chondroitin-sulfates and coralyne

Oversulfated chondroitin sulfate (OSCS) is a harmful contaminant in the pharmaceutical heparin. The development of a rapid, convenient, sensitive, and selective method is required for routine analysis of OSCS in pharmaceutical heparin. Here we report a simple, rapid, sensitive, and enzyme-free method for detecting OSCS in heparin based on the competitive binding between OSCS and the adenosine-repeated molecular beacon (MB) stem to coralyne in the presence of Ca(2+) ions. The MB (A8-MB-A8) contains a 22-mer loop, a stem of a pair of 8-mer adenosine (A) bases, a fluorophore unit at the 5'-end, and a quencher at the 3'-end. The presence of coralyne promotes these A-A mismatches to form a hairpin-shaped MB. However, this kind of MB is incapable of differentiating between heparin and OSCS because they both exhibit strong electrostatic attraction with coralyne. This study found that while Ca(2+) ions can efficiently suppress the negative charges of heparin, they do not neutralize the negative charge of OSCS. Thus, in the presence of Ca(2+) ions, OSCS can remove coralyne from the MB stem, initiating fluorescence of the MB. Under optimal conditions (10 nM A8-MB-A8, 800 nM coralyne, and 0.5 mM Ca(2+) ions), the proposed system can detect 0.01% w/w OSCS in heparin in under 5 min without enzyme treatment. This study also validates the practicality of the proposed system to determine 0.01% w/w OSCS in the pharmaceutical heparin.

Topics: Base Sequence; Berberine Alkaloids; Biosensing Techniques; Calcium; Chondroitin Sulfates; Heparin; Models, Molecular; Nucleic Acid Conformation; Oligonucleotide Probes; Time Factors

The effects of anionic chondroitin or dextran sulfates on the absorption and fluorescence properties of coralyne, a cationic benzo[c]phenanthridine type alkaloid, were studied in aqueous solution. The remarkably strong binding to both polysaccharides promotes the coralyne dimer formation, which was evidenced by the changes in the absorption and fluorescence spectra and the fluorescence decay. The extent of dimerization, induced by chondroitin, shows a significant pH dependence because the competitive protonation of the carboxylate moieties of the polymer chain decreases the number of binding sites. A larger molecular weight of dextran sulfate stabilizes the coralyne dimer more efficiently.

Topics: Anions; Berberine Alkaloids; Carbohydrate Conformation; Chondroitin Sulfates; Dextran Sulfate; Models, Molecular; Polysaccharides; Solutions; Spectrometry, Fluorescence; Spectrophotometry