Transcript Basic Physical Chemistry (12.4 MB ppt)

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Physical Chemistry
in Biochemistry
Basic Medical Chemistry
and Biochemistry
1st year
© Institute of Medical Biochemistry and Laboratory Diagnostics of the General University Hospital and of The
First Faculty of Medicine of Charles University in Prague - 2005-2016
Sylabus of the Lecture

The most important chapters of physical chemistry
 Thermodynamics, Thermochemistry, Chemical equilibrium, Kinetics
(Reaction rate, order of chemical reaction), Catalysis

Photos and films connected with history of Electrochemistry





J. Heyrovsky - Nobel Prize for polarography 1959
J. Heyrovsky – Inventor of polarography 1959
Promotion clip on polarography
Oscilopolarography (Expo 58 - Brussels)
Promotion film of J. Heyrovský Institute of Physical Chemistry of the
Academy of Sciences of the Czech Republic, v.v.i., 2007
 Promotion film of J. Heyrovský Institute of Physical Chemistry of the
Academy of Sciences of the Czech Republic, v.v.i., 2009
Phys.Chem. 2015/2016
Physical Chemistry in Biochemistry
The Most Important Topics

Physical chemistry deals with application of physics to
macroscopic, microscopic, atomic, subatomic, and particulate
phenomena in chemical systems within the field of chemistry
traditionally using the principles, practices and concepts of
thermodynamics, quantum chemistry, statistical mechanics and
kinetics.
 It is mostly defined as a large field of chemistry, in which several
sub-concepts are applied; the inclusion of quantum mechanics is
used to illustrate the application of physical chemistry to atomic
and particulate chemical interaction or experimentation.
http://en.wikipedia.org/
Phys.Chem. 2015/2016
Research Institutes dealing with
physical chemistry and biochemistry








J. Heyrovský Institute of Physical Chemistry of the
Academy of Sciences of the Czech Republic, v.v.i.
Institute of Biophysics of the Academy of Sciences of the
Czech Republic, v.v.i.
Faculty of Science, Charles University in Prague
Faculty of Science, Masaryk University in Brno
Czech University of Life Sciences, Prague
Institute of Chemical Technology, Prague
Faculty of Chemical Technology, Pardubice
…
Phys.Chem. 2015/2016
Terminology
 Physical
chemistry
 Chemical physics
 Biophysical chemistry
…
???Borders???
Application of Physical Chemistry
 User (physician) does not use the principle of the applied method
 Temperature measurement – linear thermal expansion of liquids
(mercury, ethanol), thermocouple - Physics
 Sedimentation – gravitation - Physics
 Centrifugation – centrifugal power - Physics
 Utilization of results only
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Some of the most Important Topics of
Physical Chemistry applied in Biochemistry

Thermodynamics
 Thermochemistry
 Chemical equilibrium
 Kinetics (Reaction rate, order of chemical reaction)
 Catalysis
…
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Thermodynamic laws
Thermodynamics
– science dealing with energy transports by physical and chemical
processes (shortened version)
- the study of the conversion of heat energy into different forms of
energy (in particular, mechanical, chemical, and electrical energy);
different energy conversions into heat energy; and its relation to
macroscopic variables such as temperature, pressure, and volume.
Its underpinnings, based upon statistical predictions of the
collective motion of particles from their microscopic behavior, is
the field of statistical thermodynamics, a branch of statistical
mechanics (full definition)
Phys.Chem. 2015/2016
Thermodynamic laws
Closed system – does not exchange either mass or energy (E = mc2)
Open system – exchanges mass and/or energy
vs.
Closed system – does not exchange mass; exchanges energy only
Open system – exchanges mass and/or energy
Isolated system – does not exchange either mass or energy
Vs.
Isolated Systems – matter and energy may not cross the boundary
Adiabatic Systems – heat must not cross the boundary
Diathermic Systems - heat may cross boundary
Closed Systems – matter may not cross the boundary
Open Systems – heat, work, and matter may cross the boundary (often called a
control volume in this case)
Phys.Chem. 2015/2016
Thermodynamic laws
P - pressure
Internal energy
U
Helmholtz free energy
A=U-TS
T – Temperature
Enthalpy
H=U+pV
E – potential
Gibbs free energy
G=H-TS
a – activity
V - Volume
Electrochemical potential m=m0-RT ln (a) - zFE
F – Farraday constant
First Law of Thermodynamics
Sum of all energies in closed system is constant, irrespective of
running physical or chemical processes – work is changed into
energy and energy into work (In a closed system (see below) the
total inflow of energy must equal the total outflow of energy.
dU = dw + dq (correctly should be , not d, it is not the total
differential)
U - internal energy, w - work, q – heat
Phys.Chem. 2015/2016
Thermodynamic laws II
Second law of thermodynamics
dQ
dS 
T
Q
S 
T
S…entropy (measure of disorderliness of the system – with increasing
inordinance of the system increases its entropy); Q…heat, T…temperature

The total entropy of any isolated thermodynamic system tends to increase over
time, approaching a maximum value.

