**2.2. Isolation of oat polyuronides**

Fresh oat seedlings grown for 10–12 days were thoroughly ground and hydrolyzed with 2 М NaOH (1:10 w/v) at room temperature with occasional stirring for 1 day. The hydrolysate was neutralized with concentrated HCl to pH 7.0 and filtered through filter paper. Gel filtration on Sephadex G-25 was used to remove salts and low-molecular-weight components and to isolate a high-molecular-weight fraction containing polysaccharides. The preparation collected to obtain polyuronides was fractionated by anion-exchange chromatography on DEAE cellulose. The preparation was applied onto a column with fibrous DEAE cellulose (a chloride form) equilibrated with distilled water. The column was washed consecutively with distilled water and with 0.15 and 0.5 М NaCl to the minimal A220 of the eluate . Then, polyuronides were eluted with 1.5 М NaCl. To remove NaCl, the resulting fraction was subjected to gel filtration on Sephadex G-25; the presence of chlorides was checked using silver nitrate. The preparation was dried under vacuum at 450С and proved to contain glucuronoxylans with a purity of no less than 95% and a proportion of hexuronic acids of no less than 95%. The quality of the product was checked by the Dishe test for uronic acids. The product was dissolved to 0.1 mg/ml. A 0.5-ml aliquot of the solution was combined with 3 ml of concentrated sulfuric acid containing 0.025 М sodium tetraborate and thoroughly mixed. The mixture was heated in boiling water for 10 min, chilled to room temperature, combined with 0.1 ml of 0.1% carbazole (ethanol solution), heated in boiling water for 15 min, and chilled. A530 was measured against a control sample. The polyuronide concentration (mg/ml) was estimated using a calibration plot, which was constructed using aqueous solutions of glucuronic acid.

## **2.3. Isolation of garlic glucuronoxylans**

Unpeeled garlic bulbs were thoroughly ground and hydrolyzed with 2 М NaOH (1:10 w/v) at room temperature with occasional stirring for 1 day. The hydrolysate was neutralized with concentrated HCl to pH 7.0 and filtered through filter paper. Gel filtration on Sephadex G-25 was used to remove salts and low-molecular-weight components and to isolate a highmolecular-weight fraction containing polysaccharides. The preparation collected to obtain glucuronoxylans was fractionated by anion-exchange chromatography on DEAE cellulose. The preparation was applied onto a column with fibrous DEAE cellulose (a chloride form) equilibrated with distilled water. The column was washed consecutively with distilled water and with 0.15 and 0.3 М NaCl to the minimal A220 of the eluate. Then, glucuronoxylans were eluted with 0.5 М NaCl. To remove NaCl, the resulting fraction was subjected to gel filtration on Sephadex G-25; the presence of chlorides was checked using silver nitrate. The preparation was dried under vacuum at 450С, yielding glucuronoxylans in a dry form. The quality of the product was checked by the Dishe test for uronic acids as above. The purity of the product was no less than 95%; the proportion of hexuronic acids was 20%.

## **2.4. Isolation of GAG (HA, CS, and HS) from human placenta**

Placenta was thoroughly ground and hydrolyzed with 2 М NaOH (1:10 w/v) at room temperature with occasional stirring for 3 days. The hydrolysate was neutralized with concentrated HCl to pH 7.0. To precipitate nonhydrolyzed proteins, concentrated trichloroacetic acid (TCA) was added slowly to a final concentration of 5%. The preparation was incubated at 40С for 1 h, and the precipitate was removed by centrifugation at 1500 g for 10 min. Concentrated NaOH was added to the supernatant to рН 10-11, because some proteins withstanding TCA treatment precipitated in an alkaline milieu. The precipitate was allowed to form and was removed by centrifugation (1500 g, 10 min). The supernatant was filtered through filter paper. Gel filtration on Sephadex G-25 was used to remove salts and low-molecular-weight components and to isolate a high-molecular-weight fraction containing GAG.

The preparations used to isolate HA and CS were fractionated by anion-exchange chromatography on DEAE cellulose. Each preparation was applied individually onto a column with fibrous DEAE cellulose (a chloride form) equilibrated with distilled water. The column was washed consecutively with distilled water and 0.15 М NaCl to the minimal A220 of the eluate. Then HA was eluted with 0.25 М NaCl; CS, with 0.7 М NaCl; and HS, with 1.5 М NaCl. To remove NaCl, each fraction was subjected to gel filtration on Sephadex G-25; the presence of chlorides was checked with silver nitrate. The resulting fractions were dried under vacuum at 450С to obtain HA, CS, and HS in a dry form. The purity was no less than 95%. The quality of the products was checked by the Dishe test for uronic acids as above.

