A Biotin-Conjugated Substrate Facilitating Analysis of HIV-1 Integrase Activity on Minigels

✍ Scribed by Khampoune Sayasith; Gilles Sauvé; Jocelyn Yelle

• Book ID: 102563956
• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 61 KB
• Volume: 284
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4702

✦ Synopsis

ecules may be detected at low levels. The absorption maxima (350 -360 nm) and the emission maximum of were similar to those of MANT-nucleotides. In the range pH 5.4 -9, the fluorescence yield of MANT-dextran in aqueous solution does not change by more than Ϯ7%.

An example of the usefulness of this labeling procedure is demonstrated by the gel filtration analysis of the degradation of MANT-labeled hyaluronic acid, a glycosaminoglycan, by the Streptomyces hyaluronan lyase enzyme (Fig. ). The polysaccharide was readily visualized in the intact or the partially depolymerized forms by monitoring absorbance of the chromophore at 350 nm. A distinct glycosaminoglycan, chondroitin sulfate C, was similarly labeled and then degraded with Flavobacterium chondroitin AC lyase (data not shown). Usually about 5-25 g of polymer was analyzed per run; if fluorescence detection is employed, it is expected that 10 2 -to 10 3 -fold less material would be required.

Conclusions. A major attribute of the MANT-labeling procedure is the circumvention of polysaccharide detection via extreme UV absorbance (200 -220 nm), refractive index, or radioactivity. Furthermore, the rapid reaction uses relatively mild conditions that are likely to preserve the polysaccharide's biological activity, immunogenicity, and chemiphysical properties. To assess the suitability of this labeling method for a given application, however, the fact that the MANT group covalently attached to a polymer's hydroxyl group possesses a secondary amine and a benzyl ring must be considered. The ease and cost of preparation of MANT-polysaccharides are reasonable for making customized standards for gel filtration chromatography. Additionally, polymer species in deproteinized experimental samples may also be tracked during purification and/or analysis.