PRINCIPLE: Creatinine + H 2 O Creatininase > Creatine Creatine + ATP CPK > Creatine-P + ADP ADP + PEP PK > ATP + Pyruvate Pyruvate + ß-NADH LDH > L-Lactate + ß-NAD Abbreviations used: ATP = Adenosine 5'-Triphosphate CPK = Creatine Phosphokinase Creatine-P = Phosphocreatine ADP = Adenosine 5'-Diphosphate PEP = Phospho(enol)pyruvate PK = Pyruvate Kinase ß-NADH = ß-Nicotinamide Adenine Dinucleotide, Reduced Form LDH = Lactic Dehydrogenase ß-NAD = ß-Nicotinamide Adenine Dinucleotide, Oxidized Form CONDITIONS: T = 25 C, ph = 8.0, A 340nm, Light path = 1 cm METHOD: Continuous Spectrophotometric Rate Determination REAGENTS: A. 100 mm Glycylglycine Buffer, ph 8.0 at 25 C (Prepare 100 ml in deionized water using Gly-Gly, Free Base, Sigma Prod. No G-1002. Adjust to ph 8.0 at 25 C with 1 M NaOH.) B. 12 mm ß-Nicotinamide Adenine Dinucleotide, Reduced Form, Solution (ß-NADH) (Dissolve the contents of one 10 mg vial of ß-Nicotinamide Adenine Dinucleotide, Reduced Form, Disodium Salt, Sigma Stock No. 340-110 in the appropriate volume of Reagent A. PREPARE FRESH.) SPCREA01 Page 1 of 4
REAGENTS: (continued) C. 16 mm Adenosine 5'-Triphosphate Solution (ATP) (Prepare 5 ml in Reagent A using Adenosine 5'-Triphosphate, Disodium Salt, Sigma Prod. No. A- 5394.) D. 20 mm Phospho(enol)pyruvate Solution (PEP) (Prepare 5 ml in Reagent A using Phospho(enol)pyruvate, Tri(cyclohexylammonium) Salt, Sigma Prod. No. P-7252.) E. 440 mm Creatinine Solution (Substrate) (Prepare 5 ml in deionized water using Creatinine, Free Base, Anhydrous, Sigma Prod. No. C-4255.) F. 100 mm Magnesium Chloride Solution (MgCl 2 ) (Prepare 5 ml in deionized water using Magnesium Chloride, Hexahydrate, Sigma Prod. No. M-0250.) G. PK/LDH Mixed Enzymes 1 (PK/LDH) (Use PK/LDH Enzymes Suspension, Sigma Stock No. 40-7.) H. 100 mm Glycylglycine Buffer with 200 mm Potassium Chloride, ph 8.0 at 25 C (CPK Enz Diluent) (Prepare 25 ml in deionized water using Gly-Gly, Free Base, Sigma Prod. No. G-1002 and Potassium Chloride, Sigma Prod. No. P-4504. Adjust to ph 8.0 at 25 C with 1 M NaOH.) I. Creatine Phosphokinase Enzyme Solution (CPK) (Immediately before use, prepare a solution containing 900 units/ml of Creatine Phosphokinase, Sigma Prod. No. C-3755 in Reagent H.) J. Creatininase Enzyme Solution (Creatininase) (Immediately before use, prepare a solution containing 0.30-0.50 unit/ml of Creatininase in cold Reagent A.) PROCEDURE: Pipette (in milliliters) the following reagents into suitable cuvettes: Test Blank SPCREA01 Page 2 of 4
Reagent A (Buffer) 2.30 2.30 Reagent B (ß-NADH) 0.10 0.10 Reagent C (ATP) 0.20 0.20 SPCREA01 Page 3 of 4
PROCEDURE: (continued) Test Blank Reagent D (PEP) 0.10 0.10 Reagent E (Substrate) 0.20 0.20 Reagent F (MgCl 2 ) 0.10 0.10 Reagent G (PK/LDH) 0.04 0.04 Reagent I (CPK) 0.10 0.10 Mix by inversion and equilibrate to 25 C. Monitor the A 340nm until constant, using a suitably thermostatted spectrophotometer. Then add: Reagent J (Creatininase) 0.05 ------ Reagent A (Buffer) ------ 0.05 Immediately mix by inversion and record the decrease in the A 340nm for approximately 5 minutes. Obtain the ra 340nm /minute using the maximum linear rate for both the Test and Blank. CALCULATION: Units/ml enzyme = (ra 340nm /min Test - ra 340nm /min Blank)(3.19)(df) (6.22)(0.05) 3.19 = Total Volume (in milliliters) of assay df = Dilution factor 6.22 = Millimolar extinction coefficient of ß-NADH at 340 nm 0.05 = Volume (in milliliter) of enzyme used units/ml enzyme Units/mg solid = mg solid/ml enzyme units/ml enzyme Units/mg protein = mg protein/ml enzyme UNIT DEFINITION: One unit will hydrolyze 1.0 µmole of creatinine to creatine per minute at ph 8.0 at 25 C. SPCREA01 Page 4 of 4
FINAL ASSAY CONCENTRATION: In a 3.19 ml reaction mix, the final concentrations are 88 mm glycylglycine, 0.38 mm ß-nicotinamide adenine dinucleotide, 1.0 mm adenosine 5'-triphosphate, 0.63 mm phospho(enol)pyruvate, 28 mm creatinine, 3.1 mm magnesium chloride, 28 units pyruvate kinase, 40 units lactic dehydrogenase, 90 units creatine phosphokinase, 6.1 mm potassium chloride, and 0.015-0.025 unit creatininase. REFERENCE: NOTES: Bergmeyer, H.U., Gawehn, K. and Grassl, M.(1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U. ed.) Vol. I, 2nd ed., 445-446, Academic Press, Inc., New York, NY 1. Contains not less than 700 Pyruvate Kinase units and 1000 Lactic Dehydrogenase units per ml. 2. Pyruvate Kinase Unit Definition: One unit will convert 1.0 µmole of phospho(enol)pyruvate to pyruvate per minute at ph 7.6 at 37 C. 3. L-Lactic Dehydrogenase Unit Definition: One unit will reduce 1.0 µmole of pyruvate to L-lactate per minute at ph 7.5 at 37 C. 4. Creatine Phosphokinase Unit Definition: One unit will transfer 1.0 µmole of phosphate from phosphocreatine to ADP per minute at ph 7.4 at 30 C. 5. This assay is based on the cited reference. 6. Where Sigma Product or Stock numbers are specified, equivalent reagents may be substituted. This procedure is for informational purposes. For a current copy of Sigma s quality control procedure contact our Technical Service Department. SPCREA01 Page 5 of 4