| Autor: |
Malsch, R., Guerrini, M., Torri, G., Lohr, G., Casu, B., Harenberg, J. |
| Zdroj: |
Analytical Biochemistry; March 1994, Vol. 217 Issue: 2 p255-264, 10p |
| Abstrakt: |
Heparin plays an important role in anticoagulation and several other biological processes. Cleavage of heparin by nitrous acid results in a reactive 2,5-anhydromannose (Am) which can be used to selectively insert primary and secondary amines by reductive amination. Low-molecular-mass heparin (LMMH) was bound to 4-(2-aminoethylphenol) as shown by nuclear magnetic resonance spectroscopy (NMR), high-performance size-exclusion chromatography (HPSEC), polyacrylamide gel electrophoresis (PAGE), and ultraviolet/visible (uv/vis) spectroscopy. 1H NMR spectra revealed an average sequence of (IdoA2SO3-GlcNSO36SO3)9-IdoAaSO3-Am-tyramine and a 50% binding rate of tyramine to LMMH. LMMH-Tyr had an anticoagulant activity of 108 antifactor Xa activity (aXa) U/mg and 42 antifactor IIa activity (aIIa) U/mg. The compound was neutralized by protamine. The N-alkylamine derivative was adopted to label LMMH with iodine-125 by oxidation with chloramine T. Fluorescein-5-isothiocyanate (Fitc) was used to label LMMH-Tyr with fluorescence. NMR, HPSEC, PAGE, and uv/vis spectroscopy demonstrated the binding of Fitc to LMMH-Tyr. 1H NMR spectra indicated that about 80% of the LMMH-Tyr was labeled at the secondary amino group. The fluorescent compound exhibited 70 aXa and 5 aIIa U/mg and was neutralized by protamine. The selectively bound labeled heparin derivatives are "endpoint attached" and have intact anticoagulant activity. |
| Databáze: |
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