Biochemistry in the summer semester
- MODULE A Credit
• 4 practicals (A1 – A4)
• 3 oral testings:
 Respiration, Blood and Body Fluids
 Urogenital system, Acid-Base Balance
 Digestion and Resorption, Intermediary Metabolism
Module A
st
/ 1 year
We
ek
Lecture
1
1) Transport of oxygen and carbon
dioxide / F.Duška
Introduction to practical trainings (theory:
blood collection, electrophoresis,
centrifugation, a guide to the labs) (S)
2
2) Biochemistry of blood cells /
V.Kvasnicová
Blood serum proteins electrophoresis (S/P)
A1
3
3) Biochemistry of the kidneys /
F. Duška
Hemoglobin - structure, function and
metabolism (S)
4
CONFERENCE - respiration,
blood, body fluids
5
4) Metabolism of water and
minerals / F. Duška
6
5) Acid-base balance / F.Duška
Chemical examination of the urine. Urine
sediment (P) A2
7
6) Functional biochemistry of the
digestive tract / F.Duška
Acid - base balance (S)
8
7) Energy metabolism / F. Duška
Determination of blood glucose level.
Interpretation of oGTT (P) A3
9
CONFERENCE - urogenital
system, acid-base balance
10
8) General toxicology / E.
Samcová
9) Biochemistry of
the liver /
J. Trnka
Alkali phosphatase (ALP) - determination
of serum activity (P) A4
11
10) Biotransformation of
xenobiotics/ E.Samcová
Biochemistry of the liver (S)
12
11) Vitamins in the metabolism /
J. Trnka
Biotransformation of xenobiotics (S)
13
12) Free radicals and antioxidants
/
J. Trnka
Intermediary metabolism relationships (S)
14
CONFERENCE - digestion and
resorption
15
Practical training (P) / seminar (A)
credit testing: 17.3. / 3:00 p.m.
credit testing: 21.4. / 9:40 a.m.
credit testing: 28.5. / 9:00 a.m.
Laboratory methods
in the summer semester
Vladimíra Kvasnicová
Taking a capillary blood sample
Centrifugation
= separation of particles from a solution
according to their size, shape, density,
viscosity of the medium and rotor speed
centrifugal force:
P = m
m - mass of the particle
r - radius of the centrifuge
 - angular speed
x
r
x
2
Centrifugation
• horizontal or fixed-angle rotor
• the centrifuge cups in the rotor
must be balanced
The figures were found at http://www.all-science-fair-projects.com/science_fair_projects_encyclopedia/Centrifuge
and http://www.flickr.com/photos/gonzales2010/9624402/ (Feb 2008)
Centrifugation
The figure was found at http://www.flickr.com/photos/businesscheese/1803417133/ (Feb 2008)
Centrifugation
• speed: revolutions per minute (rpm)
! the same rpm doesn´t mean the same centrifugal
force in different centrifuges !
• centrifugal force: relative centrifugal force (RCF)
 RCF = 1.12 x 10-5 x r x (rpm)2
 r = radius of the centrifuge (cm)
 units: G (= how many times is the acceleration higher
than the gravitational acceleration, G = 9.81 m x s-2)
Centrifugation
a) simple centrifuges (usual in a chemistry)
(up to 10,000 G)
b) high-speed refrigerated centrifuges
(up to 50,000 G)
c) ultracentrifuges (refrigerated + vacuum)
(up to 500,000 G)
example:
RCF at least 1000 G for 10 min will give good
separation of clotted blood from serum
The figures were found at
http://www.chem.arizona.edu/tpp/chemt/CAn/Graphics/centrifuge/blood%20centrifugation.png and
http://www.academic.marist.edu/~jzmz/HematologyI/Intro3.html (Feb 2008)
The figures were found at
http://science.tjc.edu/Course/BIOLOGY/bott/anatomy/2402/summer%202402%20%20blood%20notes/c18_02.jpg
and http://faculty.washington.edu/kepeter/119/images/hematocrit_tube_trio.jpg (Feb 2008)
A) Preparative centrifugation
•
separation of
particles from a
solution
• two fractions are
formed:
1. sediment
(pellet, solid phase)
2. supernatant
(liquid phase)
The figure was found at http://www.steve.gb.com/science/molecular_biology_methods.html (Feb 2008)
differential centrifugation
(= moving boundary, rate-zonal centrifugation)
• special kind of the preparative centrifugation
• it is used for separation of cell organelles which
differ in size and density
• large, dense structures form a sediment (pellet) in a
centrifuge tube faster (low RCF is enough for the
separation) than small, less dense ones do
supernatant obtained from a low speed centrifugation is
centrifugated again (a number of time) using a higher
RCF
Differential centrifugation
The figure was found at http://fig.cox.miami.edu/~cmallery/150/protein/c7x3fractionation.jpg (Feb 2008)
B) Analytical centrifugation
• it involves a measuring of physical properties
of the sedimenting particles
(sedimentation coefficient, MW)
• ultracentrifugation is optimal
• molecules are observed by optical system
during centrifugation and projected on to a
film or a computer
Electrophoresis
= an analytical method based on movement of
charged particles because of an external