The heat cannot spontaneously pass from the colder body to the warmer one.

The entropy of an isolated system is constant or increasing.

It is not possible to construct the periodically working machine, which would
utilize the heat from one accumulator only and which would perform the work
exactly equivalent to this heat.

It is not possible to construct perpetum mobile of the second type.

All spontaneous processes are realized with increasing entropy, with increasing
disorderliness of the system.
Phys.Chem. 2015/2016
Thermodynamic laws III
Third law of thermodynamics
lim S  0
T 0
- the entropy of all systems and of all states of a system is zero at absolute zero
- it is impossible to reach the absolute zero of temperature by any finite number of
processes
Energy types
1. Free energy – „noble“, it can be free transported, transformed
(chemical, electric)
2. Bound energy – heat, which can be transported (flow) only in
the direction of the heat gradient. Transformation to other types
of energy can be realized only, when the warmer body gives its
energy to the colder one.
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Gibbs energy
dG   SdT  VdP  Ad  ...   m~i dni
i
H…
Enthalpy H = U+pV (increase of enthalpy is equal to the heat,
which the system gains under constant pressure, and at the same
time no any other then volume work is produced)
G …
Gibbs energy G = H-TS (= Maximal reversible work other then
volume work, which the system gains (produces) by constant
temperature and pressure
γ …
surface tension
A …
area
~…
m
m
electrochemical potential
 G 
~

m i  
 ni  T , P , i ,ni  j
Negatively taken work necessary for releasing of 1 mol of
charged particles and their transport into infinitively diluted state
m~  m0  RT ln a  zFE
Phys.Chem. 2015/2016
Thermodynamic laws VI - Terminology
Reversible: system is passing through huge amount of small state
changes, by which it is always in equilibrium with surroundings; in
any moment it is possible to stop it and to change the direction of
the process
Irreversible: all changes, which differs from the reversible process
Isobaric: P = const. (pressure)
Isothermic: T = const. (temperature)
Isochoric: V = const. (volume)
Adiabatic: q = const. (heat)
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Exothermic reaction : ΔH < 0
Endothermic reaction : ΔH > 0
Spontaneous reaction: ΔG < 0 (ΔG= ΔH-TΔS)
Unspontaneous reaction: ΔG> 0
Equilibrium ΔG = 0
Thermochemistry
Reaction heat: heat, which the system gains (released), if under
constant pressure the chemical reaction in extension of 1 mol is
realized according to the given equation, provided that the
temperature of the system before the reaction is the same as after
the reaction and that reactants as well as products are in the phase
given in reaction equation.
C(s)+O2(g)=CO2(g)
Phys.Chem. 2015/2016
Thermochemical laws
First thermochemical law (Lavoisier-LaPlace’s)
The total heat released by the chemical reaction is equal to that one,
consumed by the reversed direction of the reaction.
A ↔B;ΔHA→B = -ΔHB→A
Second thermochemical law (Hess’s)
If a chemical reaction is realized in a few sequential steps, the sum of the
energy, released (consumed) in single steps, is equal to the total energy,
which would be released (consumed), if the reaction would be realized
directly in the only one step.
A→ B → C; ΔHA→C = ΔHA→B + ΔHB→C
It enables to determine the caloric value of foods by their burning up, although in human
body are they metabolized in plenty of gradual steps (glucose, lipids etc.).
Calculation of reaction heat is realized from combination heats (heat, which is released
(consumed) by formation o 1 mol of the compound directly from atoms under constant
pressure and temperature) or from combustion heats (heat, which is released, by
combustion of 1 mol of compound in pure oxygen by formation of most stable oxidizing
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products).
Heat exchange
1. Without any change of the state
T2
H  Q   c p dT   c p (T2 T 1)
i
T1
Q  m.c p (T2  T1 )
T2
c p   C p dT  A  B(T2 T 1) 
T1
C
(T2 T 1) 2
2
2. Latent heat
H Vaporization, Solidification, Liquefaction, Fusion
3. Thermochemical law
Q1  Q2
Heat of fusion = Latent heat of solidification
Latent heat of vaporization = latent heat of condensation
Phys.Chem. 2015/2016
Heat exchange - 1 kg of water
1. heating from 0 oC to 100 oC
Q  m.c p (T2  T1 )  1* 4.2 *100 kJ  420 kJ
2. Heat of vaporization (normal boiling point)
H Vaporization  2256kJ ~ heating from 0 to 540
oC
3. Melting point (normal fusion point)
H Tání  333.7kJ
Phys.Chem. 2015/2016
~ heating from 0 to 80 oC
Chemical equilibrium
A B C  D
Reactants A and B; Products C and D
In equilibrium state runs the reaction from the left side to the right
side by the same rate
K
Guldberg-Waage’s law
(1863)

