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Enzymes - Part 1 - Introduction of Enzymes
Enzymes - Part 1 - Introduction of Enzymes
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Introduction of Enzymes
Sample for enzyme estimation
1. The serum is considered to be the best sample as compared to plasma.
1. The best sample to prepare the serum is venous blood.
2. The plasma may interfere with some enzymes, like acid phosphatase and alkaline phosphatase.
3. Capillary blood may show some abnormality in Lactate dehydrogenase.
1. Capillary blood has increased total LD and abnormal LD isoenzymes.
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Introduction of Enzymes: Enzyme’s reaction equation
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Introduction of Enzymes: Mechanism of enzyme reaction
Classification of enzymes:
The enzymes are identified by the type of reaction which is catalyzed and the substrate involved. On this basis, these are classified into the following
groups:
1. Transferases: This will catalyze the transfer of moieties like glycosyl, methyl, or phosphoryl group.
2. Lyases: It will catalyze the cleavage of C–C, C–O, C–N, and other bonds by eliminating atoms, leaving double bonds.
3. Oxidoreductase: It will catalyze oxidation and reduction.
4. Hydrolases: These will catalyze and lead to hydrolytic cleavage of C–C, C–O, C–N, and other bonds.
5. Ligases: These will catalyze the joining of two molecules coupled to the hydrolysis of ATP.
6. Isomerases: These will catalyze geometric or structural changes within a molecule.
Variables For The Functional Activity Of Enzymes (Factors Affecting The Rate Of Enzyme Catalytic Reaction) Depend
Upon The Following:
1. The concentration of the enzymes:
1. The role of the enzyme depends upon the concentration of the enzymes.
2. The velocity of the reaction is directly proportional to the enzyme concentration.
3. The reaction is more rapid when there is enough amount of the enzyme to bind the abundant substrate; this is a mass action effect.
2. The concentration of the substrate:
1. It depends upon the concentration of the substrate.
2. A curve is shown in the following diagram when there is a fixed amount of enzyme against the increasing amount of substrate.
3. With a fixed enzyme and increasing substrate concentration, the reaction reaches a plateau; after that, there is no change.
4. After points C and D, the reaction reached the plateau.
5. The reaction will fall to zero.
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Enzyme and substrate relationship
3. pH:
1. The enzymes are proteins in nature, carrying a negative charge.
2. These negatively charged molecules are pH-sensitive.
3. The binding of the enzyme with the substrate is pH-dependent.
4. There is a role of pH in the enzyme reaction. Most enzymes react between a pH of 7 to 8.
5. Few react at low pHs, like pepsin at 1.5.
6. While alkaline phosphatase reacts at a pH of 10.5
7. Optimum pH is needed for various reactions.
4. Temperature:
1. Enzyme reaction depends upon the temperature.
2. The rate of the enzyme reaction velocity is proportional to the temperature.
3. The velocity of the reaction is double for every 10 °C rises.
4. The enzymes are proteins, so they are very sensitive to temperature ranges.
5. When enzymes are exposed to 60 °C and above, these are mostly denatured.
6. Mostly our enzymes are effective at 37 °C to 38 °C. Their activity decreases if the temperature goes over 42 °C to 45 °C.
7. The enzyme activity is preserved when stored at a low temperature. Enzyme activity can be maintained for months from -20 °C to -70 °C.
5. Ionic strength:
1. Most enzymes are sensitive to some of the buffer ions in the incubation mixture.
2. Some of the cations at the appropriate concentration are needed for the best action of the enzymes.
3. Presence of specific ions like activator or inhibitor.
4. Any other substance that alters the enzyme configuration affects its activity.
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Enzyme reaction and measurement of the end product
5. Enzymes are in very low concentration, so usually, these are not measured, but they are assessed by what they do.
6. So basically, to assess the enzymes, there is an assessment of the substrate decrease in the reaction or increase in the product.
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Enzymes characteristics
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1. There is increased activity in the LDH because of increased LDH in the RBCs (150 times more than the serum). So traces of hemolysis will
increase the level.
2. SGOT (AST) is also elevated due to this enzyme in the RBCs. (There is 15 times more activity in the RBCs).
3. Acid phosphatase is mildly raised due to hemolysis.
4. There is increased activity in the LDH because of increased LDH in the RBCs (150 times more than the serum). So traces of hemolysis will
increase the level.
5. SGOT (AST) is also elevated due to this enzyme in the RBCs. (There are 15 times more active in the RBCs).
6. Acid phosphatase is mildly raised due to hemolysis.
2. Effect of storage:
1. Serum or plasma should be separated as soon as possible from RBCs.
2. Enzymes are usually stable for 8 to 12 hours at room temperature in the serum or plasma.
3. While enzymes are stable for 5 to 7 days at -4 °C.
4. When kept at -20 °C, then stable from weeks to months.
5. At -70° C, these are stable for an indefinite period.
3. Patient variables:
1. A fasting sample is always preferred.
2. The alkaline phosphatase level is elevated after eating the food (the Intestine has a high alkaline phosphatase level).
3. The alkaline phosphatase level is very high at birth and in growing children. It comes to the adult level by 15 to 17 years in females and 18
to 20 years in males. Overall there is a variation of these enzymes with age.
4. SGOT (AST) and SGPT (ALT) are raised at birth and reach a normal level by 2 to 4 months.
5. Gamma GT (glutamyltransferase) is raised at birth and comes to the adult level by 6 months of age.
6. Aldolase is also raised at birth and in young children. It comes to a normal level by the age of 16 years.
7. Creatinine kinase is higher in males than females.
8. AST, Creatinine kinase, and LDH may be raised after the exercise.
9. Intramuscular injection may increase the value of Creatinine Kinase, AST, and LDH.
4. Leakage of the enzyme from the cells.
5. Altered enzyme production.
6. Clearance of the enzyme.
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2. The catalytic activity of the enzymes show:
1. Their presence.
2. Help in their detection.
3. Provides the basis for enzyme-linked immunoassay.
3. Assay of the plasma enzymes helps in:
1. Diagnosis of the diseases like myocardial infarction and viral hepatitis.
2. Help to evaluate prognosis.
4. Most enzymes are assayed spectrophotometrically.
5. Enzymes are present in all body cells, and each one functions in a specific reaction.
6. Enzymes catalyze all essential reactions like oxidation, reduction, hydrolysis, esterification, and synthesis that supply the energy and /or
chemical changes necessary for vital activities.
7. Enzymes help in:
1. Nerve conduction.
2. Muscle contraction.
3. Digestion.
4. Respiration.
5. Maintenance of body temperature.
6. Reproduction.
7. Growth.
8. Digestion.
8. Enzymes are under the control of genes.
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