**3. Results**

268 The Complex World of Polysaccharides

**2.7. NA radiolabeling** 

**2.8. Dot hybridization** 

**2.9. Neutral sugar assays** 

dried and exposed with a РМ-В X-ray film for 1-7 days.

calibration plot, which was constructed with galactose solutions.

recorded.

The annealing of double-stranded DNA with the corresponding polysaccharide was carried out in a closed quartz cuvet with a light path of 2 mm. The cuvet containing DNA and the polysaccharide in certain proportions was heated in boiling water for 2 min and immediately chilled in ice-cold water (0оС) for 30 s. Then the absorbance or CD spectra were

The absorbance spectra were obtained using a Specord M-40 spectrophotometer. The CD spectra of complexes were recorded with an SKD-2 portable dichrometer (Institute of Spectroscopy, Russian Academy of Sciences, Troitsk). The CD spectra were presented as a

NA were 5'-endlabeled *in vitro* with -[32P]ATP and T4 polynucleotide kinase (Maxam A., et al. 1977). Dephosphorylation was carried out in 50 mM Tris-HCL (pH 9.0), 10 mM MgCl2, 10 mM spermidine, 1 mM ZnCl2, 0.1 units of alkaline phosphatase at 370С. The enzyme was removed by treating the samples twice with phenol. NA was precipitated with ethanol and collected by centrifugation in a minicentrifuge at 12,000 rpm for 5 min. The pellet was washed with 70% ethanol to remove phenol, dried, and dissolved in a necessary volume of bidistilled water. The resulting solution was combined with a kinase buffer (50 mM Tris-HCl, pH 7.6), 10 mM MgCl2, 50 mM dithiothreitol, 1 mM spermidine), 1-2 MBq of -[32P]ATP, and 2 units of T4 polynucleotide kinase. The mixture was incubated at 37С for 30 min. The reaction was terminated by adding EDTA to 20 mM, and the enzyme was removed by phenol treatment. NA was precipitated with ethanol, and the nonbound label was removed by repeated precipitation with ethanol or by gel filtration on Sephadex G-25.

BA83 and BA85 filters (Schleicher & Shuell, Germany) with immobilized DNA and polysaccharides (20 g per dot) were hybridized with radiolabeled NA probes at 37C for 18-24 h. The hybridization buffer contained 5 х Denhardt's solution (0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 0.1% SDS (Denhardt D.T. 1966). Before being applied onto a filter, DNA was denatured in boiling water for 5 min. Filters were

Neutral sugars (monosaccharides contained in protein-polysaccharide complexes) were assayed by the anthrone method according to (Jermyn M.A. 1975, Chaplin M.F., et al. 1986). A sample (0.5 ml) was combined with 4 ml of an anthrone reagent (20 mg of anthrone, 100 ml of 80% sulfuric acid). The mixture was heated in boiling water for 5 min, chilled in water to room temperature, and tested for A630. As a control, we used an aqueous solution water treated with the anthrone reagent as above. Neutral sugars were quantitated using a

wavelength dependence of the CD value А = A**L** – A**R** (Dunin V.V., et al. 1979).
