Enzymes Lab # 7

Enzymes: Definition Enzymes are highly specific biologic catalysts that greatly speed up the rate of a chemical reaction occurring in living cells

Measurement of enzymes activity Quantitations of enz. involve measurement of its catalytic activity & relate this activity to enz. conc Substrate Product Enzyme Coenzyme

Cont. Look for - ↑ in product conc. - ↓ in substrate conc. - ↓ in coenzyme conc. - ↑ in altered coenzyme conc. Unit of enzymes activity International unit (IU) The amount of an enzyme that convert one micro-mole of a substrate per minute in an assay system

Techniques for determination of enzyme activity I. Fixed time reaction 1- Incubate the sample with substrate for a fixed time 2- Stop the reaction 3- Measure the amt. of product or substrate II. Kinetic assay (continuous monitoring) Multiple measurements of absorbance are made during the reaction

Kinetic or rate reaction assay: The use of coenzymes NAD NADH Absorbance Wavelength λ Pyruvate + NADH + H + LDH Lactate + NAD +

Techniques for determination of enzyme activity Radioimmunoassay Measures the enzyme conc.

Enzymes classification Oxidoreductases: ex. glucose oxidase Oxidoreductases: ex. glucose oxidase Transferases: ex. aminotransferases Transferases: ex. aminotransferases Hydrolases: ex.lipase Hydrolases: ex.lipase Lyases: ex. decarboxylases Lyases: ex. decarboxylases Isomerases: ex. racemases Isomerases: ex. racemases Ligases or synthetases Ligases or synthetases

Factors that influence enzymatic reaction Substrate concentration Substrate concentration Enzyme concentration Enzyme concentration Product concentration Product concentration PH PH Temperature Temperature Activators & Co-enzymes Activators & Co-enzymes Inhibitors Inhibitors Specificity of enzymes Specificity of enzymes

Determination of lactate dehydrogenase (LDH) LDH is a Hydrogen-transfer enzyme Distribution Heart > liver > SM > erythrocytes > pancreas Principle Pyruvate + NADH + H + LDH L-lactate + NAD +

Determination of LDH: cont. Reference range 200 – 480 U/L (37° C) Wave length: Wave length: 340 nmProcedure

Determination of LDH: Cont. Calculation ∆ A /min=( (A 0 – A 1 ) + (A 1 – A 2 ) + (A 2 – A 3 ) ) / 3 LDH activity (U/L) = ∆ A /min X 9690 Clinical significance ↑LDH could be due to: - Myocardial infarction (MI) - Pernicious anemia - Hepatitis - Skeletal muscles diseases

Determination of Aminotransferases 1- Aspartate aminotransferase (AST) OR glutamate oxaloacetate aminotransferase (GOT) Tissues sources Heart > liver > SM > kidney > pancreasPrinciple L-aspartate +  -ketoglutarate AST oxaloacetate + L-glutamate H + + Oxaloacetate+ NADH + H + malate dehydrogenase L-malate +NAD +

Determination of AST: Cont. Normal range Up to 37 U/L at 37°C Wave length: Wave length: 340 nmProcedure

Determination of AST: Cont. Calculation ∆ A /min=( (A 0 – A 1 ) + (A 1 – A 2 ) + (A 2 – A 3 ) ) / 3 AST activity (U/L) = ∆ A /min X 1750 Clinical significance ↑AST activity could be due to: - Myocardial infarction - Hepatobiliary diseases

Aminotransferases: AST 2- Alanine amino transferase (ALT) OR glutamate pyruvate transaminase (GGT) Tissues sources Liver > heart > kidney > SM > spleenPrinciple pyruvate L-alanine +  -ketoglutarate ALT pyruvate + L-glutamate Pyruvate ++ Pyruvate + NADH +H + LDH L-lactate + NAD +

Determination of ALT: Cont. Normal range Up to 40 U/L at 37°C Wave length: Wave length: 340 nmProcedure

Determination of ALT: Cont. Calculation ∆ A /min=( (A 0 – A 1 ) + (A 1 – A 2 ) + (A 2 – A 3 ) ) / 3 ALT activity (U/L) = ∆ A /min X 1750 Clinical significance ↑ALT activity could be due to: - Myocardial infarction - Liver diseases (hepatocellular disorders)