The separation of lactatdehydrogense (LDH) isoenzymes by agarose electrophoresis MUDr. Michal Jurajda Svatava Tschöplová Šárka Kuchtíčková Pavla Součková

The objectives of the practical training n Revision of enzymology n Evaluation of activity of enzymes in bilogic samples n LDH isoenzymes assay

The basic characteristics of the enzymes A E B

Enzymes = biocatalyzers n Enzymes Lower the Activation Energy of Reactions n Speed up chemical reactions n Equilibrium is not influenced n Michaelis-Menten Equation n K m is defined as the [S] that results in half-maximal rate.

Units n Catal is the unit of enzymatic activity 1cat =activity which converts 1mol of substrate to product during 1second n Katal characterizes amount of enzyme not properties of that (Km)

Enzymes  proteins n Most biological enzymes are proteins. They speed up chemical reactions in biological systems. (the exception is catalytic RNA).enzymes proteins n 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 substratesactive site

Izoenzymes n They catalyze the same reaction but they are different in the structure  physical-chemical characteristics n Primarny - different genes n Secondary - one gene produce different enzymes by different posttranslation alterations (acetylation, cleavage)

Regulation of enzymatic activity in the biological systems n Regulation of transcription n Activation of proenzymes n Inhibition by specific inhibitors (competitive, non-competitive) n Metabolic pathways

Genetics

n Genetic polymorfism multigene diseases Genetic polymorfism multigene diseases n Rare alleles (mutation) hereditary enzymopathies Rare alleles (mutation) hereditary enzymopathies n Consequences: alterations of structure and/or concentration

Metabolic consequences A E B C A B E

Clinical medicine

Enzymes in blood plasma n Functional plasmatic enzymes enzymes of blood clotting, lipoprotein lipaze, ceruloplasmin n Non-functional plasmatic enzymes 1.enzymes from exocrine glands (amylase) 2.intracellular enzymes

The concentration of enzymes in the blood plasma n The level of enzymatic activity of individual enzymes in the cell n The localization of the enzyme in the cell n The extent of cellular damage n The number of damaged cells n The elimination rate of the enzyme

Liver, kidney Inhibitors

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

The separation of isoenzymes n Electrophoresis or chromatography n Activity assay under different conditions pH, temperature, different substrates

LDH - tetramer n H unit and M unit n LDH 1 HHHH heart, brain, kidney n LDH 2 HHHM heart n LDH 3 HHMM smooth muscle n LDH 4 HMMM skeletal muscle n LDH 5 MMMM skeletal muscle, liver

LDH - tetramer n H unit aerobic metabolism lactat pyruvate n M unit anaerobic metabolism pyruvate lactat

LDH izoenzymes n 1. Heat deactivation - LDH 5 is termo- instable when heated to 57  C n 2. afinity to hydroxybutyrat - myocardial fraction (LDH 1 LDH 2 ) catalyze hydroxybutyrat dehydrogenation LD/HBD low = myocardial affection, LD/HBD high = hepatal affection

LDH n Lactat dehydrogenase: hemolysis causes false increase of LDH

Electrophoretic separation of LDH in agarose gel n Agarose in barbital buffer n Visualization: n 1. lithium lactat n 2. p-iodonitroterazoluim violet - colour substantion, blue when reduced n 3. NAD + n 4. KCN n 5. Fanezinmethosulfat electron transducer from NADH n 5% acetic acid

Normal levels of LDH isoenzymes

Automatic pipette

The gel pouring

Agarose gel

The sample loading

Agarose gel

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

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

Matrixmetalloproteinases- zymography n SDS elektrophoresis - SDS coats proteins and form polyanionts, elektrophoresis runs toward anode. n SDS is removed with Triton and proteins renaturate their enzymatic activity is restored.

Matrixmetalloproteinasy- zymografie n Elektrophoresis - PolyAacrylamidGelElectrophoresis with gelatine. n Coomasie blue staining

Zymogram pro MMP-2 MMP-2

Sources n n Biochemie v obrazech, J. Musil, O.Nováková, Avicenum 1990 n Enzymologie jaterních nemocí, J. Pojer, SZN 1968 n Enzymologie srdečního infarktu, J. Pojer, SZN 1963

Multifactorial diseases n Atherosclerosis n Diabetes mellitus n Allergy n Tumors Back

Hereditary enzymopathies n Fenylketonuria n Alkaptonuria n Thesaurismosy: glykogenozy, mukopolysacharidozy, glykosfingolipidozy Back