Bz-Ile-Glu-Gly-Arg-pNA.HBr (S-2222

Bz-Ile-Glu-Gly-Arg-pNA.HBr (S-2222, CAS 102601-24-1) is the hydrobromide salt form of the chromogenic tetrapeptide substrate optimized for Factor Xa amidolytic activity assays in hemostasis and thrombosis research. Factor Xa hydrolyzes the arginyl-p-nitroanilide bond, releasing yellow p-nitroaniline (pNA, ε405 nm = 12,700 M⁻¹cm⁻¹) for precise kinetic measurement in microplate readers, supporting anti-Xa heparin/LMWH/DOAC potency testing and coagulation factor diagnostics. The HBr salt offers enhanced stability and solubility compared to HCl/acetate forms, making it ideal for pharmaceutical QC, clinical labs, and high-throughput screening of serine protease inhibitors with validated ISTH/Ph. Eur. performance.

Appearance

• White to off-white crystalline lyophilized powder, highly hygroscopic.
• Readily soluble in water (5-15 mg/mL), DMSO (>100 mg/mL); clear solutions.

Source

• Synthesized via Fmoc-SPPS: sequential Bz-Ile, Glu(γ-OR 50:50 mix), Gly, Arg-pNA coupling, followed by HBr precipitation for purity.
• Bulk production for research/diagnostic reagents by specialized peptide manufacturers.

Molecular Weight and Structure

• C32H44BrN9O9, 778.66 g/mol; free base C32H43N9O9 = 697.75 g/mol.
• N-benzoyl-Ile-Glu(γ-OR)-Gly-Arg-p-nitroanilide hydrobromide; cleavable Arg-pN

Appearance

• White to off-white crystalline lyophilized powder, highly hygroscopic.
• Readily soluble in water (5-15 mg/mL), DMSO (>100 mg/mL); clear solutions.

Source

• Synthesized via Fmoc-SPPS: sequential Bz-Ile, Glu(γ-OR 50:50 mix), Gly, Arg-pNA coupling, followed by HBr precipitation for purity.
• Bulk production for research/diagnostic reagents by specialized peptide manufacturers.

Molecular Weight and Structure

• C32H44BrN9O9, 778.66 g/mol; free base C32H43N9O9 = 697.75 g/mol.
• N-benzoyl-Ile-Glu(γ-OR)-Gly-Arg-p-nitroanilide hydrobromide; cleavable Arg-pNA.

Sugar Specificity

• None (synthetic peptide chromogen; no glycan components).

Biological Activity

• Factor Xa Km = 0.4-0.9 mM (Tris 0.05M pH 8.4); Vmax 4 µkat/L linearity; 150x selective vs. thrombin.
• pH optimum 7.5-8.5, 37°C; also substrates plasma kallikrein, FXIIa.

Purity and Microbial Contamination

• ≥99% HPLC purity; free pNA <0.01%; single impurity <0.1%.
• Endotoxin <0.05 EU/mg; sterile packaging; no microbial growth (USP <71>).

Identity and Quality Control

• RP-HPLC (>99%), LC-MS (m/z 698.3 [M+H]+), AA hydrolysis (1:1:1:1 ratio), ΔA405 functional assay.
• [α]D -20°±5° (c=1, H2O); NMR/IR; Ph. Eur. reference standard equivalence.

Shelf Life and Storage

• 4-6 years lyophilized ≤-20°C desiccated; reconstituted 3 months -70°C or 10 days 2-8°C.
• Light/moisture protected; stable working solutions 24h RT in assay buffer.

Application

• Quantitative anti-Xa activity (UFH, LMWH, rivaroxaban, apixaban); Factor X potency.
• Prothrombinase complex assays; trypsin-like protease screening; coagulation diagnostics.

Key Characteristics

• Superior solubility/stability vs. HCl salt; detection limit 0.05 pM FXa; automation validated.
• Minimal plasma interference; linear to 10 µkat/L; cost-effective bulk formats.

Citation Links

• http://www.geneseqtools.com/cn/product03_cid_217_pid_1085.html
• https://www.hepattack.com/en/product03_cid_217_pid_1085.html
• https://www.hepattack.com/en/product02_page_2.html
• https://www.sigmaaldrich.com/VN/en/product/sigma/f3301
• https://www.chemicalbook.com/ChemicalProductProperty_EN_CB53310510.htm
• https://chromogenicsubstrates.com/methods/factor-x/
• https://pubmed.ncbi.nlm.nih.gov/21811937/
• https://pubmed.ncbi.nlm.nih.gov/26177660/
• https://www.glpbio.com/ga21213.html
• https://www.sigmaaldrich.com/US/en/product/sigma/f3301