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

AE

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 polymorfismmultigene diseases Rare alleles (mutation)hereditary

enzymopathies

Consequences:alterations of structure and/or concentration

Metabolic consequences

AE

BC

A B

E

Clinical medicine

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

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

Liver, kidney

Inhibitors

Inhibitors

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 metabolismlactat pyruvate

M unit anaerobic metabolismpyruvate lactat

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

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

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

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

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

Matrixmetalloproteinases

enzymes capable to cleave ECM release of growth and motility factors

from ECM activity regulation (transkription,

plasmin) tissue inhibitors of MMPs (TIMPs)

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.

Matrixmetalloproteinasy-zymografie

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

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Hereditary enzymopathies

Fenylketonuria Alkaptonuria Thesaurismosy: glykogenozy,

mukopolysacharidozy, glykosfingolipidozy

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