Enzyme Concentration and Reaction Rates
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Questions and Answers

Which characteristic is unique to the chemiluminescence method described?

  • Measurable concentrations limited to 1 decade
  • Unaffected by interfering substances
  • Absorbance less than 0.8 at 340 nm (correct)
  • Uses Pyruvate instead of Glutamate
  • What role does glutamate oxidase play in the chemiluminescence method?

  • Catalyzes glutamate to generate hydrogen peroxide (correct)
  • Increases pyruvate concentration in the sample
  • Replaces the need for hydrogen peroxide
  • Is an interfering substance in the assay
  • Which of the following statements about the advantages of the method is NOT true?

  • It has a low background signal level.
  • It is highly sensitive.
  • It is completely free from interference from drugs. (correct)
  • It has a wide range of measurable concentrations.
  • Why should hemolyzed samples be avoided in this assay?

    <p>RBCs can falsely elevate ALT activity</p> Signup and view all the answers

    Which compound at elevated levels can interfere with ALT activity?

    <p>Pyruvate</p> Signup and view all the answers

    How does an increase in enzyme concentration affect the rate of a reaction?

    <p>It speeds up the reaction since more enzymes are available.</p> Signup and view all the answers

    What is the primary advantage of using a continuous-reading spectrophotometer?

    <p>It provides instantaneous data during the reaction.</p> Signup and view all the answers

    What effect does temperature have on enzyme activity?

    <p>Enzyme activity increases with a rise in temperature.</p> Signup and view all the answers

    At what pH range do most enzyme-catalyzed reactions occur optimally?

    <p>7.0 - 8.0</p> Signup and view all the answers

    How does extreme pH affect enzymes?

    <p>It can lead to denaturation of enzymes.</p> Signup and view all the answers

    What defines a multi-enzyme method in a biochemical pathway?

    <p>A series of enzymes that work sequentially in reactions.</p> Signup and view all the answers

    How does measuring absorbance at different intervals benefit linearity assessment?

    <p>It ensures accurate verification of the reaction's linearity.</p> Signup and view all the answers

    What is the relationship between substrate concentration and reaction rate?

    <p>Increase in substrate concentration can lead to increased reaction rate until a maximum is reached.</p> Signup and view all the answers

    What factor should be used to correct AST results obtained at 30 degrees Celsius when reporting at 37 degrees Celsius?

    <p>1.43</p> Signup and view all the answers

    Which of the following substances does NOT falsely increase AST activity?

    <p>Acetaminophen</p> Signup and view all the answers

    What volume of Bilirubin Activator is used in each tube?

    <p>0.04 mL</p> Signup and view all the answers

    What is the reference value for AST at 37 degrees Celsius?

    <p>Up to 40 IU/L</p> Signup and view all the answers

    How should a sample with AST values greater than 500 IU/L be processed?

    <p>Dilute 1:1 with saline and multiply results by 2</p> Signup and view all the answers

    What is the purpose of covering the tubes with parafilm after adding reagents?

    <p>To prevent evaporation</p> Signup and view all the answers

    What is the wavelength used to zero the spectrophotometer in this procedure?

    <p>540 nm</p> Signup and view all the answers

    What effect does chronic alcoholism have on AST activity?

    <p>Increases AST activity</p> Signup and view all the answers

    During strenuous exercise, how does AST activity change?

    <p>Threefold increase</p> Signup and view all the answers

    How long should the samples stand at room temperature after mixing?

    <p>90 seconds</p> Signup and view all the answers

    Which of these factors contributes to a 5-10% variation in AST levels?

    <p>Race</p> Signup and view all the answers

    Which component is not included in the patient sample tube?

    <p>Calibrator Reagent</p> Signup and view all the answers

    What is the detection limit for the AST reagent?

    <p>500 IU/L</p> Signup and view all the answers

    What does the calculation of concentration of the unknown require?

    <p>Absorbance of the Standard</p> Signup and view all the answers

    What is the total volume of reagents required for the control sample?

    <p>0.1 mL</p> Signup and view all the answers

    What are the reference values for Total Bilirubin at birth?

