Enzymatic Assay of Thrombin
1. OBJECTIVE
To standardize a procedure for the method of Thrombin enzymatic activity determination.
2. SCOPE
This procedure applies to all Thrombin products released by Sigma Chemical under NIH standard unit specifications.
3.1. Purified Water - water from a deionizing system, resistivity ~18MΩ•cm @ 25ºC
3.2. Unit Definition – Thrombin activity is expressed in NIH units obtained by direct comparison to a NIH Thrombin Reference Standard.
4. DISCUSSION
Fibrinogen Thrombin > Fibrin
5. RESPONSIBILITIES
It is the responsibility of all trained Analytical Services laboratory personnel to follow this procedure as written.
6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.1. CONDITIONS: T=37ºC, pH=7.35
7.2. METHOD: Fibrometer
7.3 REAGENTS:
7.3.1. 28.56 mM Barbital Buffer with 1% (w/v) Bovine Serum Albumin, and 0.5% (w/v) Polyethylene Glycol (Enzyme Diluent)
7.3.1.1. Prepare 1L of Barbital Buffer using a Sigma Aldrich Product Number such as B5934 by dissolving 1 vial of B5934 to 1L with purified water.
7.3.1.1.1. Dilute 47.6 ml of Reagent 7.3.1.1 to 100 ml with purified water.
7.3.1.1.2. Reconstituted Barbital Buffer, Reagent 7.3.1.1, can be stored at 2-8ºC for 1 month.
7.3.1.2. Prepare in Reagent 7.3.1.1.1 a 10 mg/ml solution of Bovine Serum Albumin, using a Sigma-Aldrich product such as A4378 with 5 mg/ml solution Polyethylene Glycol, using a Sigma-Aldrich product such as P2139.
7.3.1.3. Adjust the pH to 7.35 at 37ºC with 1.0N NaOH.
7.3.1.4. Allow to equilibrate to room temperature prior to use.
7.3.2. 0.85% (w/v) Sodium Chloride Solution Use Sigma Aldrich Product Number such as S0817. Allow to warm to room temperature before using.
7.3.3. Plasma Solution (Plasma)
7.3.3.1. Immediately before use, reconstitute 1 vial of lyophilized Normal Human Plasma, using Sigma-Aldrich product number Z9824 or equivalent, with 2.0 ml of purified water. Allow to reconstitute for 5 minutes, then add 2.0 ml of Reagent 7.3.2.
7.3.3.2. Allow to equilibrate to room temperature prior to use.
7.3.3.3. Being a substrate specific assay, the most current qualified lot of Normal Human Plasma, and its corresponding composite curve based on NIH standard, must be used to convert clotting times, in seconds, to thrombin activity, in units/ml.
7.3.4. Thrombin Enzyme Solution (Thrombin)
7.3.4.1. If sample is to be assayed per vial, immediately before use, reconstitute one Thrombin vial with 1.0 ml of ambient Reagent 7.3.1 (Enzyme Diluent). Then immediately dilute to 2.5 to 4.0 units/ml of Thrombin using room temperature Reagent 7.3.1 (Enzyme Diluent) in polypropylene tubes.
7.3.4.2. If sample is to be assayed as units/mg solid, immediately before use,prepare a 10 mg/ml solution in a polypropylene tube with ambient Reagent 7.3.1 (Enzyme Diluent). Then immediately dilute to 2.5 to 4.0 units/ml of Thrombin using room temperature Reagent 7.3.1 (Enzyme Diluent) in polypropylene tubes.
7.3.4.3. Serial dilutions should be made in Reagent 7.3.1 (Enzyme Diluent) so that the final clotting times are between 15 and 25 seconds. If the clotting time is too short, decrease the Thrombin concentration. If the time recorded is too long, increase the Thrombin concentration.
7.3.4.4. Store concentrated solution, which can be used over 4 hours, on ice.
7.4. ASSAY
7.4.1. Accurately pipette (in milliliters) the following reagents into fibrometer cup containers, which have been pre-warmed to 37ºC.
| Test | |
| Reagent 7.3.3 (Plasma) | 0.20 |
7.4.2. Allow to stand at 37ºC for exactly thirty seconds, then add:
| Reagent 7.3.4 (Thrombin) | 0.10 |
7.4.3. Record the clotting time using a suitably thermostatted Fibrometer. A 0.3 ml Fibrometer probe should be used for this assay.
7.4.4. Compare the clotting times of the test against a NIH Standard Curve of Thrombin clotting times vs. units of Thrombin/ml.
7.4.5. Determine the units of Thrombin of the Test from the Standard Curve.
7.5 CALCULATIONS
7.5.1 Calculations for Units/mg solid
| 7.5.1.1. | Units/ml enzyme = | (Units of Thrombin from Standard Curve)(df) |
where df = dilution factor
| 7.5.1.2. | Units/mg solid = | Units/ml enzyme |
| Mg solid/ml enzyme |
| 7.5.1.3. | Units/mg protein = | Units/ml enzyme |
| Mg protein/ml enzyme |
7.5.2. Calculations for Units/Vial
| 7.5.2.1. | Units/vial = | (Units of Thrombin from Standard Curve)(df) |
where df = dilution factor
| 7.5.2.2. | Units/mg protein = | Units/vial |
| Mg protein/vial |
7.6 FINAL ASSAY CONTENTRATION
In a 0.3 ml reaction mix, the final concentrations are 0.33% (w/v) Bovine Serum Albumin, 9.52mM Barbital Buffer, 0.17% (w/v) Polyethylene Glycol, 0.28% (w/v) Sodium Chloride, 3% (w/v) Plasma and 0.25–0.4 unit Thrombin.
8.1 Replaces BEPLAS01.
8.2. Human Blood Coagulation, Haemostasis, and Thrombosis (1976) p.721-722, 2nd ed., R. Biggs, ed., Blackwell Scientific Publications, Philadelphia, PA
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