Title: The separation of lactatdehydrogense (LDH) isoenzymes by agarose electrophoresis
1The separation of lactatdehydrogense (LDH)
isoenzymes by agarose electrophoresis
- MUDr. Michal Jurajda
- Svatava Tschöplová
- Šárka Kuchtícková
- Pavla Soucková
2The objectives of the practical training
- Revision of enzymology
- Evaluation of activity of enzymes in bilogic
samples - LDH isoenzymes assay
3The basic characteristics of the enzymes
4Enzymes 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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8Units
- 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)
9Enzymes ? 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
10Izoenzymes
- 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)
11Regulation of enzymatic activity in the
biological systems
- Regulation of transcription
- Activation of proenzymes
- Inhibition by specific inhibitors (competitive,
non-competitive) - Metabolic pathways
12Genetics
13- Genetic polymorfismmultigene diseases
- Rare alleles (mutation)hereditary enzymopathies
- Consequencesalterations of structure and/or
concentration
14Metabolic consequences
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15Clinical medicine
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17Enzymes in blood plasma
- Functional plasmatic enzymesenzymes of blood
clotting, lipoprotein lipaze, ceruloplasmin - Non-functional plasmatic enzymes1.enzymes from
exocrine glands (amylase)2.intracellular enzymes
18The 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
19Inhibitors
Inhibitors
Liver, kidney
20Diagnostics
- CK creatinkinase, CK-MB myocardial band
- AST aspartate aminotransferase (mit.)
- ALT alaninaminotransferase
- LDH laktatedehydrogenase
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23The separation of isoenzymes
- Electrophoresis or chromatography
- Activity assay under different conditions pH,
temperature, different substrates
24LDH - 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
25LDH - tetramer
- H unit aerobic metabolismlactat
pyruvate - M unit anaerobic metabolismpyruvate
lactat
26LDH izoenzymes
- 1. Heat deactivation - LDH5 is termo-instable
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
27LDH
- Lactat dehydrogenase hemolysis causes false
increase of LDH
28Electrophoretic 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
29Normal levels of LDH isoenzymes
30Automatic pipette
31The gel pouring
32Agarose gel
33The sample loading
34Agarose gel
35The Sample loading
36The sample loading
37The sample loading
38Gel with samples in elfo tank
39Electrophoresis
40Paper bridges in elfo tank
41Visualization
42Line and peak detection
43Densitometric evaluation
44Normal levels of LDH isoenzymes
45Matrixmetalloproteinases
- 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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47Matrixmetalloproteinases-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.
48Matrixmetalloproteinasy-zymografie
- Elektrophoresis - PolyAacrylamidGelElectrophoresis
with gelatine. - Coomasie blue staining
49Zymogram
pro MMP-2
MMP-2
50Sources
- http//esg-www.mit.edu8001/esgbio/7001main.html
- Biochemie v obrazech, J. Musil, O.Nováková,
Avicenum 1990 - Enzymologie jaterních nemocí, J. Pojer, SZN 1968
- Enzymologie srdecního infarktu, J. Pojer, SZN
1963
51Multifactorial diseases
- Atherosclerosis
- Diabetes mellitus
- Allergy
- Tumors
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52Hereditary enzymopathies
- Fenylketonuria
- Alkaptonuria
- Thesaurismosy glykogenozy, mukopolysacharidozy,
glykosfingolipidozy
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