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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Purification and Biochemical Characterization of Lectin from Viscum album

겨우살이 Lectin의 정제 및 생화학적 특성

  • Published : 1999.10.01
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Abstract

The lectin was purified through 0.15 M NaCl extraction, ammonium sulfate precipitation, sepharose 4B affinity chromatography and gel filtration using sephadex G-150 from the leaves of Visum album collected in Mt. Duk Yu. The final gel filtration step resulted in 11.64 folds purification with 0.14% of recovery yield. We also performed biochemical characterization of the purified Visum album lectin. HPLC analysis of lectin purified by gel filtration revealed a singel peak. The analysis of the purified lectin by SDS-PAGE showed a tetramer composed of two identical subunits with molecular weights of 32 and 30 kDa. The lectin was a glycoprotein containing 14.4% carbohydrate, which consist of glucose, fructose, arabinose and xylose, and the amino acids such as phenylalanine, lysine and tyrosine. The purified lectin agglutinated human red blood cell types with similar potency, but when tested against red blood cells from mouse, bovine, rabbit, chicken and porcine, significant difference in potency were observed. Hemaggluting activity was inhibited by D-galactose, D-mannose, D-lactose and D-raffinose, but not by D-glucose, D-glucosamine, D-mannosamine, L-fructose, D-xylose, D-arabinose, D-galacturonic acid, D-fructose, L-rhamnose and N-acetyl-D-galactosamine. The optimal pH and thermal stability of the purified lectin were pH 4.0-7.0 and 20-5$0^{\circ}C$, respectively.

겨우살이가 가지고 있는 lectin의 약리작용의 가능성에 대한 연구의 일환으로, lectin을 분리 . 정제하였으며, 분자량, subunit수, 탄수화물의 조성, 단백질의 아미노산 조성, 적혈구 응집력, 탄수화물 특이성, pH 및 열 안정성등의 생화학적 특성을 연구하였다. Lectin의 분리는 0.15 M NaCl에 의한 추출, $(NH_4)_2SO_4$ 침전, sepharose 4B affinity chromatography 및 sephadex G-150 gel filtration에 의해 11.64배 정제에 0.14%의 수율을 획득하였으며, HPLC와 전기영동을 이용하여 순도를 검정하였다. Gel filtration에 의해 분석된 lectin의 분자량은 124,000 Da이였고, SDS-PAGE에 의해 분자량이 30,000과 32,000 Da인 2개의 band로 나타나서 각각 2개의 subunit를 갖는 tetramer로 확인되었다. 겨우살이 lectin의 탄수화물은 glucose, fructose, arabinose 및 xylose를 함유하고 있다. 또한 phenlylalanine, lysine, tyrosine, aspartic acid가 비교적 풍부하였고 methionine, alanine, arginine은 비교적 적었다. Lectin은 적혈구에 종 특이성을 나타내는데 겨우살이의 혈구응집반응에서는 사람혈액형간의 차이는 없었으며, 쥐에서 가장 높았고 소에서 가장 낮았으므로 동물 종간에 대한 특이성은 뚜렸하였다. Lectin은 당 특이성을 가지는데, D-galactose, D-mannose, D-lactose 및 D-raffinose에 대해 특이성을 나타내었다. 겨우살이 lectin은 pH4~7에서 안정하였으며, 5$0^{\circ}C$까지는 10~30분 열처리해도 활성이 유지되는 열 안정성 단백질이었다.

Keywords

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