### **2.5. NA and amylose**

266 The Complex World of Polysaccharides

**2.2. Isolation of oat polyuronides**

various initial approximations of the complex structure.

constructed using aqueous solutions of glucuronic acid.

**2.3. Isolation of garlic glucuronoxylans** 

was 20%.

the Newton-Raphson method with an energy gradient of 0.010 kcal/molÅ, starting from

Fresh oat seedlings grown for 10–12 days were thoroughly ground and hydrolyzed with 2 М NaOH (1:10 w/v) at room temperature with occasional stirring for 1 day. The hydrolysate was neutralized with concentrated HCl to pH 7.0 and filtered through filter paper. Gel filtration on Sephadex G-25 was used to remove salts and low-molecular-weight components and to isolate a high-molecular-weight fraction containing polysaccharides. The preparation collected to obtain polyuronides was fractionated by anion-exchange chromatography on DEAE cellulose. The preparation was applied onto a column with fibrous DEAE cellulose (a chloride form) equilibrated with distilled water. The column was washed consecutively with distilled water and with 0.15 and 0.5 М NaCl to the minimal A220 of the eluate . Then, polyuronides were eluted with 1.5 М NaCl. To remove NaCl, the resulting fraction was subjected to gel filtration on Sephadex G-25; the presence of chlorides was checked using silver nitrate. The preparation was dried under vacuum at 450С and proved to contain glucuronoxylans with a purity of no less than 95% and a proportion of hexuronic acids of no less than 95%. The quality of the product was checked by the Dishe test for uronic acids. The product was dissolved to 0.1 mg/ml. A 0.5-ml aliquot of the solution was combined with 3 ml of concentrated sulfuric acid containing 0.025 М sodium tetraborate and thoroughly mixed. The mixture was heated in boiling water for 10 min, chilled to room temperature, combined with 0.1 ml of 0.1% carbazole (ethanol solution), heated in boiling water for 15 min, and chilled. A530 was measured against a control sample. The polyuronide concentration (mg/ml) was estimated using a calibration plot, which was

Unpeeled garlic bulbs were thoroughly ground and hydrolyzed with 2 М NaOH (1:10 w/v) at room temperature with occasional stirring for 1 day. The hydrolysate was neutralized with concentrated HCl to pH 7.0 and filtered through filter paper. Gel filtration on Sephadex G-25 was used to remove salts and low-molecular-weight components and to isolate a highmolecular-weight fraction containing polysaccharides. The preparation collected to obtain glucuronoxylans was fractionated by anion-exchange chromatography on DEAE cellulose. The preparation was applied onto a column with fibrous DEAE cellulose (a chloride form) equilibrated with distilled water. The column was washed consecutively with distilled water and with 0.15 and 0.3 М NaCl to the minimal A220 of the eluate. Then, glucuronoxylans were eluted with 0.5 М NaCl. To remove NaCl, the resulting fraction was subjected to gel filtration on Sephadex G-25; the presence of chlorides was checked using silver nitrate. The preparation was dried under vacuum at 450С, yielding glucuronoxylans in a dry form. The quality of the product was checked by the Dishe test for uronic acids as above. The purity of the product was no less than 95%; the proportion of hexuronic acids The single-stranded probe d(T)16 was obtained from Sintol (Russia). Garlic DNA was isolated from root meristem tissue of seedlings. Human DNA was isolated from the placenta by phenol-detergent extraction (Belozersky A.N. 1970). The purity and concentration of DNA were checked by optical density and by Hoechst 33258 fluorescence, using a DNA minifluorimeter (Hoefer Scientific Instruments, United States). Fragmented calf thymus double-stranded DNA (MW ~1 x 106 Da) and single-stranded polynucleotides d(A)350, d(C)350, and d(GC)350 were purchased from Amersham Biosciences. Amylose was purchased from Serva Feinbiochemica GMBH.

### **2.6. Spectrophotometric assays**

Working solutions of polynucleotides were prepared in a quartz cuvet with a light path of 2 mm. Small (5-25 l) aliquots of a stock polynucleotide solution were added to 0.3М NaCl, 0.002М Na-phosphate (рН 6.8) to the final volume 400 l. After recording the absorption or circular dichroism (CD) spectra of a polynucleotide solution, polynucleotide-polysaccharide complexes were obtained. For this purpose, 400 l of the polynucleotide solution were combined in the cuvet with an aliquot of a stock polysaccharide solution with continuous stirring. Then the absorbance or CD spectra were recorded again.

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 recorded.

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