Transcript ELEKTROMAGNETSKO ZRAČENJE

SPECTROSCOPIC
METHODS FOR
STRUCTURAL ANALYSIS
OF BIOLOGICAL
MACROMOLECULES
D. Krilov
20.10. 2008.
Interactions in biological
macromolecules
 Van der Waals's forces; hydrogen bond; hydrophobic




interactions; ionic bonds
interactions between atomic groups in macromolecule,
between macromolecule and smaller molecules or
macromolecule and water
these interactions are of electrostatic nature
they are about 20 times weaker than covalent bond
they determine the secondary and tertiary structure of
macromolecules
Van der Waals's forces
 attractive interactions between molecules with closed
shells (even number of electrons in outer shell):
 a) nonpolar molecules - dispersion interactions between
transient dipoles induced by fluctuation of electrons
 b) polar molecules - interactions dipole-charge, dipoledipole, induced dipole-dipole, induced dipole-induced
dipole
B
U r    6
r
 potential energy
 the bond is multi directional and unsaturated
 (one molecule can form several such bonds with surrounding
molecules)
Energies of attractive interactions
 at 25°C the average
energy of interaction is
- 0,07 kJ mol-1 (kinetic
energy is 3,7 kJ mol-1)

energy of interaction is
- 0,8 kJ mol-1
 energy of interaction is
about - 5 kJ mol-1; it
depends on molecule
polarizability
 repulsive interactions: at very short
distances the repulsive forces
predominate - forces between
atomic nuclei and between electronic
clouds:
U r   A e r r0
 for numerical calculations the
potential is:
U r  
A
r 12
Hydrogen bonds
 attractive interactions between molecules with closed
shells, of specific structure: A - H ··· B
 A and B are strongly electronegative elements
(usually N, O, F)
 B must have a free electron pair
 due to electronegativity of A atom, the hydrogen atom
tends to localize between A and B; in that way H
becomes partially positive and B partially negative
 this bond is unidirectional and saturated

(one hydrogen atom can form only one hydrogen bond)
 bond energy is about 20 kJ mol-1
Examples of hydrogen bond
 a) between atoms in adjoining
molecules
 the bond is stronger when the
atoms are aligned
 hydrogen bond in biological
molecules:
 between two amino acids in
polypeptide chain,
 between pairs of bases in
nucleic acids
0,2 nm
 b) in water
 each molecule capable of creation
the hydrogen bond with another
molecule, creates such bond also
with molecules of water
 that is the reason why the hydrogen
bond between two molecules
becomes weaker when they are
dissolved in water
 among water molecules there is a
network of hydrogen bonds which is
responsible for the specific
properties of water
 the hydrogen bonds exist between
the surface of macromolecule and
surrounding water molecules
 the layer of partially immobilized
molecules of water arround a
macromolecule is called hydration
shell
elastine
Hydrophobic interactions
 the hydrophobic groups are forced
by water to stick together in order to
minimize their influence on hydrogen
bonding network
 this assembling is described as
hydrophobic bond, but actually these
are the repulsive interactions
between molecules of water and
hydrophobic groups
 such ordering diminishes the total
energy and increases the entropy of
the system
Ionic bonds
 a) in macromolecules
 ionic electrostatic interactions are
present between charged groups;
they are strong in the absence of
water molecules
 b) in aquaeous solutions
 ionic interactions are less strong
and ionic bonds are weak,
especially when there are
dissolved salts in water
 enzyme (-) is bound to the
substrate (+)
ELECTROMAGNETIC
RADIATION
 electromagnetic waves – communication with the outer
world: sight, the sense of heat, communication facilities
(radio, TV, cell phones …)
 interaction with matter: information about structure and
dynamics of molecules; conformations of
macromolecules and their interaction with environment
 the sources: natural (atoms, molecules, cosmic rays,
stars); artificial (aerials, lamps, X-ray tube, cobalt bomb)
Electromagnetic spectrum
Interaction of electromagnetic field
with matter
 it is explained by particle nature of radiation:




