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 @ 25 ºC

3.2 Unit Definition: One unit will produce a ΔA_600nm of 0.001 per minute per milliliter at pH 8.0 at 37 ºC using a suspension of Micrococcus lysodeikticus as substrate.

4. Discussion

Micrococcus lysodeikticus cells (intact)    ^{Achromopeptidase}   > Micrococcus lysodeikticus cells (lysed)

5. Responsibilities

Analytical Services laboratory personnel should 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 = 37 ºC

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

7.3.1.1    Prepare a solution in purified water containing 1.21 mg/mL Trizma Base, Product No. T1503, and 0.584 mg/mL sodium chloride, Product No. S9888.

7.3.1.2    Adjust the pH to 8.0 at 37 ºC using 1 N Hydrochloic Acid, Product No. H3162.

7.3.2     Micrococcus lysodeikticus Cell Suspension (Substrate)

7.3.1.2    Adjust the pH to 8.0 at 37 ºC using 1 N Hydrochloic Acid, Product No. H3162.

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, Product No. M3770.

7.3.2.2    The A_600nm 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, place samples on ice for 20-30 minutes after the addition of Reagent 7.3.1 (Buffer).

7.4 PROCEDURE

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

	Test	Blank
Reagent 7.3.2 (Substrate)	2.90	2.90

7.4.2     Equilibrate the cuvettes to 37 ºC. Monitor the A_600nm 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 A_600nm for approximately 15 minutes. Obtain the maximum linear rate (ΔA_600nm/minute) for the test using at least a one minute interval and a minimum of four data points. Record the blank rate for the same time interval as the test.

7.5 CALCULATIONS

7.5.1

where:    df = Dilution Factor

     3 = Volume (in milliliters) of reaction mix
     0.001 = Change in absorbance at A_600nm as per the Unit Definition
     0.10 = Volume (in milliliters) of enzyme used

7.5.2

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.