Purification and Characterization of Mannose-Specific Lectin Isolated from the Rhizomes of Georgian Endemic Plant Polygonatum obtusifolium Miscz.

摘要

Mannose-Specific Lectin (SABA-1) was isolated from the rhizomes of Georgian endemic medicinal plant - mountainous Solomon's seal. Isolation and identification of lectin includes the following stages: 1. Obtaining of protein extract from the rhizome; 2. Chromatography on Toyopearl HW-SS column; 3. Fractionation using ammonium sulphate; 4. Thermal treatment at +60°C for 30 minutes; 5. Treatment with acetone; 6. Chromatography on Toyopearl HW-55 column; 7. Affinity chromatography on rabbit erythrocyte column, fixed with glutar aldehyde. SABA-1 was purified 789 times, hemagglutination activity corresponds to 0.000095 mg/ml. As a result of fractionation of summary proteins on Toyopearl HW-55 column 9 protein fractions with different molecular masses were obtained. Hemagglutination activity was expressed only in the third peak, eluted from the column corresponding to 0.0080 mg/1. Using affinity chromatography on the column, stabilized with trypsin-treated rabbit erythrocytes, 2 protein fractions were obtained, of which only the fraction, eluted with 50 mM D-mannose, possessed hemagglutination activity, which corresponds to 0.000096 mg/ml. Using the marker proteins and analytical gel-filtration, molecular weight of the native SABA-1 protein was established, which corresponded to 30 000 Da molecular weight protein fraction. Using electrophoresis of marker proteins on polyacrylamide gradient gel (10-25%), in the presence of SDS, it was established that SABA-1, dissociated into subunits, migrates as a single strip and its molecular mass corresponds to 15 000 Da. Thus, a new mannose specific lectin was identified from the rhizome of Georgian endemic medicinal plant - mountainous Solomon's seal. It is shown, that its native molecular mass is 30 000 Da and it is composed of two equal molecular mass -15 000 Da - polypeptide subunits.