

66
Q

beta sheet vibrational spectra

A

1630

67
Q

nmr spectroscopy is used for the investigation of…

A

structural, mechanistic, and dynamical properties of biomolecules under physiological conditions

68
Q

alpha spin

A

the magnetic moment is oriented with the external magnetic field

69
Q

beta spin

A

the magnetic moment is oriented against the external magnetic field

70
Q

nmr spectroscopy is based on the measurement of

A

the energy difference between these two states by absorption or emission of electromagnetic radiation on the radiowave scale

Biophysical Chem (2 decks)

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