Transcript Enzymes
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 Enzyme Coenzyme Product 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 Pyruvate + NADH + H+ LDH Lactate + NAD+ NAD NADH Absorbance 1.5 1 0.5 256 340 Wavelength λ Techniques for determination of enzyme activity Radioimmunoassay Measures the enzyme conc. Enzymes classification Oxidoreductases: ex. glucose oxidase Transferases: ex. aminotransferases Hydrolases: ex.lipase Lyases: ex. decarboxylases Isomerases: ex. racemases Ligases or synthetases Factors that influence enzymatic reaction Substrate concentration Enzyme concentration Product concentration PH Temperature Activators & Co-enzymes Inhibitors 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: 340 nm Procedure Determination of LDH: Cont. Calculation ∆ A /min=( (A0 – A1) + (A1 – A2) + (A2 – A3) ) / 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 > pancreas Principle L-aspartate + a-ketoglutarate AST oxaloacetate + L-glutamate Oxaloacetate+ NADH + H+ malate dehydrogenase L-malate +NAD+ Determination of AST: Cont. Normal range Up to 37 U/L at 37°C Wave length: 340 nm Procedure Determination of AST: Cont. Calculation ∆ A /min=( (A0 – A1) + (A1 – A2) + (A2 – A3) ) / 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 > spleen Principle L-alanine + a-ketoglutarate ALT pyruvate + L-glutamate Pyruvate + NADH +H+ LDH L-lactate + NAD + Determination of ALT: Cont. Normal range Up to 40 U/L at 37°C Wave length: 340 nm Procedure Determination of ALT: Cont. Calculation ∆ A /min=( (A0 – A1) + (A1 – A2) + (A2 – A3) ) / 3 ALT activity (U/L) = ∆ A /min X 1750 Clinical significance ↑ALT activity could be due to: - Myocardial infarction - Liver diseases (hepatocellular disorders)