Enzymatic Assay of Achromopeptidase

1. Objective

The objective of this procedure is to standardize a method for the enzymatic assay of Achromopeptidase.

2. Scope

The scope of this procedure includes products that have a specification for achromopeptidase activity.

3. Definitions

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

3.2 Unit Definition: One unit will produce a ΔA600nm of 0.001 per minute per milliliter at pH 8.0 at 37ºC using a suspension of Micrococcus lysodeikticus as substrate.

4. Discussion

Micrococcus lysodeikticus Cells (Intact)    Achromopeptidase   > Micrococcus lysodeikticus Cells (Lysed)

5. Responsibilities

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

6. Safety

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

7. Procedure

7.1 CONDITIONS:

7.1.1     Temperature = 37ºC

7.1.2     pH = 8.0

7.1.3     Absorbance = 600 nm

7.1.4     Light Path = 1 cm

7.2 METHOD:

7.2.1     Turbidimetric Rate Determination

7.3 REAGENTS:

7.3.1     10mM Tris HCl with 10mM Sodium Chloride, pH 8.0 at 37ºC (Buffer)

7.3.1.1    Prepare a solution in purified water containing 1.21 mg/ml Trizma Base, Sigma-Aldrich Product Number , T1503, and 0.584mg/ml Sodium Chloride, Sigma-Aldrich Product Number , S9888.

7.3.1.2    Adjust the pH to 8.0 at 37ºC using 1N Hydrochloic Acid, Sigma-Aldrich Product Number , H3162.

7.3.2     Micrococcus lysodeikticus Cell Suspension (Substrate)

7.3.2.1    Prepare a 0.20 mg/ml suspension of cells in reagent 7.3.1(Buffer) using Micrococcus lysodeikticus, ATCC 4698 lyophilized cells, Sigma-Aldrich Product Number , M3770.

7.3.2.2    The A600nm of this suspension should be 0.60-0.70 versus a buffer blank. This is typically a 0.02% (w/v) solution, but could vary based on the purity of the cell suspension used. Add Reagent 7.3.1 (Buffer) as needed to adjust the absorbance into the appropriate range.

7.3.3     Achromopeptidase Enzyme Solution (Enzyme).

7.3.3.1    Immediately before use, prepare a solution containing 350-700 units/ml of Achromopaptidase in cold Reagent 7.3.1 (Buffer).

7.3.3.2    For crude products, let samples sit on ice for 20-30 minutes after the addition of Reagent 7.3.1 (Buffer).

7.4 PROCEDURE

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

  Test Blank
Reagent 7.3.2 (Substrate) 2.90 2.90

 

7.4.2     Equilibrate the cuvettes to 37ºC. Monitor the A600nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Reagent 7.3.1 (Buffer) 0.10 ------
Reagent 7.3.3 (Enzyme) ------- 0.10

 

7.4.3     Immediately mix by inversion and record the decrease in A600nm for approximately 15 minutes. Obtain the maximum linear rate (ΔA600nm/minute) for the test using at least a one minute interval and a minimum of 4 data points. Record the blank rate for the same time interval as the test.

7.5 CALCULATIONS

7.5.1 Units/ml solid = (ΔA600nm/min Test - ΔA600nm/min Blank)(df)(3)
(0.001)(0.10)(mg sample used)

 

where:    df = Dilution Factor
     3 = Volume (in milliliters) of reaction mix
     0.001 = Change in absorbance at A600nm as per the Unit Definition
     0.10 = Volume (in milliliters) of enzyme used

7.5.2 Units/mg protein= Units/mg solid
(%protein/100)

8. References & Attachments

8.1 Ezaki, T and Suzuki, S. (1982) Journal of Clinical Microbiology 16, 844-846

8.2 This procedure is based on OP SPMICR01.

9. Approval

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

Materials

     
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