C D

AB
K ... equilibrium constant of the reaction
K ... depends on T, P etc.
In words: The product of molar concentrations of products of the reaction divided by the
product of molar concentrations of reactants is in equilibrium state constant in closed
system.
Le Chatelier's principle
If a chemical system at equilibrium experiences a change in concentration, temperature,
volume, or partial pressure, then the equilibrium shifts to counter-act the imposed change.
Phys.Chem. 2015/2016
Chemical equilibrium II
[C].[D] = [B].[A]=> K=1
[C].[D] > [B].[A] => K>1 – prevailing products
[C].[D] < [B].[A] => K<1 –prevailing reactants
Oscillating reactions: e.g., Zhabotinsky
To influence the course of the reversible reaction (its direction), it
is necessary to work in open system.
If one component of the reaction is removed, the system produces
the removed amount continuously to reach (restore) the
equilibrium. Thereby we can reach practically total realization of
the reaction in the direction, in which it practically does not run in
closed system (K<<1).
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Possibilities of influencing of steady state (equilibrium)

C D
1. Decrease of final products quantity
K 
2. Increase of starting compounds quantity
AB
3. By inequal number of molls of starting compounds and final product
in gaseous system (2A + B = C) the change of pressure
4. Change of temperature (exothermic – the rate decreases with
increasing temperature; endothermic – the rate increases with
increasing temperature)
All reactions in living systems are realized in open systems,
consequential, consecutive reaction takes off products of previous
reaction, whereby the equilibrium (steady) state is disturbed and so
influences the course of the reaction.
A+B=C+D → D+E=F+G → G+H=I+J → J+K=L+M
The compound M is as the final product of the metabolism removed from
the (living) system away, e.g., by respiration or excretion.
Catalyzers influence the reaction rate, but not the equilibrium. They are
enabling other reaction way, energetic of the reaction, but not the
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equilibrium!!!
Reaction rate, order of chemical reaction
d [ A] d [C ]

v 

 k C 
dt
dt
AC
A B  C  D
 
v  k C D 


 v K  k  C D
k AB 
 
v  k  AB 
Equilibrium:
1st order reaction
2nd order reaction

v
 
v  k  A
AC  D
  2
v  k  A
2A  C  D
 
v  k  AB  A  B  C  D
Reaction of more than 2nd order is realized in fact stepwise,
gradually, as the reaction composed of more reaction substeps. For the reaction rate is the controlling the slowest one).
Phys.Chem. 2015/2016
Influence of the Temperature on the Reaction Rate
Increase of the temperature increases the reaction rate. Their
relationship is given by Arhenius equation:
 E

