毛細管電泳在蜂王漿蛋白質分析上之應用   全文下載

The Application of Capillary Electrophoresis on the Characterization of Protein in Royal Jelly

本研究採本省中南部以生產蜂王漿為主之義大利蜂種所生產之新鮮蜂王漿，進行蛋白質特性之探討。蜂王漿中總氮含量為2.46%，其中來自總胺基酸的氮含量為2.34%；而來自游離胺基酸的氮含量為0.11%，胺基態氮的含量為0.20%，顯示蜂王漿中的含氮化合物大部分是以蛋白質的型態存在。蜂王漿經離心、沉澱和透析等分離及純化後，可得65±5%之水溶性蛋白質，再經DEAE-Sephacel離子交換管柱分離，得到二部分之區分物質，F1和F2。將F1和F2分別以SDS-PAGE電泳分析，F1部分含二個染色帶，分子量分別為50及44 KDa，F2部分則僅含一個染色帶，分子量為55 KDa。毛細管電泳之分析模式，由於分離機制與解析度各不相同，可將蜂王漿蛋白質分離成不同區分。將F1和F2經毛細管區帶電泳分析，F1出現四個吸收峰，F2則有二個吸收峰；而經毛細管凝膠電泳分析，F1部分出現二個吸收峰，分子量分別為59及73 KDa，F2則僅出現一個吸收峰，分子量為118 KDa；但經毛細管等電聚焦來分離F1和F2，F1則出現六個吸收峰，等電點分別為6.9，6.7，6.3，5.9，5.7及5.5，F2出現二個吸收峰，等電點為4.8及4.7。 The objective of this study was to characterize the protein fractions in royal jelly made from Apis mellifera ligustica from middle and southern areas of Taiwan. The total nitrogen content of fresh royal jelly was 2.46 %, and the total amino acid nitrogen was 2.34 %, suggesting that the nitrogen compound in royal jelly was mostly derived from protein . The nitrogen content of free amino acid in royal jelly was 0.11 %, and the amino type nitrogen was 0.20 %, indicating that the protein in royal jelly existed mainly in the form of large moleculars. To characterize the protein, royal jelly was dissolved in 0.1 M phosphate buffer(pH 7.0), followed by centrifugation, ammonia sulfate precipitation and dialysis to separate the protein into water soluble and water insoluble fractions. Water soluble fraction accounts for more than 60 % of the total protein in royal jelly, and was further investigated by DEAE-Sephacel, SDSPAGE and capillary electrophoresis. By DEAE-Sephacel, two fraction peaks (F1 and F2) were identified and collected. By SDS-PAGE, F1 fraction was further separated into two bands, and the molecular weight was determined to be 50 KDa and 44 KDa, whereas F2 fraction was shown to have only one band with molecular weight of 55 KDa. By capillary zone electrophoresis, four poorly-separated peaks were observed in F1 fraction, and two well-separated peaks in F2 fraction. By capillary gel electrophoresis, two peaks were identified in F1 fraction, of which the molecular weight was estimated to be 59 KDa and 73 KDa. By contrast, only one peak was identified in F2 fraction, of which the molecular weight was estimated to be 118 KDa. However, by capillary isoelectric focusing, 6 peaks were identified in F1 fraction with pi of 6.9, 6.7, 6.3, 5.9, 5.7 and 5.5, respectively. Of that, pi of 4.8 and 4.7 were identified in F2 fraction."

The objective of this study was to characterize the protein fractions in royal jelly made from Apis mellifera ligustica from middle and southern areas of Taiwan. The total nitrogen content of fresh royal jelly was 2.46 %, and the total amino acid nitrogen was 2.34 %, suggesting that the nitrogen compound in royal jelly was mostly derived from protein . The nitrogen content of free amino acid in royal jelly was 0.11 %, and the amino type nitrogen was 0.20 %, indicating that the protein in royal jelly existed mainly in the form of large moleculars. To characterize the protein, royal jelly was dissolved in 0.1 M phosphate buffer(pH 7.0), followed by centrifugation, ammonia sulfate precipitation and dialysis to separate the protein into water soluble and water insoluble fractions. Water soluble fraction accounts for more than 60 % of the total protein in royal jelly, and was further investigated by DEAE-Sephacel, SDSPAGE and capillary electrophoresis. By DEAE-Sephacel, two fraction peaks (F1 and F2) were identified and collected. By SDS-PAGE, F1 fraction was further separated into two bands, and the molecular weight was determined to be 50 KDa and 44 KDa, whereas F2 fraction was shown to have only one band with molecular weight of 55 KDa. By capillary zone electrophoresis, four poorly-separated peaks were observed in F1 fraction, and two well-separated peaks in F2 fraction. By capillary gel electrophoresis, two peaks were identified in F1 fraction, of which the molecular weight was estimated to be 59 KDa and 73 KDa. By contrast, only one peak was identified in F2 fraction, of which the molecular weight was estimated to be 118 KDa. However, by capillary isoelectric focusing, 6 peaks were identified in F1 fraction with pi of 6.9, 6.7, 6.3, 5.9, 5.7 and 5.5, respectively. Of that, pi of 4.8 and 4.7 were identified in F2 fraction.