Determination of Concentration of Soybean Nodule Leghemoglobin Components by Capillary Electrophoresis

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  • 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
  • キャピラリー デンキ エイドウホウ ニヨル ダイズ コンリュウ レグ ヘモグロ

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Abstract

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.

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