k  A. exp 

a
RT 
k... rate constant, A… function factor; T … absolute temperature; Ea…
activation energy; R universal gas constant
It follows that the increase of the
temperature essentially increases
the reaction rate – exponentially.
activity
This fact is commonly used by
homonotermn organisms, e.g., by
defense reactions, such reactions
run at higher temperature faster and
they are more effective.
Coeffitient Q10 = how many times changes the reaction rate by
the change of the temperature by 10 grades ~ 2
Phys.Chem. 2015/2016
Electric doublelayer
Electrode
Electrode
Diffusion part
Helmholtz part
Use:
a) Electrolysis of the solutions
b) Electroplating
c) Tooth cell – improper materials
d) Voltammetry
e) Power sources
Phys.Chem. 2015/2016
Solution
Oxidation - reduction reactions (redox)
Example
Galvanic cell - spontaneous
Anode: Zn=Zn2++2e1st redox system E0(Zn2+/Zn)=-0.76 V
Cathode: Cu2++2e-=Cu
2nd redox system E0(Cu2+/Cu)=0.34 V
Zn(s) + Cu2+ = Cu(s) + Zn2+ U=Ec-Ea
Measurement of redox potential (Secondary school)
Electrolytic cell – Inserted voltage
Anode: Cu = Cu2+ + 2e1st redox system
Cathode: Zn2+ + 2e- = Zn
2nd redox system
Cu(s) + Zn2+ = Zn(s) + Cu2+ U=Ea-Ec
Reduction is always realized at cathode!
E1  E10 
a
a
RT
RT
RT a1red
ln
U  E1  E 2  E10 - E 02 
ln 1red 
ln 2red
nF a1ox
nF
a1ox
nF
a 2ox
Phys.Chem. 2015/2016
Oxidation - reduction reactions (redox)
Redox pair
[V]
Redox pair
[V]
Li+/Li (s)
- 3.04
Co2+/Co (s)
- 0.28
K+/K (s)
-2.92
Ni2+/Ni (s)
- 0.25
Na+/Na (s)
- 2.71
Sn2+/Sn (s)
- 0.14
Ca2+/Ca (s)
-2.50
Pb2+/Pb (s)
- 0.13
Al3+/Al (s)
- 1.66
2H+/H2 (g)
+0.00
Mn2+/Mn (s)
- 1.18
Sn4+/Sn2+
+0.15
Zn2+/Zn (s)
- 0.76
Cu2+/Cu (s)
+0.34
Cr3+/Cr (s)
- 0.74
Ag+/Ag (s)
+0.80
Fe2+/Fe (s)
- 0.44
Cl2/2Cl-(g)
+1.36
Cd2+/Cd (s)
- 0.40
Au+/Au (s)
+1.50
Tl+/Tl (s)
- 0.34
Phys.Chem. 2015/2016
Oxidation - reduction reactions (redox) I
Standard electrode potentials at 25 oC in aqueous solutions
Phys.Chem. 2015/2016
Oxidation - reduction reactions (redox) II
The redox pair with the higher standard potential is the
oxidant of the redox pair with the lower standard potential.
Voltage change:
1. Connection of two different metals
2. Connection of the same metals dipped in two different
electrolytes (different concentration)
Electric current direction
a) Galvanic cell b) electrolytic cell
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Oxidation - reduction reactions (redox) III
Comparison of efficiency of energy production by microorganisms. In
parentheses are numbers corresponding to ΔGO’ in kJ.mol-3, cytFe3+ and cytFe2+
are oxidized and reduced forms of cytochroms
Phys.Chem. 2015/2016
Redox reactions in living organism
Living organisms commonly use redox reactions as energy
sources. A number of organic compounds exist in oxidized form as
well as in reduced form and therefore they can be involved in the
transport of electrons. In these processes the organisms gain the
energy necessary for life. Transferred electrons enable, e.g., transport
of protons (H+) through membranes and enable the changes of pH.
Accumulated protons by reverse transport through the membrane can
supply the energy for the transport of other compounds or for the
synthesis of ATP.
From redox potential of two equal redox systems we can
calculate ΔGo of the chemical reaction ΔGo = -zF ΔE’o (z – number
of transported electrons) by the change of the redox potential ΔE’o
(ΔE’o - „biologic“ standard reduction potential – standard system
state for pH=7) [Ared]=[Aox].
Phys.Chem. 2015/2016
Redox reactions in living organism II
During the aerobe transformation of compounds (metabolism) is
realized (in principle) strong exergonic reaction (and exothermic)
redox reaction: 2H2+O2→2H2O. The high energetic electrons of
hydrogen are transported on oxygen in many sequential steps. Their
energy is used for living processes of the cell (organism). These
processes are studied in biochemistry. As electron carrier are often
used metals bound on peptides.
Spontaneous reaction = exergonic (exothermic)
Non-spontaneous reaction = endergonic (endothermic)
2H2(g)+O2(g)→2H2O(g) ΔGo =-242 kJ.mol-1 – exothermic (burning)
2H2O(g) → 2H2(g)+O2(g) ΔGo =242 kJ.mol-1 – endothermic,
equilibrium is shifted to the left, only by high temperature and
decreased pressure starts the decomposition (at 2100 oC and 0.1 MPa
reacts 2 % of molecules only) .
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Scheme of the Polarographic Device
W - Working electrode:
Polarography – dropping (mercury) electrode
Voltammetry – stationary surface - Hanging mercury drop electrode
Solid2015/2016
electrode
Phys.Chem.
First polarograms
Phys.Chem. 2015/2016
First paper on polarography – 1922
in journal “Chemicke listy”
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December 10th, 1959 (37 years after discovery of polarography)
was awarded
Prof. Jaroslav Heyrovský by the Swedish king
Gustavo Adolph VI. in Stockholm by Nobel prize for chemistry
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Phys.Chem. 2015/2016
Order for production of the first polarograph
and one of the first polarographic machines
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Mercury fountain (Spain) Barcelona, Foundation Miró
Mercury mine Almaden
Phys.Chem. 2015/2016
Modern polarographic device – small amount of mercury
(produced in Czech republic 21st century)
Phys.Chem. 2015/2016
Inorganic analysis
H
Li
Be
B
C
N
O
F
Na
Mg
Al
Si
P
S
Cl
K
Ca
Sc
Ti
V
Cr
Mn
Fe
Co
Ni
Cu
Zn
Ga
Ge
As
Se
Br
Rb
Sr
Y
Zr
Nb
Mo
Te
Ru
Rh
Pd
Ag
Cd
In
Sn
Sb
Te
I
Cs
Ba
La
Hf
Ta
W
Re
Os
Ir
Pt
Au
Hg
Tl
Pb
Bi
Po
At
Fr
Ra
Ac
Ce
Pr
Nd
Pm
Sm
Eu
Gd
Tb
Dy
Ho
Er
Tm
Yb
Lu
Th
Pa
U
Np
Pu
Am
Cm
Bk
Cf
Es
Fm
Md
No
Lr
(Elements determined by DC polarography are in full lined boxes, by ASV in
double boxes, and by AdSV are underlined )
Phys.Chem. 2015/2016
Applications of polarography (voltammetry)

