Enzymatic Assay of Myokinase (EC 2.7.4.3)

Document History

Replaces OP SP-ADP01. New document assigned to conform to current document numbering system. Refer to CR SOP-DEK-ENZ47.

1. Objective

To standardize a procedure for the enzymatic assay of myokinase.

2. Scope

This procedure applies to all products that have a specification for myokinase.

3. Definitions

3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC

3.2. Myokinase Unit Definition – One unit will convert 2.0 μmoles of ADP to ATP + AMP per minute at pH 7.6 at 37°C.

3.3. Hexokinase Unit Definition – One unit will phophorylate 1.0 μmole of D-glucose per minute at pH 7.6 at 25ºC.

3.4. Glucose-6-Phosphate Dehydrogenase Unit Definition – One unit will oxidize 1.0 μmole of D-glucose 6-phosphate to 6–phospho-D-gluconate per minute in the presence of NADP at pH 7.4 at 25ºC.

3.5. ADP = Adenosine 5'-Diphosphate

3.6. AMP = Adenosine 5'-Monophosphate

3.7. ATP = Adenosine 5'-Triphosphate

3.8. G-6-P = Glucose 6-Phosphate

3.9. G-6-PDH = Glucose-6-Phosphate Dehydrogenase

3.10. β-NADP = β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form

3.11. β-NADPH = β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form

4. Discussion

2 ADP + H2O    Myokinase   > AMP + ATP

β-D(+)Glucose + ATP    Hexokinase   > G-6-P + ADP

G-6-P + β-NADP    G-6-PDH   > 6-Phosphoglucose-γ-Lactone + β-NADPH

5. Responsibilities

It is the responsibility of all Analytical Services laboratory personnel to follow this procedure as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 37°C, pH = 7.6, A340nm, Light path = 1 cm

7.2 METHOD:
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1    250 mM Glycylglycine Buffer (Buffer)
Prepare a 33 mg/ml solution in purified water using Gly-Gly, Free Base, Sigma-Aldrich Product Number G1002. Adjust to pH 7.6 at 37°C.

7.3.2.    40 mM Adenosine Diphosphate Solution (ADP)
Immediately before use, prepare a 19.9 mg/ml solution in cold purified water using Adenosine 5' Diphosphate, Sodium Salt, Sigma-Aldrich Product Number A2754. Prepare fresh for each test solution since ADP is not stable in dilute solutions.

7.3.3.    20 mM β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form Solution (β-NADP)
Prepare a 16.7 mg/ml solution in cold purified water using β-Nicotinamide Adenine Dinucleotide Phosphate, Sodium Salt, Sigma-Aldrich Product Number N0505.

7.3.4     300 mM Magnesium Chloride Solution (MgCl2)
Prepare 10 ml in purified water using Magnesium Chloride, Hexahydrate, Sigma-Aldrich Product Number M0250 or Magnesium Chloride, 1.0 M Solution, Molecular Biology Reagent, Sigma-Aldrich Product Number M1028 .

7.3.5.    1 M β-D(+)Glucose Solution (Glucose)
Prepare a 180 mg/ml solution in purified water using β-D(+)Glucose, Sigma-Aldrich Product Number G5250.

7.3.6.    Hexokinase and Glucose-6-Phosphate Dehydrogenase Solution (Hex/G-6-PDH)
Immediately before use, prepare a solution containing a minimum of 20 units/ml of Hexokinase activity and a minimum of 10 units/ml of Glucose-6-Phosphate Dehydrogenase activity in cold purified water using Hexokinase and Glucose-6 Phosphate Dehydrogenase , Sigma-Aldrich Product Number H8629.

7.3.6.1    Note that if the combined Hex/G-6-PDH Sigma-Aldrich Product Number H8629 is not available, use Hexokinase, Sigma-Aldrich Product Number H5625 and Glucose-6-Phosphate Dehydrogenase, Sigma-Aldrich Product Number G4134 at the above concentrations.

7.3.7.    0.1% (w/v) Bovine Serum Albumin Solution (BSA)
Prepare 100 ml in Reagent 7.3.1 using Albumin, Bovine, Sigma-Aldrich Product Number A4503.

7.3.8.    Myokinase Enzyme Solution (Myokinase)
Immediately before use, prepare a solution containing 0.2 - 0.6 unit/ml of Myokinase in cold Reagent 7.3.7

7.4 TEST METHOD

7.4.1    Pipette (in milliliters) the following reagents in the following sequence into suitable cuvettes:

  Test Blank
Purified Water 1.57 1.57
Reagent 7.3.1 (Buffer) 0.60 0.60
Reagent 7.3.3 (β-NADP) 0.35 0.35
Reagent 7.3.4 (MgCl2) 0.10 0.10
Reagent 7.3.5 (Glucose) 0.03 0.03
Reagent 7.3.6 (Hex/G-6-PDH) 0.10 0.10

 

7.4.2    Mix by inversion and equilibrate to 37°C. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer.

7.4.3    Then add (in milliliters) the following regents

Reagent 7.3.2 (ADP) 0.15 0.15
Reagent 7.3.8 (Myokinase) 0.10 -----
Reagent 7.3.7 (BSA) ----- 0.10

 

7.4.4    Immediately mix by inversion and record the increase in A340nm for approximately 5 minutes. Obtain the ΔA340nm/minute using the maximum linear rate for both the Test and Blank.

7.5 CALCULATIONS

7.5.1 Units/ml enzyme = (ΔA340nm/min Test - ΔA340nm/min Blank)*(3)*(df)
(6.22)(0.1)

 

where:
     3 = Total volume (in milliliters) of assay
     df = Dilution factor
     6.22 = Millimolar extinction coefficient of β-NADPH at 340 nm
     0.1 = Volume (in milliliter) of enzyme used

7.5.2 Units/mg solid = Units/ml enzyme
mg solid / ml enzyme

 

7.5.3 Units/mg protein = Units/ml enzyme
mg protein / ml enzyme

 

7.6 FINAL ASSAY CONTENTRATION:
In a 3.00 ml reaction mix, the final concentrations are 58 mM glycylglycine, 2.0 mM adenosine 5’-diphosphate, 2.3 mM β-nicotinamide adenine dinucleotide phosphate, 10 mM magnesium chloride, 10 mM glucose, 2 units of hexokinase, 1 unit glucose-6- phosphate dehydrogenase, 0.003% (w/v) bovine serum albumin, and 0.02-0.06 unit myokinase.

8. References & Attachments

Bergmeyer, H. U. (1974) Methods of Enzymatic Analysis, 2nd ed., Volume II, 486

9. Approval

Review, approvals and signatures for this document will be generated electronically using DocCompliance (QUMAS). Print a “For Use” copy if hardcopy with signature verification is required.

Materials

     
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