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2.20: Enzymes Rate, Creatine Kinase (CK)

  • Page ID
    120625
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    RELATED READING: Chapter 31. See Methods on CD-ROM for Creatine kinase.

    OBJECTIVES

    Upon completion of this exercise and appropriate discussion and related reading, the student will be able to:

    1. Perform a multipoint enzyme rate reaction for CK.
    2. Calculate CK values using reaction rate and molar absortivity constant.
    3. Explain the effect of time on enzyme activity.
    4. Explain the effect of temperature on enzyme activity.
    5. Explain the effect of pH on enzyme activity.

    PRINCIPLE

    Creatine phosphate and ADP are converted to creatine and ATP by the enzyme Creatine Kinase. This conversion can be measured by utilizing two additional enzymatic reactions that result in the conversion of NAD to NADH with a resulting increase in absorbance.

    \[\text{creatine phosphate} + \text{ADP} \rightarrow \text{creatine} + \text{ATP}\]

    \[\text{glucose} + \text{ATP} \xrightarrow{\text{Hexokinase}} \text{glucose-6-phosphate} + \text{ADP}\]

    \[\text{glucose-6-phosphate} + NAD^{+} \xrightarrow{\text{glucose-6-phosphate dehydrogenase}} \rightarrow \text{6-phosphogluconate} + H^{+} + \text{NADH}\]

    Enzyme activity can be calculated using the following formula:

    \[F=\frac{V_{r}}{V_{s}} + \frac{1}{\epsilon} + \frac{1}{PL} + 10^{6}\]

    where F = Enzyme factor, Vr = Total reaction volume (mL), Vs = Sample volume (mL), \(\epsilon\) = Molar absorptivity constant, PL = Path length of cuvette in cm

    For CK measurement by the following procedure:

    \[F = \frac{2.1}{0.1} + \frac{1}{6.22 \times 10^{3}} + \frac{1}{1} + 10^{6} = 3375\]

    Activity is expressed in International Units/liter (U/L) Further, \(\Delta\)A/min X F = CK activity in Units/Liter.

    MATERIALS

    • UV Spectrophotometer
    • Pipets
    • Cuvettes
    • 37°C Water bath
    • CK Reagent

    PROCEDURE

    1. Pipet 2.0 mL of CK reagent into a clean dry cuvette for each control and serum sample to be tested.
    2. Incubate the reagent at 37°C for 5 minutes to prewarm.
    3. Adjust your spectrophotometer wavelength to 340 nm and set zero absorbance (100% T) with distilled water.
    4. Add 100 \(\mu\)L of sample (control or serum) to the warned reagent, mix by inversion, and incubate for 2 mm at 37°C.
    5. Read the absorbance every 2 min. for a total of 10 min., incubating between readings. Record your results on the data sheet.
    6. Repeat steps 4 and 5 for each additional material to be tested. With practice, multiple samples can be spaced at 1 minute intervals.
    7. Calculate the average \(\Delta\)A/min. during the maximum reaction rate and record your answer on the data sheet.
    8. Calculate the CK for each sample and control activity using the formula given previously.
    9. Cover your sample tubes with parafilm and save as instructed for further testing.

    OPTIONAL EXERCISES

    1. Effect of Temperature
      1. Repeat the CK analysis on one of the samples or controls, but perform the incubation at room temperature.
      2. Calculate the CK activity, measure the room temperature and record results on the data sheet.
    2. Effect of pH
      1. Using a pH meter, measure the pH of the CK reagent and then adjust the pH of the reagent to approximately pH 6.0 with 0.1 M HC1 or pH 7.5 with 0.1 M NaOH.
      2. Repeat the CK analysis of one of the samples or controls at 37°C.
      3. Calculate the CK activity and record it and the pH of the reagent on the data sheet.
    DATA SHEET, EXERCISE #20

    NAME: ___________

    DATE: ___________

    RESULTS

    Time (Minutes) Average \(\Delta\)A/min. CK (U/L)
    2 4 6 8 10
    Normal Control
    Abnormal Control
    Sample # 1
    Sample # 2

    Note

    Dilute samples with isotonic saline if calculated CK value exceeds the assay’s linear range as determined by your instructor.

    OPTIONAL EXERCISES

    1. Room temperature_____°C
      CK activity of sample #________________at room temperature was____U/L.
    2. pH of unadjusted CK reagent = _____
      pH of adjusted CK reagent = _____
      CK activity of sample #_____using the adjusted reagent was ___________U/L.

    Discussion Questions

    1. Under what conditions would you expect the \(\Delta\)A for each time interval to be approximately equal?
    2. If a sample had a high CK value, what might you observe in your results in addition to the high \(\Delta\)A value?
    3. What is the effect of decreasing the incubation temperature on CK activity? What do you think might be the affect of increasing the incubation temperature on the activity of CK?
    4. What was the effect of changing the pH of the CK reagent on the measured CK activity? Why does this occur.

    2.20: Enzymes Rate, Creatine Kinase (CK) is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by LibreTexts.

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