To calculate Creatine Kinase (CK) activity, replace [(A340)40 min – (A340)20 min] with [(A340)25 min – (A340)20 min] and replace the factor 150 with 600 in the standard calculation equation. One unit of CK activity is defined as the amount of enzyme that will transfer 1.0 μmole of phosphate from phosphocreatine to ADP per minute at pH 6.0.
This implies that CK activity calculation revolves around monitoring the change in absorbance at 340 nm (A340) over a specific time interval and applying a specific factor based on the assay conditions. The calculation reflects the enzyme's ability to catalyze the transfer of phosphate, which is directly related to its activity.
Here's a breakdown:
- Measure Absorbance: You need to measure the absorbance at 340 nm (A340) at two time points: 20 minutes and 25 minutes in this case.
- Calculate the Difference: Subtract the absorbance at 20 minutes from the absorbance at 25 minutes: [(A340)25 min – (A340)20 min]. This gives you the change in absorbance over 5 minutes.
- Apply the Factor: Multiply the difference in absorbance by the factor 600. This factor incorporates various parameters, including the path length of the cuvette, the molar absorptivity of the product, and the reaction volume, converting the change in absorbance to units of CK activity (U/L).
Therefore, the simplified equation is:
*CK Activity (U/L) = [(A340)25 min – (A340)20 min] 600**
Key Considerations:
- Temperature: The reaction is typically performed at a specific temperature (e.g., 37°C).
- pH: The reaction is performed at a specific pH (e.g., pH 6.0).
- Units: The units of CK activity are expressed as U/L (units per liter), where one unit (U) is defined as the amount of enzyme that catalyzes the transfer of 1.0 μmole of phosphate from phosphocreatine to ADP per minute under specified conditions.
- Assay-Specific Factor: The factor (600 in this instance) is crucial and dependent on the specific assay kit or method used. Always refer to the manufacturer's instructions or the established protocol for the correct factor. The factor typically compensates for the light path length, molar absorptivity of NADH (or NADPH), and the sample dilution.
In summary, accurately determining CK activity involves precise absorbance measurements at specific time points, followed by applying a predetermined assay-specific factor to calculate the enzyme activity in U/L. Remember to adhere to the established protocol for accurate results.
