항 방사선 인삼단백분획의 sepharose 4B column chromatography에 의한 분리

Abstract

인삼단백질을 부분적으로 정제하기 위하여 Tris-HCl 완충액(pH7.6)추출, 황산암모늄 분별침전, heat inactivation, CM-cellulose 이온교환 컬럼 크로마토그래피 및 Sephadex G75컬럼 크로마토그래피를 실시하여 방사선 방어효과가 있다고 보고된 GI분획을 얻었다. GI분획 단백질을 Sepharose 4B 컬럼 크로마토그래피를 실시한 결과 SⅠ, SⅡ 그리고 SⅢ의 세분획을 얻었다. 이 분획들의 단백질 구성 및 subunit 조성을 알아보고 그들의 분자량을 측정하기 위하여 gradient gel을 사용한 native-polyacrylamide gel electrophoresis(PAGE) 와 SDS-PAGE를 실시하였다. 그 결과는 다음과 같다. SⅠ분획은 native-PAGE결과 한개의 단백질 band (분자량 : 574,000)가 검출되었고, SDS-PAGE결과 두개의 Subunit band(subunit분자량 : 43,500 , 13,500)가 나타났다. SⅡ분획은 native-PAGE결과 세개의 단백질 band (분자량: 514,000 , 124,000 , 57,800)가 측정되었고, SDS-PAGE결과 다섯개의 subunit band(subunit 분자량 : 223,000 , 145,000 , 37,300 , 30,100 , 14,800)가 측정되었다. 그리고 SⅢ분획을 native-PAGE한 결과 세개의 단백질 band(분자량 : 159,000 , 90,500 , 59,400)가 나타났고, SDS-PAGE결과 세개의 Subunit band(Subunit 분자량 : 47,700 , 26,000 , 15,900)가 측정되었다.;The ginseng proteins were partially purified by Tris-HCI buffer (pH 7.6) extraction, ammonium sulfate fractionation, heat inactivation, carboxymethyl-cellulose ion exchange column chromatography and Sephadex G75 column chromatography to obtain GI and GⅡ fractions. Of the two, GI fraction, having the radiation-protective effect, was further purified by Sepharose 4B column chromatography and three fractions (SI, SⅡ and SⅢ) were obtained. These three fractions were subjected to native-polyacrylamide gradient gel electrophoresis (native-PAGE) to estimate molecular weight of native proteins and to SDS-polyacrylamide gradient gel electrophoresis (SDS-PAGE) to estimate molecular weight of subunits. The molecular weight of proteins and subunits were calculated by their Rm values from regression lines for HMW calibration kit proteins. Results are as follows: SI showed one band by native-PAGE with M. W. of 574,000 and two bands by SDS-PAGE with M. W. of 43,500 and 13,500 and SⅡ showed three bands by native-PAGE with M. W. of 514,000, 124,000 and 57,800 and five bands by SDS-PAGE with MoW. of 223,000, 145,000, 37,300, 30,100 and 14,800. SⅢ showed three bands by native-PAGE with M. W. of 159,000, 90,500 and 59,400 and three bands by SDS-PAGE with M. W. of 47,700. 26,000 and 15,900.