The separation of lactatdehydrogense (LDH) isoenzymes by agarose electrophoresis

1
The separation of lactatdehydrogense (LDH)
isoenzymes by agarose electrophoresis

• MUDr. Michal Jurajda
• Svatava Tschöplová
• Šárka Kuchtícková
• Pavla Soucková

2
The objectives of the practical training

• Revision of enzymology
• Evaluation of activity of enzymes in bilogic
  samples
• LDH isoenzymes assay

3
The basic characteristics of the enzymes
4
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.

5
(No Transcript)
6
(No Transcript)
7
(No Transcript)
8
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)

9
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

10
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)

11
Regulation of enzymatic activity in the
biological systems

• Regulation of transcription
• Activation of proenzymes
• Inhibition by specific inhibitors (competitive,
  non-competitive)
• Metabolic pathways

12
Genetics
13

• Genetic polymorfismmultigene diseases
• Rare alleles (mutation)hereditary enzymopathies
• Consequencesalterations of structure and/or
  concentration

14
Metabolic consequences
E
E
B
A
A
B
C
15
Clinical medicine
16
(No Transcript)
17
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

18
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

19
Inhibitors
Inhibitors
Liver, kidney
20
Diagnostics

• CK creatinkinase, CK-MB myocardial band
• AST aspartate aminotransferase (mit.)
• ALT alaninaminotransferase
• LDH laktatedehydrogenase

21
(No Transcript)
22
(No Transcript)
23
The separation of isoenzymes

• Electrophoresis or chromatography
• Activity assay under different conditions pH,
  temperature, different substrates

24
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

25
LDH - tetramer

• H unit aerobic metabolismlactat
  pyruvate
• M unit anaerobic metabolismpyruvate
  lactat

26
LDH 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

27
LDH

• Lactat dehydrogenase hemolysis causes false
  increase of LDH

28
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

29
Normal levels of LDH isoenzymes
30
Automatic pipette
31
The gel pouring
32
Agarose gel
33
The sample loading
34
Agarose gel
35
The Sample loading
36
The sample loading
37
The sample loading
38
Gel with samples in elfo tank
39
Electrophoresis
40
Paper bridges in elfo tank
41
Visualization
42
Line and peak detection
43
Densitometric evaluation
44
Normal levels of LDH isoenzymes
45
Matrixmetalloproteinases

• enzymes capable to cleave ECM
• release of growth and motility factors from ECM
• activity regulation (transkription, plasmin)
• tissue inhibitors of MMPs (TIMPs)

46
(No Transcript)
47
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.

48
Matrixmetalloproteinasy-zymografie

• Elektrophoresis - PolyAacrylamidGelElectrophoresis
  with gelatine.
• Coomasie blue staining

49
Zymogram
pro MMP-2
MMP-2
50
Sources

• 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

51
Multifactorial diseases

• Atherosclerosis
• Diabetes mellitus
• Allergy
• Tumors

Back
52
Hereditary enzymopathies

• Fenylketonuria
• Alkaptonuria
• Thesaurismosy glykogenozy, mukopolysacharidozy,
  glykosfingolipidozy

Back