H-D-Phe-Pip-Arg-pNA dihydrochloride  (Synonyms: S-2238 dihydrochloride)

H-D-Phe-Pip-Arg-pNA (S-2238) dihydrochloride, a chromogenic substrate, is patterned after the N-terminal portion of the A alpha chain of fibrinogen, which is the natural substrate of thrombin. H-D-Phe-Pip-Arg-pNA dihydrochloride is specific for thrombin and is used to measure antithrombin-heparin cofactor (AT-III). The AT-III assay using H-D-Phe-Pip-Arg-pNA dihydrochloride is sensitive, accurate, and easy to perform^[1][2].

Guidelines (The following is our recommended protocol; this protocol is for guidance only and should be modified according to your specific needs)^[1].
1.1 Preparation of Stock Solution
Dissolve one bottle of 25 mg H-D-Phe-Pip-Arg-pNA dihydrochloride in 40 mL of sterile distilled water to prepare a 1.0 mmol/L stock solution.
Note: The H-D-Phe-Pip-Arg-pNA dihydrochloride stock solution is recommended to be aliquoted and stored at 4°C in the dark.
1.2 Preparation of Working Solution
1) Measure 7.5 mL of H-D-Phe-Pip-Arg-pNA dihydrochloride stock solution.
2) Add 1.0 mL of 3 mg/mL Polybrene (HY-112735) solution.
3) Add 1.5 mL of sterile distilled water.
4) Mix thoroughly to prepare a 0.75 mM H-D-Phe-Pip-Arg-pNA dihydrochloride working solution.
5) Store at 4°C; the shelf life is 1 week.
1.3 Specific Steps
1) Take 50 μL of the plasma to be tested and dilute with 3.50 mL of heparin-containing buffer.
2) Take 400 μL of the diluted plasma and pre-incubate at 37°C for 2-6 min.
3) Add 100 μL of 10-NIH U/ml thrombin solution and mix well.
4) After precisely 30 seconds, add 300 μL of pre-warmed H-D-Phe-Pip-Arg-pNA dihydrochloride working solution and mix vigorously.
5) After another precisely 30 seconds, add 300 μL of 50% acetic acid to immediately stop the reaction.
6) Store the reaction mixture in the dark until spectrophotometric measurement.
7) Measure the absorbance at 405 nm using a spectrophotometer.
8) Plot a standard curve and calculate the AT-III concentration of the plasma to be tested.
9) Blank Control Settings:
Normal plasma blank: 400 μL of 1.00 U/ml AT-III standard solution + 300 μL of 50% acetic acid + 400 μL of distilled water, mix well.
Test plasma blank: 400 μL of diluted test plasma + 300 μL of 50% acetic acid + 400 μL of distilled water, mix well.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

625.55

C₂₇H₃₈Cl₂N₈O₅

62354-65-8

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Goodnight SH Jr, et al. Measurement of antithrombin III in normal and pathologic states using chromogenic substrate S-2238. Comparison with immunoelectrophoretic and factor Xa inhibition assays. Am J Clin Pathol. 1980;73(5):639-647.  [Content Brief]

  [2]. Voorthuizen H, Kluft C. Improved assay conditions for automated antithrombin III determinations with the chromogenic substrate S-2238. Thromb Haemost. 1984;52(3):350-353.  [Content Brief]