Metals (cations): Cd, Cu, Zn, Pb, Sn, Tl, Mo, Cr, Ni,
Ag, V, Hg, …Speciation (different valences!!!)
 Anions: Chlorides, bromides, iodides, iodates, sulphates,
phosphates, nitrates, nitrites
 Organic compounds:
 Amino acids (cysteine, cystine,…)
 Glutathione, metallothioneins
 Nukleic acids (adenine, guanine, thymine, cytosine, uracil)
 Nitro compounds (TNT, nitrobenzene)
 Thiodiglycolic acid
 Phenylglyoxylic acid
 Carcinogens, toxic compounds, medicals, pesticides …
Phys.Chem. 2015/2016
Main fields of application of
polarography (voltammetry) today
1.






Mechanistic studies esp. of organic
substances - importance for
basic research
structure activity relationship
clue for biological processes
supramolecular interactions
electrosynthesis
electroanalysis
Phys.Chem. 2015/2016
Main fields of application of
polarography (voltammetry) today
Trace metal and inorganic determination and speciation

bioavailability studies

water analysis

soil analysis
3. Trace organic analysis

Pharmaceuticals

Preparations

Biological samples

Metabolites

Herbicides and pesticides

Explosives

Ecotoxic substances

Dyes

Chemical carcinogens
2.
Phys.Chem. 2015/2016
LDR of various
polarographic and
voltammetric
techniques
Phys.Chem. 2015/2016
Phospholipid bilayers
Real
Model
Aqueous Outer area
Hydrophillic part
Phospholipid bilayer
Hodrophobic part
Aqueous Inner area
Phys.Chem. 2015/2016
Types of experimental phase interphases
Free
RE1
Stabilized
WE1
(Supported)
RE2
WE2
Phys.Chem. 2015/2016
Metallic electrode or polymer
Patch Clamp Technique
Classic P-C
technique
Planar P-C
technique
Phys.Chem. 2015/2016
DNA
Adenine:
• 6-electron reduction
including deamination
• Under normal conditions 4electron
Cytosine:
• 3-electron reduction
(deamination + dimerization)
Phys.Chem. 2015/2016
OSCILOGRAFIC POLAROGRAPHY
Controlled insertion of auxiliary current
dE/dt
dsDNA
ssDNA
CA
Cathodic
part
Poorly soluble
compound with Hg
Anodic
part
G
E
J.N.Davidson and E.Chargraff: The Nucleic Acids, Vol.1, Academic Press, New York 1955
Palecek E.: Oszillographiche Polarographie der Nucleinsauren und ihrer Bestandteile; Naturwiss. 45 (1958) 186
Palecek E.: Oscillographic polarography of highly polymerized deoxyribonucleic acid; Nature 188 (1960) 656
Phys.Chem. 2015/2016
Various techniques of analysis
ADSORPTIVE STRIPPING
ADSORPTIVE
TRANSFER
STRIPPING
Nucleic acid in the cell with electrolyte
is accumulated at the electrode surface
Nucleic acid is
accumulated at the small
electrode from very small
drop of the sample (3-10
ml)
Phys.Chem.
2015/2016
The sample (nucleic acid) is
transferred into the pure
eelctro;yte
Cytosine on Silver solid amalgam electrode
m-AgSAE
2.10-5 M; 0,1 M acetate buffer; pH 4,8
-1600
Adenin - Rce 31 (Id=Id; Ic=0; Ik=0; Iir=0)
-600
-1000
-100
-1100
-1200
-1300
-1400
-1500
-18000
400
Rce 23 (Id=Id; Ic=0; Ik=0)
E [mV]
-12000
adenin 1.10-4 M + cytosin 1.10-4 M
v=80-160-320-640 mV.s-1;
I [nA]
I [nA]
-1100
I ref - 20-40-80-160
20-40-80-160
I ref - 40-80-160-320
40-80-160-320
I ref - 80-160-160-320
80-160-160-320
-6000
rce (23)
0
-1100
-1300
-1500
-1700
E [mV]
Phys.Chem. 2015/2016
Mixture of adenine a guanine
-800
blank
Ade
Gua
I [nA]
-600
mixture
adenine : guanine
58 : 42
-400
-200
0
-700
-900
E [mV]
-1100
adenine
guanine CT DNA
p DNA
Critical level nmol.L-1
0.67
0.82
0.86
0.75
Limit of detection nmol.L-1
0.95
1.85
1.75
1.25
Limit of determination nmol.L-1
3.06
6.70
5.99
6.93
RSD %
1.28
1.83
0.47
0.57
Phys.Chem.
2015/2016
CT - Calf thymus DNA; p DNA - plazmidová
DNA;
C. Interaktion of damaged (denaturated) DNA
with osmium complexex
OsO4 (L)
O
+
N
-O
Os
L = Pyridine
-O
1,10–Fenantroline
Bipyridyl
Phys.Chem. 2015/2016
O
+
N
Pyrimidina bases – OsO4 – Pyridine
O
O
CH3
NH
O
O
CH3
OsO4(Py)
O
NH
O
NH
+
N
Os
NH
O
O
Thymine
+
N
NH2
NH2
N
T>>C,U>>A,G
O
O
O
NH
Uracil
O
O
+
O
N
O
N
O
OsO4(Py)
NH
Os
NH
O
NH
Cytosine
O
N
OsO4(Py)
+
N
O
NH
O
+
Os
NH
Phys.Chem. 2015/2016
O
O
+
N
DNA modiffied with Os(py) on HMDE a m-AgSAE
DNA 2 mg.l-1; 0,1 M acetate buffer; pH 4,8
