Bz-Ile-Glu-Gly-Arg-pNA.HCl (S-2222)

Bz-Ile-Glu-Gly-Arg-pNA.HCl (S-2222, CAS 60457-00-3) is a chromogenic tetrapeptide hydrochloride substrate essential for measuring Factor Xa activity in coagulation diagnostics and research. Factor Xa cleaves the C-terminal arginyl-p-nitroanilide bond, releasing quantifiable p-nitroaniline (pNA) with absorbance at 405 nm (ε 12,700 M⁻¹cm⁻¹), enabling precise kinetic evaluation of anticoagulant therapies like heparins and direct oral anticoagulants (DOACs). Its high specificity and stability make it a gold standard for prothrombin activation assays, plasma amidolytic activity, and serine protease profiling in clinical labs and pharma screening.

Appearance

• White to off-white lyophilized hygroscopic powder or crystalline solid.
• Highly soluble in water (4-10 mM) and DMSO (>50 mM); slow dissolution aided by sonication.

Source

• Produced by solid-phase Fmoc/tBoc peptide synthesis: Bz-Ile-OH coupling to H-Glu(γ-OR)-Gly-Arg(H)-pNA·HCl, where R= H/Me (50:50).
• Commercial grades from Chromogenix/DiaPharma, bulk suppliers.

Molecular Weight and Structure

• C32H43N9O9·HCl, 734.20 g/mol (calculated); empirical often lists minor variants.
• N-benzoyl-L-ileucyl-L-glutamyl(γ-OR)-glycyl-L-arginyl-p-nitroanilide HCl.

Sugar Specificity

• None (non-glycosylated synthetic peptide substrate).

Biological Activity

• Factor Xa Km=0.5-1.0 mM (pH 8.4, I=0.25); linearity to 4 µkat/L; also active on trypsin (Km=0.1 mM).
• Reaction: Bz-Ile-Glu-Gly-Arg-pNA → Bz-Ile-Glu-Gly-Arg-OH + pNA.

Purity and Microbial Contamination

• >98-99% by RP-HPLC; free pNA <0.05%; single peak ≥98.5%.
• Endotoxin-free (<0.1 EU/mg); sterile, protected against microbial hydrolysis.

Identity and Quality Control

• HPLC/MS (m/z 698.3 [M+H]+ free base), AA analysis (I:E:G:R=1:1:1:1), specific activity assay (ΔA405).
• [α]D, IR, NMR; functional test per Ph. Eur./ISTH standards.

Shelf Life and Storage

• >3-5 years dry powder at -20°C; solutions 6 months 2-8°C or 1 month -20°C aliquots.
• Hygroscopic: store desiccated, sealed; avoid >3 freeze-thaws.

Application

• Anti-Xa assays for UFH/LMWH/DOACs; Factor Xa in ATIII deficiency; prothrombinase complex.
• Trypsin amidase activity; kallikrein/FXIIa screening; coagulation factor potency.

Key Characteristics

• 100-200x selective for FXa vs. thrombin; no plasma protein interference.
• Automation-ready; low Km for high sensitivity (0.1 pM detection); validated diagnostic reagent.

Citation Links

• https://www.dcchemicals.com/coa/COA_DC11455.html
• https://www.dcchemicals.com/product_show-S2222.html
• https://tribioscience.com/product/life-sciences/protein-and-peptide/bz-ile-glu-gly-arg-pna-hcl-tbp0116/
• https://diapharma.com/wp-content/uploads/inserts/S820316_301925R2_chromogenix_S2222_insert.pdf
• http://geneseqtools.com/en/product03_cid_217_pid_1086.html
• https://www.vulcanchem.com/product/vc2043663
• https://pubchem.ncbi.nlm.nih.gov/compound/Bz-Ile-glu-gly-arg-pna
• https://www.chemicalbook.com/ChemicalProductProperty_EN_CB95540878.htm
• https://www.glpbio.com/ga21213.html
• https://www.benchchem.com/pdf/An_In_Depth_Technical_Guide_to_Bz_IEGR_pNA_Acetate_in_Protease_Research.pdf