흰강남콩 (Phaseolus vulgaris)의 추출물을 DEAE Sephadex A-50과 hydroxyapatite column을 통과시켜 적혈구 및 립프구 응집효과를 갖는 EL-PHA를 얻었다. EL-PHA로부터 적혈구 응집효과만 가진 E-PHA을 분리하기 위해 EL-PHA를 4M urea로 처리한 다음 CM-Sephadex column을 통과시켜 림프구 응집력만 가진 L-PHA, 적혈구 응집력만 가진 EI-PHA, 적혈구 응집력과 약간의 림프구 응집력을 가진 ElI-PHA를 얻었다. EI-, EII-PHA의 생물물리화학적 성상을 구명코저 activity test, 전기영동, SDS-전기영동, 당류시험, 면역화학적 실험 등을 수행하였다. 전기영동에서 EL-PHA는 3개의 band를 나타내었고, EI-, EII-PHA는 1개의 뚜렷한 band를 나타내었으며, 분자량은 각각 130,000과 125,000 daltons으로 추정되었다. SDS-PAGE 결과 EI-PHA는 분자량이 약 34,000인 subunits 1종으로 나타났고, EII-PHA는 41,000과 30,000정도의 2종의 서로 다른 subunits로 나타났다. 당류에 의한 activity의 저해 효과는 0.3% D-mannose와 N-acetyl-D-galactosamine의 경우에 현저 하였고, D-galactose, L-fucose, N-acetylneuraminic acid, N-acetyl-D-glucosamine의 경우는 저해 효과가 없었다. 한편, EE-, EII-PHA 당류를 미량 함유한 당단백질임을 알 수 있었다. 면역 화학적인 연구의 하나로 immunodiffusion을 한 결과 EI-PHA는 antigen-antibody 반응을 잘 나타내 주었다.
Salt fractionated extracts of Phaseolus vulgaris was purified successively through ion exchange chromatography on DEAE Sephadex A-50, hydroxyapatite column and EL-PHA was obtained. EL-PHA was treated with 4M urea just prior to the application into CM-Sephadex C-50 column. This column was eluted with 10 mM (pH 5.7), 20 mM (pH 6.6) and 30mM (pH 8.0) phosphate buffer, each containing 4M urea. In order to identify isolation of E-and L-PHA, erythroagglutination test with human blood A group and lymphoagglutinating test with mouse spleen lymphocytes were measured on each fraction and found three active fractions, L-PHA, having lymphoagglutinating activity only, EI-PHA, having high erythroagglutinating activity only and EII-PHA, having erythroagglutinating activity along with weak lymphoagglutinating activity. In order to characterize biophysicochemical properties of the EI-and EII-PHA, polyacrylamide disc gel electrophoresis, SDS gel electrophoresis, sugar inhibition test, carbohydrate test were performed. In polyacrylamide disc gel electrophoresis, EL-PHA was observed as three bands, EI-and EII-PHA were observed as clear single band with a very faint minor band. Molecular weight of EI-and EII-PHA were estimated at about 130,000 daltons and 125,000 daltons, respectively by PAGE. The results of SDS-PAGE shown that EI-PHA consists of four homogenous subunits having molecular weight of 34,000 per unit and EII-PHA consists of two different subunits with 41,000 arid 30,000 daltons. Erythroagglutination by EI-and EII-PHA was inhibited with 0.3% D-mannose and N-acetyl-D-galactosamine, but not with N-galactose, L-fucose, N-acetylneuraminic acid and N-acetyl-D-glucosamine. Sugar components of EI-and EII-PHA were recognized by anthrone test, but no spot was observed in paper chromatography. For immunochemical studies, antisera to EI-PHA were prepared by immunizing rabbit and EI-PHA formed precipitin bands against various antisera in Ouchterlony double immunodiffusion.