-5500
20 mV/s
40 mV/s
80 mV/s
160 mV/s
320 mV/s
640 mV/s
-4500
i [nA]
-3500
-2500
-1500
-500
-1200
-1250
-1300
-1350
-1400
-1450
-1500
-1500
E [mV]
-1300
2) A- +H+  HAAds
-1100
i [nA]
1)
HAAds+e-A-+Hads
-900
-700
-1550
-1600
Os-DNA_A2
20 mV/s
40 mV/s
80 mV/s
160 mV/s
320 mV/s
640 mV/s
-500
-300
-100
-900
-1000
Phys.Chem. 2015/2016
-1100
-1200
-1300
E [mV]
-1400
-1500
-1600
Hybridization of DNA (RNA) is based on the principle of
double helix formation from two complementary
strands
hybridization probe
CGAATACGACCTTA
Sequence of the probe is arranged
(synthetized) with respect to the DNA
sequence, which is
CGAATACGACCTTA
GCTTATGCTGGAAT
GCTTATGCTGGAAT
Target DNA
is detected using the probe
Phys.Chem. 2015/2016
Hybridization of DNA (RNA) is based on the principle of
double helix formation from two complementary
strands
This principle is used in various moidification in routine
analysis:
• Detection of some nucleotide sequences
• Detection of mutations, „polymorphisms“ in some sequences of genome
• Determination of gen expressions
Phys.Chem. 2015/2016
From practical reason is one end of the probe
connected to the solid surface
Immobilized probe is exposed to the analyzed DNA (RNA) sample
If the sample contains the chain of DNA (RNA) with
complementary sequence to the probe („target sequence “), the
double helix („duplex“, „hybrid“) is formed on the solid surface
Phys.Chem. 2015/2016
From practical reason is one end of the probe connected to the solid
surface
If the sample contains the chain of DNA (RNA) with
complementary sequence to the probe („target sequence “), the
double helix („duplex“, „hybrid“)
is formed on the solid surface
Phys.Chem. 2015/2016
Non-specific DNA molecules are removed (washed off)
If the sample contains the chain of DNA (RNA) with
complementary sequence to the probe („target sequence “), the
double helix („duplex“, „hybrid“) is formed on the solid surface
Phys.Chem. 2015/2016
Non-specific DNA molecules are removed (washed off)
The detection step follows
It is advantageous to mark DNA with some detectable sensor
(radionuclide, fluoroform…)
Phys.Chem. 2015/2016
DNA („arrays“):
• Simultaneous application of a lot of probes
• Application of different (of different “colors“) fluorescence
probes
• Commercial available devices (Affymetrix…)
Phys.Chem. 2015/2016
Electrochemical sensor for DNA hybridization:
electrode with hybridization probe on the surface
I
hybrid
samotná sonda
Phys.Chem. 2015/2016
E
Double surface strategy:
• Hybridization is realized on one surface (H), which was
optimized for these purposes; it is not necessary to be an
electrode
• separation of target DNA
• target DNA from the surface H is released and
electrochemically determined
Detection electrode
surface H
Phys.Chem. 2015/2016
Double surface strategy: :
• Hybridization is realized on one surface (H), which was
optimized for these purposes; it is not necessary to be an
electrode
• separation of target DNA
• target DNA from the surface H is released and
electrochemically determined
detekční elektroda
Phys.Chem. 2015/2016
Double surface strategy: the use of magnetic beads
Surface H
Detection electrode
detection
cílová DNA
Nonspecific
DNA
magnetic beads with
hybridization probe ~1µm
hybridization
separation
magnet
Phys.Chem. 2015/2016
Release of target
DNA
Double surface strategy:
•Enzymatic probes („biocatalytic amplification of the signal“)
•Usually using biotin marked DNA in combination with streptavidin
conjugates with enzymes (Streptavidin (STV) - alcali phosphatase (ALP))
detection electrode
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
P
OH
OH
O
OH
Surface H
Phys.Chem. 2015/2016
Wang, J.; Xu, D. K.; Erdem, A.;
Polsky, R.; Salazar, M. A. Talanta
2002, 56, 931-938.
O P
alkalic
phosphatase OH
Palecek, E.; Billova, S.; Havran, L.;
Kizek, R.; Miculkova, A.; Jelen, F.
Talanta 2002, 56, 919-930.
Phys.Chem. 2015/2016
Double surface strategy:
• reporter probes, RP
povrch H
Phys.Chem. 2015/2016
Catalysis