electric field
• velocity of a particle depends on the:
a) size, shape and charge of the particle
b) given applied voltage
Electrophoresis
• anion - negatively charged ion, it moves to the
anode (+)
• cation - positively charged ion, it moves to the
catode (-)
• amphoteric - a substance that can have a
positive, zero, or negative charge, depending
on conditions (e.g. proteins)
Classification of electrophoretic
techniques
1. free-boundary electrophoresis
 separation is carried out entirely in a
liquid phase, i.e. no support is used
(capillary electrophoresis)
2. electrophoresis in a supporting medium
 paper, gel (agarose, polyacrylamide)
 it can be done horizontally or vertically
Capillary electrophoresis
The figure was found at
http://www.hood.edu/images/content/academics/instruments/Agilent_Capillary_Electrophoresis_System.JPG
(Feb 2008)
Capillary electrophoresis
The figure was found at http://en.wikipedia.org/wiki/Capillary_electrophoresis (Feb 2008)
Gel electrophoresis - horizontal
The figure was found at http://www.mun.ca/biology/desmid/brian/BIOL2250/Week_Three/electro4.jpg (Feb 2008)
Gel electrophoresis - vertical
SDS-PAGE – animation
The figure was found at http://fig.cox.miami.edu/~cmallery/150/protein/page.jpg (Feb 2008)
Effects of electrophoretic parameters
on separation
• pH changes charge of analyte and hence its mobility, it can
affect structure of analyte (denaturing, dissociating)
• ionic strength changes voltage or current: increased ion. str.
usually reduces migration velocity and increases heating
• temperature: overheating can denaturate (precipitate) proteins;
lower t. reduces diffusion but also reduces migration velocity,
no effect on resolution
• current: too high current causes overheating
• voltage: migration velocity is proportional to voltage
• time: resolution (separation of bands) increases linearly with
time, but dilution of bands (diffusion) increases with the square
root of time
• medium: major factors are endosmosis and pore-size effects,
which affect migration velocities
Process of electrophoresis
1. sample application
2. adjustment of voltage or current - DIRECT CURRENT !
(gel-electrophoresis about 70 - 100 volts,
capillary electrophoresis about 20,000 volts)
3. separation time: minutes
(e.g. gel-electrophoresis of serum proteins 30 min.)
4. electrophoresis in supporting medium: fixation, staining
5. evaluation:
 qualitative (standards)
 quantitative (densitometry)
Equipment used for the gel electrophoresis
in the practical training A1
power suply
(direct current)
electrophoresis
chamber
containers for staining
and destaining gel
applicator
Electrophoresis
– examples from clinical medicine
• separation of
serum proteins,
isoenzymes,
nucleic acids
• immunoelectrophoresis
(immunoglobulins)
The figure was found at http://www.sebia-usa.com/images/controlGel1.jpg (Feb 2008)
Electrophoresis
– examples from clinical medicine
• separation of
serum proteins,
isoenzymes,
nucleic acids
• immunoelectrophoresis
(immunoglobulins)
The figure was found at http://www.sebia.com/V2/php/index.php?tpc=1&nv=0,2&page=contenu&id_prod=39# (Feb 2008)
The use of protein electrophoresis in
diagnostics of diseases
• electrophoretic patern is constant under
physiological conditions (intensity of bands)
• spectrum of plasma proteins changes under
various diseases (their ratio)
evaluation of electrophoretic patern
(bands or peaks)
Principal proteins of each fraction
immunoglobulins:
IgG, IgA, IgM
2-macroglobulin
haptoglobin
1-antitrypsin
transferrin
orosomucoid
C3-complement
Electrophoresis of serum proteins
on agarose gel – 6 bands
hypergammaglobulinemia
normal patern
The figure is from http://www.sebia-usa.com/products/proteinControl.html (Feb 2007)
Electrophoresis of serum proteins
on agarose gel – 5 bands
A. normal patern
B. acute response
C. paraproteinemia
D. fraction of fibrinogen
if plasma is analyzed
instead of serum
Evaluation by densitometry - peaks
60% 3% 9% 12% 16%
The figures are from http://www.sebia-usa.com/products/hyrys2.html
and http://erl.pathology.iupui.edu/LABMED/GENER27.HTM respectivelly (Feb 2007)
The figure is from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed.
Wiley-Liss, Inc., New York, 1997. ISBN 0-471-15451-2
The figure is from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed.
Wiley-Liss, Inc., New York, 1997. ISBN 0-471-15451-2
The figure is from http://www.sebia-usa.com/products/reagents.html (Feb 2007)
The figure is from http://www.sebia-usa.com/products/reagents.html (Feb 2007)
IMMUNOFIXATION
- paraprotein specification (monoclonal Ig)
The figure is from http://www.sebia-usa.com/products/reagents.html (Feb 2007)