Enzymatic Assay of Plasmin with D-Val-Leu-Lys-p-Nitroanilide Dihydrochloride

1. Objective

To standardize a procedure for the determination of the enzymatic assay of plasmin.

2. Scope

This scope of this procedure applies to products that have a specification for the enzymatic activity of plasmin.

3. Definitions

3.1 VALY = D-Val-Leu-Lys-p-Nitroanilide Dichloride

3.2 Purified water = water from a deionizing system, resistivity ~18MΩ•cm @ 25 ºC

4. Discussion

VALY ^Plasmin; > p-Nitroanilide + D-Val-Leu-Lys

5. Responsibilities

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

6. Safety

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

7. Procedure

7.1 CONDITIONS: T = 37 ºC, pH = 7.5, A_405nm, Light path = 1 cm

7.2 METHOD: Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1. 10 mM Potassium Phosphate, 70 mM Sodium Phosphate, 100 mM Lysine Buffer, pH 7.5 at 37 ºC (Buffer)
Prepare a 1.361 mg /mL solution of Potassium Phosphate, Product Number such as P5379, with 9.94 mg/mL Sodium Phosphate, Product Number such as S0876, with 18.3 mg/mL Lysine, Product Number L5626, in purified water. Adjust pH to 7.5 at 37 ºC with 1N NaOH.

7.3.2. 6.5 mM D-Val-Leu-Lys-p-Nitroanilide Dihydrochloride Solution, pH 7.8 at 25 ºC (VALY)
Prepare a 3.58 mg/mL solution of VALY, Product Number V0882, in Reagent 7.3.1.

7.3.3. Plasmin Test (Plasmin)
Immediately before use, prepare a 0.025-0.10 Unit/mL solution in cold purified water

7.4 TEST METHOD

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

	Test	Blank
Reagent 7.3.1 (Buffer)	1.00	1.00
Reagent 7.3.2 (VALY)	0.25	0.25
Purified Water	----	0.10

7.4.2. Mix by inversion and incubate at 37 ºC using a suitable thermostatted spectrophotometer for 3-5 min. Then add:

Reagent 7.3.4. (Plasmin)

0.10

Immediately mix by inversion and record the increase in A_405nm for approximately 10 minutes. Obtain the ΔA_405nm / minute over a five minute interval for both the Test and the Blank.

7.5 CALCULATIONS

7.5.1.	Units / mL enzyme =	(ΔA405nm /min Test-ΔA405nm /min Blank)(1.35)(df)
		(10.5)(0.1)

where,
1.35 = Total volume (in milliliters) of assay
df = Dilution Factor
10.5 = Micromolar extinction coefficient for p-Nitroanilide at 405nm
0.1 = Volume (in milliliters) of enzyme used

7.5.2.	Units / mg solid =	Units/mL enzyme
		mg solid/mL enzyme

7.5.3.	Units / mg protein =	Units/mg solid
		mg protein/mL enzyme

7.6 UNIT DEFINITION
One unit will produce one umole of p-Nitroanilide from D-Val-Leu-Lys-p-Nitroanilide at pH 7.5 at 37 ^ºC

7.7 FINAL ASSAY CONTENTRATION:
In a 1.35 mL reaction mix, the final concentrations are 9.25 mM potassium phosphate, 64.8 mM sodium phosphate, 92.6 mM lysine, 1.3 mM D-Val-Leu-Lys-p-Nitroanilide, and 0.0025 to 0.010 units of plasmin.

8. References & Attachments

8.1. Replaces SPVALY05

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.