Transcript Stanovení isoenzymů laktátdehydrogenázy (LDH) elektroforezou

The separation of
lactatdehydrogense (LDH)
isoenzymes by agarose
electrophoresis
MUDr. Michal Jurajda
Svatava Tschöplová
Šárka Kuchtíčková
Pavla Součková
The objectives of the practical
training



Revision of enzymology
Evaluation of activity of enzymes in
bilogic samples
LDH isoenzymes assay
The basic characteristics of the
enzymes
A
E
B
Enzymes = biocatalyzers

Enzymes Lower the Activation Energy
of Reactions
Speed up chemical reactions
Equilibrium is not influenced

Michaelis-Menten Equation

Km is defined as the [S] that results in
half-maximal rate.


Units

Catal is the unit of enzymatic activity
1cat =activity which converts 1mol of
substrate to product during 1second

Katal characterizes amount of enzyme
not properties of that (Km)
Enzymes  proteins

Most biological enzymes are proteins .
They speed up chemical reactions in
biological systems. (the exception is
catalytic RNA).

The segment of the enzyme molecule
that does the work is called the active
site . The amino-acid residues in this
site are arranged in specific 3D
conformation enabling interaction with
substrates
Izoenzymes



They catalyze the same reaction but
they are different in the structure 
physical-chemical characteristics
Primarny - different genes
Secondary - one gene produce different
enzymes by different posttranslation
alterations (acetylation, cleavage)
Regulation of enzymatic activity
in the biological systems




Regulation of transcription
Activation of proenzymes
Inhibition by specific inhibitors
(competitive, non-competitive)
Metabolic pathways
Genetics



Genetic polymorfism
multigene diseases
Rare alleles (mutation)
hereditary enzymopathies
Consequences:
alterations of structure and/or
concentration
Metabolic consequences
E
E
A
A
B
B
C
Clinical medicine
Enzymes in blood plasma


Functional plasmatic enzymes
enzymes of blood clotting, lipoprotein
lipaze, ceruloplasmin
Non-functional plasmatic enzymes
1.enzymes from exocrine glands
(amylase)
2.intracellular enzymes
The concentration of enzymes in
the blood plasma





The level of enzymatic activity of
individual enzymes in the cell
The localization of the enzyme in the
cell
The extent of cellular damage
The number of damaged cells
The elimination rate of the enzyme
Inhibitors
Inhibitors
Liver, kidney
Diagnostics




CK creatinkinase, CK-MB myocardial
band
AST aspartate aminotransferase (mit.)
ALT alaninaminotransferase
LDH laktatedehydrogenase
The separation of isoenzymes


Electrophoresis or chromatography
Activity assay under different conditions
pH, temperature, different substrates
LDH - tetramer






H unit and M unit
LDH1 HHHH heart, brain, kidney
LDH2 HHHM heart
LDH3 HHMM smooth muscle
LDH4 HMMM skeletal muscle
LDH5 MMMM skeletal muscle, liver
LDH - tetramer


H unit aerobic metabolism
lactat
pyruvate
M unit anaerobic metabolism
pyruvate
lactat
LDH izoenzymes


1. Heat deactivation - LDH5 is termoinstable when heated to 57C
2. afinity to hydroxybutyrat - myocardial
fraction (LDH1 LDH2 ) catalyze
hydroxybutyrat dehydrogenation
LD/HBD low = myocardial affection,
LD/HBD high = hepatal affection
LDH

Lactat dehydrogenase: hemolysis
causes false increase of LDH
Electrophoretic separation of
LDH in agarose gel


Agarose in barbital buffer
Visualization:

1. lithium lactat
2. p-iodonitroterazoluim violet - colour substantion, blue when
reduced
3. NAD+
4. KCN
5. Fanezinmethosulfat electron transducer from NADH

5% acetic acid




Normal levels of LDH
isoenzymes
LDH 1
LDH 2
LDH 3
LDH 4
LDH 5
Range [%]
31,0 - 49,0
38,0- 58,0
5,5 -16,5
0,0 - 7,0
0,0 - 1,5
mean  standard deviation
39,15,10
47,75,30
10,93,20
2,22,02
0,240,36
Automatic pipette
The gel pouring
Agarose gel
The sample loading
Agarose gel
The Sample loading
The sample loading
The sample loading
Gel with samples in elfo tank
Electrophoresis
Paper bridges in elfo tank
Visualization
Line and peak detection
Densitometric evaluation
Normal levels of LDH
isoenzymes
LDH 1
LDH 2
LDH 3
LDH 4
LDH 5
Range [%]
31,0 - 49,0
38,0- 58,0
5,5 -16,5
0,0 - 7,0
0,0 - 1,5
mean  standard deviation
39,15,10
47,75,30
10,93,20
2,22,02
0,240,36
Matrixmetalloproteinases




enzymes capable to cleave ECM
release of growth and motility factors
from ECM
activity regulation (transkription,
plasmin)
tissue inhibitors of MMPs (TIMPs)
Matrixmetalloproteinaseszymography


SDS elektrophoresis - SDS coats
proteins and form polyanionts,
elektrophoresis runs toward anode.
SDS is removed with Triton and proteins
renaturate their enzymatic activity is
restored.
Matrixmetalloproteinasyzymografie


Elektrophoresis PolyAacrylamidGelElectrophoresis with
gelatine.
Coomasie blue staining
Zymogram
pro MMP-2
MMP-2
Sources




http://esg-www.mit.edu:8001/esgbio/7001main.html
Biochemie v obrazech, J. Musil, O.Nováková,
Avicenum 1990
Enzymologie jaterních nemocí, J. Pojer, SZN 1968
Enzymologie srdečního infarktu, J. Pojer, SZN 1963
Multifactorial diseases




Atherosclerosis
Diabetes mellitus
Allergy
Tumors
Back
Hereditary enzymopathies



Fenylketonuria
Alkaptonuria
Thesaurismosy: glykogenozy,
mukopolysacharidozy, glykosfingolipidozy
Back