Catalysis is the process, in which the rate (not equilibrium) of a chemical reaction is
increased (decreased, respectively) by means of a chemical substance known as a
catalyst.

Unlike other reagents that participate in the chemical reaction, a catalyst is not
consumed.

The catalyst may participate in multiple chemical transformations, although in
practice catalysts are secondary processes.

it changes the reaction mechanism, it changes the activation energy, it is involved in
the formation of the activation complex
A+B→AB vs.
A+B+K→ABK→AB+K
EAB
EAB – activation energy without catalysis
EABK – activation energy with catalysis
GAB – Gibbs energy of the reaction
Phys.Chem. 2015/2016
EABK
GAB
products
Example:
Catalytic production of sulphuric acid
Sulfuric acid is produced from sulfur, oxygen and water via the contact process.
In the first step, sulfur is burned to produce sulfur dioxide.
(1) S(s) + O2(g) → SO2(g)
This is then oxidised to sulfur trioxide using oxygen in the presence of a
vanadium(V) oxide catalyst.
(2) 2 SO2 + O2(g) → 2 SO3(g) (in presence of V2O5)
Finally the sulfur trioxide is treated with water (usually as 97-98% H2SO4
containing 2-3% water) to produce 98-99% sulfuric acid.
(3) SO3(g) + H2O(l) → H2SO4(l)
Phys.Chem. 2015/2016
Reaction rate
E  S 
 ES
k1 , k 1
k 1  k 2
Km 
k1
k2

