Creatine Kinase - Assay
Creatine Kinase Assay
Creatine kinase activity is determined in a coupled enzyme system utilizing pyruvate kinase (PK) and lactate dehydrogenase (LDH). The following procedure is essentially that described by Tanzer and Gilvarg (1959). One Unit is defined as the conversion of one micromole of creatine to creatine phosphate per minute at 25°C and pH 8.9 under the specified conditions.
- Reagent solution -- the required amount of solution should be prepared containing:
ATP 8.5 mM NADH 1.22 mM PEP 2.0 mM LDH 15.0 u/ml PK 7.0 u/ml MgSO4 28.0 mM Glutathione (reduced) 26.0 mM pH adjusted to 7.4 - Buffered Creatine: 0.40 M Glycine containing 53.2 mM creatine and 62 mM potassium carbonate. Adjust the pH to 8.9 with NaOH.
- Enzyme diluent: Freshly prepared 5 mM glycine pH 9.0
Prepare mg/ml in 5 mM glycine, pH 9.0.
Dilute to 0.1-10 μg/ml in glycine buffer.
Adjust spectrophotometer to 25°C and 340 nm. Pipette into each cuvette as follows:
Reagent solution 0.7 ml Buffered creatine 2.2 ml
Incubate in spectrophotometer at 25°C for 3-5 minutes to achieve temperature equilibrium and establish blank rate, if any. Add 0.1 ml diluted enzyme and record decrease in A340 for 5-8 minutes. An initial lag period may occur. Determine ΔA340 from linear portion of the curve.