    <p>Up to 5 mg/dL</p> Signup and view all the answers

    How does muscle mass influence creatinine kinase activity between males and females?

    <p>Males have higher creatinine kinase related to greater muscle mass.</p> Signup and view all the answers

    What is the effect of competitive inhibition on Km when an inhibitor is present?

    <p>Km is increased.</p> Signup and view all the answers

    How does the presence of noncompetitive inhibition affect Vmax?

    <p>Vmax is reduced.</p> Signup and view all the answers

    What physiological factor contributes to the differences in alcohol metabolism between males and females?

    <p>Variations in alcohol dehydrogenase level.</p> Signup and view all the answers

    In which scenario would creatinine kinase levels likely be higher?

    <p>Among ambulatory patients.</p> Signup and view all the answers

    What happens to Km and Vmax in the presence of uncompetitive inhibition?

    <p>Km is unaffected, Vmax is reduced.</p> Signup and view all the answers

    What is a characteristic of lower creatinine kinase levels found in a population?

    <p>Less frequent exercise among individuals.</p> Signup and view all the answers

    Which demographic might exhibit higher creatinine kinase levels due to muscle mass characteristics?

    <p>African American individuals compared to Caucasian individuals.</p> Signup and view all the answers

    Study Notes

    Enzyme Activity Factors

    • Enzyme Concentration: Higher enzyme levels accelerate reactions due to increased availability for substrate interaction.
    • Temperature Impact: For every 10°C increase, enzyme activity doubles, enhancing molecular movement and reaction speed.
    • pH Levels: Optimal activity occurs between pH 7.0-8.0; extreme pH can denature enzymes or alter amino acid states, affecting active sites.

    Multi-Enzyme Methods

    • Enzymes work sequentially in biochemical pathways; the product of one reaction acts as the substrate for the next.
    • Measuring the final target substrate indicates the overall efficiency of the pathway.

    Serum Bilirubin Determination Experiment Steps

    • Label test tubes for Reagent Blank, Standard, Patient Sample, and Control.
    • Add specified volumes of Total Bilirubin Reagent, Bilirubin Activator, and corresponding samples, then mix.
    • Let the mixture stand for 90 seconds before zeroing the spectrophotometer and measuring absorbance at 540 nm.

    Calculation and Reference Values

    • Total Bilirubin Calculation: Uses absorbance values of unknown relative to the standard.
    • Reference values at birth: up to 5 mg/dL and up to 85.5 μmol/L.

    Influence of Inhibitors on Enzyme Kinetics

    • Competitive Inhibition: Increases Km, Vmax remains unchanged.
    • Noncompetitive Inhibition: Reduces both Km and Vmax.
    • Uncompetitive Inhibition: Km unchanged, Vmax reduced.

    Factors Affecting AST Activity

    • Elevated AST levels can result from various conditions including lipemic samples, high pyruvate levels, anabolic steroids, and chronic alcoholism.
    • Exercise: Strenuous physical activity can significantly increase AST levels.

    Temperature Correction

    • For reactions performed at different temperatures, apply correction factors (e.g., multiply by 1.43 for results at 30°C to report at 37°C).

    Detection Limits and Sample Considerations

    • Reagents have a linear detection limit up to 500 IU/L; samples exceeding should be diluted and results adjusted accordingly.
    • Serum is preferred for testing but plasma may be acceptable; avoid hemolyzed samples as they affect results.

    Advantages of Kinetic Methods

    • Kinetic assays, especially those using spectrophotometry, provide more accurate assessment of enzymatic reactions compared to colorimetric methods.
    • Allows for continuous monitoring improving the accuracy of linearity assessments across reactions.

    Interfering Substances

    • Pyridoxal phosphate can artificially elevate ALT values by stimulating apoenzyme activation.
    • High levels of pyruvate (> 0.2 mmol/L) can similarly interfere with results.

    Specimen Considerations

    • Serum is ideal; plasma may be used but must be free of hemolysis to ensure reliable activity measurement.

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    Description

    This quiz explores the relationship between enzyme concentration and the rate of biochemical reactions. Understand the impact of higher enzyme levels on reaction speed and the advantages of continuous measurement techniques. Perfect for students studying biochemistry concepts.

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