H-D-Phe-Pip-Arg-pNA acetate

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Cat No.

HDPG-001

Description

H-D-Phe-Pip-Arg-pNA acetate is a chromogenic peptide substrate modeled on the N-terminal region of the fibrinogen Aα chain, the natural substrate of thrombin.

Applications

Chromogenic measurement of antithrombin-heparin cofactor (AT-III) using thrombin.

Appearance

White to yellow solid

Product Overview

This substrate is specific for thrombin and is used in an AT-III assay based on chromogenic spectrophotometric detection. The assay is described as sensitive, accurate, and straightforward to perform.

Form

Solid

CAS_No

115388-96-0

Molecular Weight

612.68

Purity

99.81%

Specificity

Thrombin

Storage

Sealed storage, protected from moisture.
Powder: -80°C for 2 years; -20°C for 1 year.
In solvent: -80°C for 6 months; -20°C for 1 month.

Preparation Instructions

Stock solution volume guide:
1 mM: 1 mg in 1.6322 mL; 5 mg in 8.1609 mL; 10 mg in 16.3217 mL.
5 mM: 1 mg in 0.3264 mL; 5 mg in 1.6322 mL; 10 mg in 3.2643 mL.
10 mM: 1 mg in 0.1632 mL; 5 mg in 0.8161 mL; 10 mg in 1.6322 mL.
Select the solvent according to the solubility data. Aliquot prepared solutions to avoid repeated freeze-thaw cycles. If water is used for the stock solution, dilute it to the working solution and then sterilize through a 0.22 μm filter before use.

Function

Chromogenic substrate for thrombin in AT-III measurement.

Synonyms

S-2238 acetate

Targets

Thrombin

Molecular Formula

C₂₉H₄₀N₈O₇

Solubility

DMSO: 100 mg/mL (163.22 mM); ultrasonication is required. Hygroscopic DMSO can markedly affect solubility; use newly opened DMSO.
H₂O: ≥ 100 mg/mL (163.22 mM); saturation is unknown.

In vitro

Stock solution preparation:
1. Dissolve 25 mg of H-D-Phe-Pip-Arg-pNA acetate in 40 mL of sterile distilled water to prepare a 1.0 mmol/L stock solution.
2. Aliquot the stock solution and store at 4°C protected from light.

Working solution preparation:
1. Measure 7.5 mL of the stock solution.
2. Add 1.0 mL of 3 mg/mL Polybrene solution.
3. Add 1.5 mL of sterile distilled water.
4. Mix thoroughly to prepare a 0.75 mM working solution.
5. Store at 4°C; the working solution is stable for 1 week.

AT-III assay:
1. Dilute 50 μL of test plasma with 3.50 mL of heparin-containing buffer.
2. Transfer 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 exactly 30 s, add 300 μL of pre-warmed working solution and mix vigorously.
5. After another exactly 30 s, add 300 μL of 50% acetic acid to stop the reaction immediately.
6. Keep the reaction mixture protected from light until measurement.
7. Measure absorbance at 405 nm.
8. Plot a standard curve and calculate the AT-III concentration of the test plasma.
9. Blank controls: 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; test plasma blank = 400 μL of diluted test plasma + 300 μL of 50% acetic acid + 400 μL of distilled water.

In vivo

Prepare a clear stock solution using an in vitro dissolution method, then add co-solvents sequentially. Prepare the in vivo working solution fresh and use it on the same day. If precipitation or phase separation occurs, heating and/or sonication may be used to aid dissolution.

Protocol 1: 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline. Solubility: ≥ 2.5 mg/mL (4.08 mM), clear solution. For 1 mL, add 100 μL of 25.0 mg/mL DMSO stock solution to 400 μL PEG300 and mix; add 50 μL Tween-80 and mix; then add 450 μL saline to 1 mL. Saline preparation: dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL.

Protocol 2: 10% DMSO + 90% of 20% SBE-β-CD in saline. Solubility: ≥ 2.5 mg/mL (4.08 mM), clear solution. For 1 mL, add 100 μL of 25.0 mg/mL DMSO stock solution to 900 μL of 20% SBE-β-CD in saline and mix. Prepare 20% SBE-β-CD in saline by dissolving 2 g SBE-β-CD in 10 mL saline; store at 4°C for up to 1 week.

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Project Description:

For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.

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