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The separation of lactatdehydrogense (LDH)
isoenzymes by agarose electrophoresis
MUDr. Michal Jurajda
Svatava Tschöplová
Šárka Kuchtíčková
Pavla Součková
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The objectives of the practical training
Revision of enzymology Evaluation of activity of enzymes in
bilogic samples LDH isoenzymes assay
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The basic characteristics of the enzymes
AE
B
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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.
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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)
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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
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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)
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Regulation of enzymatic activity in the biological systems
Regulation of transcription Activation of proenzymes Inhibition by specific inhibitors
(competitive, non-competitive)
Metabolic pathways
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Genetics
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Genetic polymorfismmultigene diseases Rare alleles (mutation)hereditary
enzymopathies
Consequences:alterations of structure and/or concentration
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Metabolic consequences
AE
BC
A B
E
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Clinical medicine
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Enzymes in blood plasma
Functional plasmatic enzymesenzymes of blood clotting, lipoprotein lipaze, ceruloplasmin
Non-functional plasmatic enzymes1.enzymes from exocrine glands (amylase)2.intracellular enzymes
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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
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Liver, kidney
Inhibitors
Inhibitors
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Diagnostics
CK creatinkinase, CK-MB myocardial band
AST aspartate aminotransferase (mit.) ALT alaninaminotransferase LDH laktatedehydrogenase
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The separation of isoenzymes
Electrophoresis or chromatography Activity assay under different conditions
pH, temperature, different substrates
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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
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LDH - tetramer
H unit aerobic metabolismlactat pyruvate
M unit anaerobic metabolismpyruvate lactat
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LDH izoenzymes
1. Heat deactivation - LDH5 is termo-instable when heated to 57C
2. afinity to hydroxybutyrat - myocardial fraction (LDH1 LDH2 ) catalyze hydroxybutyrat dehydrogenation LD/HBD low = myocardial affection, LD/HBD high = hepatal affection
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LDH
Lactat dehydrogenase: hemolysis causes false increase of LDH
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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
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Normal levels of LDH isoenzymes
Range [%] mean standard deviationLDH 1 31,0 - 49,0 39,15,10LDH 2 38,0- 58,0 47,75,30LDH 3 5,5 -16,5 10,93,20LDH 4 0,0 - 7,0 2,22,02LDH 5 0,0 - 1,5 0,240,36
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Automatic pipette
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The gel pouring
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Agarose gel
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The sample loading
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Agarose gel
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The Sample loading
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The sample loading
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The sample loading
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Gel with samples in elfo tank
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Electrophoresis
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Paper bridges in elfo tank
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Visualization
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Line and peak detection
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Densitometric evaluation
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Normal levels of LDH isoenzymes
Range [%] mean standard deviationLDH 1 31,0 - 49,0 39,15,10LDH 2 38,0- 58,0 47,75,30LDH 3 5,5 -16,5 10,93,20LDH 4 0,0 - 7,0 2,22,02LDH 5 0,0 - 1,5 0,240,36
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Matrixmetalloproteinases
enzymes capable to cleave ECM release of growth and motility factors
from ECM activity regulation (transkription,
plasmin) tissue inhibitors of MMPs (TIMPs)
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Matrixmetalloproteinases-zymography
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.
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Matrixmetalloproteinasy-zymografie
Elektrophoresis - PolyAacrylamidGelElectrophoresis with gelatine.
Coomasie blue staining
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Zymogram
pro MMP-2
MMP-2
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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
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Multifactorial diseases
Atherosclerosis Diabetes mellitus Allergy Tumors
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Hereditary enzymopathies
Fenylketonuria Alkaptonuria Thesaurismosy: glykogenozy,
mukopolysacharidozy, glykosfingolipidozy
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