@article{oai:niigata-u.repo.nii.ac.jp:00004074,
 author = {佐藤, 孝 and 西脇, 俊和 and 大竹, 憲邦 and 大山, 卓爾},
 issue = {5},
 journal = {日本土壌肥料學雜誌, 日本土壌肥料學雜誌},
 month = {Oct},
 note = {分子量とアミノ酸組成が類似しているダイズ根粒レグヘモグロビン(Lb)の 4つの成分(Lba, Lbc_1,Lbc_2,Lbc_3)のキャビラリー電気泳動法による定量方法を検討し,3品種のダイズにおけるLb成分含有量と存在割合を比較した.根粒の凍結乾燥試料粉未20mgを1mLの0.1M リン酸カリウム緩衝液(pH6.8)で抽出した.夾雑物質を除去するために,ヒドロキシアパタイトのミニカラムで粗精製を行った.粗精製液25μLにフェリシアン化カリウム・ニコチン酸溶液を5μL と 2gL^-1アジ化ナトリウム溶液20μLを加え分析に供した.キャピラリー電気泳動分析では,キャピラリーは長さ 100cm, 内径75μmのフューズドシリカ管を用いた.電気泳動用緩衝液は,75mMリン酸緩衝液(pH2.0)を用い,印加電圧25kV,温度20゜C の条件で泳動し,185nmの吸光度を検出した.上記の条件で分析した結果,ダイズ根粒のレグヘモグロビンの各成分は完全に分離できた.精製したレグヘモグロビンを用いて,ピークの同定を行ったところ,移動時間の早い順に,  Lbc_2, Lbc_3, Lba, Lbc_1が溶出した.また,ビドロキシアパタイトにより夾雑成分が除去されバックグラウンドノイズが減少した.さらに,等濃度のLbaとLbcでは吸光度に差がないことから,定量の標準物質上しては,精製したLbaを使用した.播種後40日目のダイズ品種, Williams, エンレイ,T202の根粒(粒径3～5mm)のレグヘモグロビン各成分を分析したところ,全Lb含有量は,エンレイが最も高く,他の2品種の約2倍含んでいた.エンレイでは,Lbaが半分近くを占め, Lbc_1, Lbc_2, Lbc_3, の順に減少した. Williamsと T202では, Lbaの割合が40%以下であり, Lbc_1よりもLbc_2を多く含んでいた., Soybean nodule contains four leghemoglobin components, Lba, Lbc_1, Lbc_2 and Lbc_3, whose molecular weights and amino acid sequences are similar to each other. An analytical method for determining the concentrations of the four Lb components was developed using capillary electrophoresis, and the concentrations of each component were compared among three soybean cultivars. Soybean nodules were freeze-dried and ground into fine powder. Twenty milligrams of sample powder was extracted with 1 mL of 100 mM potassium phosphate buffer (pH 6.8). The extract was passed through a hydroxyapatite mini column to eliminate the interfering substances. Twenty five microliters of the partially purified solution was mixed with 5 μL of a potassium ferricyanide (10 g kg^<-1>) and nicotinic acid (10 g kg^<-1>) solution, plus 20 μL of a sodium azide (2 g kg^<-1>) solution. For analysis, we used a fused-silica capillary with a 1 m length and 75 μm internal diameter. 75 mM sodium phosphate buffer (pH2.0) was used as the electrolite and charged with 25 kV at 20℃. Leghemoglobin components were detected by optical density at 185 nm. The Lb components were purified and migration time of each Lb component identified. By the analytical conditions described above, four peaks were completely separated and the migration times of Lbc_2, Lbc_3, Lba, Lbc_1 were faster in this sequence. The peak heights of each Lb component was the same, so we used purified Lba as the standard for calibration. The concentrations of each Lb component in nodules with diameters 3-5 mm were analyzed for soybean cultivars "Williams", "Enrei" and "T202" at 40 d after planting. The Enrei nodules contained a higher concentration of total Lb components than the other two cultivars. The relative compositions of the four components were different among the cultivars.},
 pages = {521--526},
 title = {キャピラリー電気泳動法によるダイズ根粒レグヘモグロビン成分の定量},
 volume = {68},
 year = {1997}
}