wavepacket – photon (Einstein 1905.)
natural and artificial sources of radiation are not
simple harmonical oscillators – the emitted
waves are in the narrow range of frequencies
arround w0:
w = w0  w, w << w0
the interference of the waves of close
frequencies results in energy localization in the
form of the wave packet; its energy is E=h0
energy is transferred to matter in quanta
The concept of wavepacket (quantum of
energy)
Nonionizing interactions
 after absorption of
incident photon, atom or
molecule is raised to
higher energy state or
there is an increase in
overall translational motion
- heating of the matter
 elastic scattering of
incident photon at atom
Elastic incoherent scattering of photon
– Compton's effect
 collision of photon with atom
results in ejection of
electron from outer shell;
the scattered photon has
lower energy and different
direction
 the recoil electron can
induce further ionizations
 the remaining cation is
relaxed by emission of
secondary photon
 the interaction is more
probable for photons with
energy much higher from
the ionization energy of
electron in atom
Absorption of photon – photoelectric
effect
 the incident photon which
collides with atom is completely
absorbed and electron is ejected
from an inner shell
 the recoil electron can
induce further ionizations
 the remaining cation is relaxed
by emission of secondary photon
the probability of interaction is
higher for the photons with lower
energy
2
h   
m vmax
2
A. Einstein 1905.
Pair production
 in the vicinity of heavy
nucleus photon with energy
higher than 1 MeV can be
transformed into the pair
of particles:
electron - positron
h  2me c 2
 the heavy nucleus takes
over the part of photon 's
momentum
Spectroscopy
 the methods are based on
interaction of electromagnetic
radiation with matter
 the molecule will absorb photon if
its energy is equal to energy
difference of two energy states in
molecule:

h   En  Ev
the properties of
molecule are changed: electrons
distribution, electric dipole
momentum, magnetic momentum
of nucleus or electron ...
molecule will emit photon if it is in
excited state, i.e. with excess of
energy
In each spectroscopy method the photons will interact with matter if
their energy corresponds to the energy differences determined by the
structure and properties of molecular pattern of the sample.
Attenuation of electromagnetic
radiation in matter
Due to interaction of photons with
molecules the intensity of the beam
is decreasing along its path through
the sample
I1
 -
I = I2-I1= k I1 x
 - dI = k I dx
I2
I
I0
x
dI
  k dx 
I
I

I0
x
dI
  k  dx
I
0
I x   I 0 e
k x
 k() is attenuation coefficient which depends on the
medium and wavelength of radiation
 when the radiation is passing through the solution:
 k (,c) = () c
molar absorption
coefficient
concentration
 transmittance
T = I / I0
 absorbance
A = - log I = log I0 / I
Characteristical spectral parameters
 Spectrum is the distribution of spectral radiancy (I ) (or absorbance,




or molar absorpton coefficient…) over energy (or wavelength, or
frequency, or wave number)
The line position reflects the transition energy between two states
The line intensity is the measure of the number of equal transitions
The line width depends on dynamics of the environment of
investigated molecule; the higher is the number of collisions with
other molecules, the shorter is the lifetime of excited state; the
spectral line is broadened
The ground state of molecule is the state with minimal energy; in all
spectroscopy ranges it is predominantly populated. That means that
the process of absorption of photon is always possible.
Spectroscopic techniques
 Absorption
 Emission
transmission
It
I0
emission
I0
excitation
partial
absorption
Ie
Basic spectroscopic methods in
biology and medicine
 Absorption spectroscopies:
 1. Optical or electron spectroscopy – electron transitions between
molecular orbitals; the change in electron distribution; spectra in
visible and ultraviolet range (100-700 nm)
 2. Infrared spectroscopy – transitions between vibrational states;
change in the value of electric dipole momentum; spectra in infrared
range (800-10000 nm)
 3. Electron spin resonance– transitions between electron spin states
in external magnetic field; the change of magnetic spin momentum of
electron; spectra in microwave range (1-10 cm)
 4. Nuclear magnetic resonance - transitions between nuclear spin
states in external magnetic field; the change of magnetic spin
momentum of nucleus; spectra in radiowave range (1 – 10 m)
 Emission spectroscopies:
 Fluorescence – molecules are excited to higher energy
state by ultraviolet or laser radiation; in the process of
relaxation to the ground state they emit the radiation in
visible range; molecules or supramolecular structures
which don't possess intrinsical fluorophores are labeled
by covalently bonded fluorescence probes