E  P
Michaelis constant Km
dP
[S]
 vo  Vmax
dt
[S]  K m
Michaelis–Menten equation
The rate of production of the product, is referred to as the reaction
rate, V in enzyme kinetics.
Vmax = maximal rate for the given catalyst concentration
(Double) Reciprocal expression
K
1
1
1

 m 
v0 Vmax Vmax [S]
Phys.Chem. 2015/2016
Graphical expression of
Michaelis–Menten equation
1st order
kinetic
vo
[S]  K m  vo  Vmax
0 order
kinetic
Vmax
Vmax
2
vo  Vmax
[S]
[S] V
[S]
Vmax K  max
v2o 
[S]  [S]
2[Sm]
Vmax
[S]
[S]
 Vmax
 Vmax  k[S]0
[S]  K m
[S]
[S]
[S] Vmax
 Vmax

[S]  [S]
2[S] 2
Area
of catalyst
enzym
nasycen
saturation by a
substrátem
substrate
[S] mol/l
Km
Phys.Chem. 2015/2016
Reaction rate in Linearized Graph
1/vo
Km 1
1
1



v0 Vmax Vmax [S]
1/v0 vs. [S]
1 / V m ax
1/[S]
- 1 / Km
Phys.Chem. 2015/2016
Reaction rate in Linearized Graph
1/vo
Km 1
1
1



v0 Vmax Vmax [S]
1/v0 vs. [S]
1 / V m ax
1/[S]
- 1 / Km
Phys.Chem. 2015/2016
Competitive inhibition
Some molecules inhibit catalysis by competing for the active sites. The
strongest inhibitors are called poisons.
CH3OH  HCOOH
C2H5OH  CH3COOH
Alcohol dehydrogenase
The catalyst is competitively inhibited by a non-toxic substrate to the
prejudice of toxic substrate



The maximal rate is reached at higher [S] values
Vmax is not changed
Km is increased
Phys.Chem. 2015/2016
Competitive inhibition
Vmax
1/vo
vo
1 / Vmax 1 / Vmax
[S]-1
Km Km inh
[S]
mol.l-1
- 1/Km - 1/Km
Phys.Chem. 2015/2016
Non-competitive inhibition

The inhibitor (substrate) binds to the enzyme at a site other than the
catalyst's active site (this other site is called an allosteric site).

Km is not changed (active site is free for the substrate)

Vmax is decreased, because the concentration of E-S complex decreases

In this mode of inhibition, there is no competition between the
inhibitor and the substrate, so increasing the concentration of the
substrate still does not allow the maximum enzyme activity rate to be
reached.
Phys.Chem. 2015/2016
Non-competitive inhibition
v0
Vmax
1/vo
Vmax inh
1 / Vmax inh
1 / Vmax
1/[S]
Km
Km inh
[S] mol.l-1
- 1 / Km
- 1 / Km
Phys.Chem. 2015/2016
Research topics in Physical Chemistry
www.jh-inst.cas.cz
•
Development of new fluorescence methods and their application in the research of
structure, functionality and dynamics of biomembranes; single molecule spectroscopy in
biological systems, dynamics characterization in model and biomembranes on the
picosecond to millisecond time scale; characterisation of DNA condensation processes
relevant to gene therapy; advanced in vivo fluorescence microscopy.
•
Elucidation of the function of biologically active molecules based on their electrochemical
reactivity. The interim aims will be the elucidation of (i) creation and stability of
monolayers on polarized interfaces and (ii) charge transfer reactions.
•
preparation and characterization of phospholipid bilayers at stabilized phase boundaries
enabling studies of transfer of charged and uncharged species between the two phases
•
studies of transfer of charged and uncharged species between the two phases
•
transport of compounds across the model membrane using cell permeable peptides
•
studies on electrochemical properties of biologically important species
•
development of new types of electrochemical sensors, devices and methods for following
DNA, proteins and other biologically active substances;
•
novel techniques of DNA damage detection;
Phys.Chem. 2015/2016
Research topics in Physical Chemistry
www.jh-inst.cas.cz
•
research on function and structure of metallothioneins and phytochelatins (bioligands);
•
biophysical and biochemical problems accompanying utilization of electrochemical
techniques;
•
state of solutions and its effect on electrode processes;
•
bioavailable forms of trace elements present in soil solution and their availability to
plants.
•
Molecular sieve chemistry and catalysis
•
Synthesis of zeolites, zeotypes, mesoporous molecular sieves and
•
hierarchic materials combining micro and meso porosity
•
Design and characterization of new photosensitisers and investigation of their
interactions with target biological macromolecules
Phys.Chem. 2015/2016