Determination of concentration of soyabean nodule leghaemoglobin components by capillary electrophoresis.

摘要

An analytical method for determining the concentrations of the 4 leghaemoglobin (Lb) components (Lba, Lbc1, Lbc2 and Lbc3) in soyabean nodules was developed using capillary electrophoresis. The concentrations of each component were compared among 3soyabean cultivars. Soyabean 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. 25 鎙 of the partially purified solution was mixed with 5 鎙 of a potassium ferricyanide (10 g kg-1) and nicotinic acid (10 g kg-1) solution, plus 20 鎙 of a sodium azide (2 g kg-1) solution. For analysis, a fused-silica capillary with a 1 m length and 75 鎚 internal diameter was used. 75 mM sodium phosphate buffer (pH 2.0) was used as the electrolyte and charged with 25 kV at 20鳦. Leghaemoglobin 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 Lbc2, Lbc3, Lba, Lbc1 were faster in this sequence. The peak heights of each Lb component was the same, so purified Lba was used as the standard for calibration. The concentrations of each Lb component in nodules with diameters 3-5 mm were analyzed for soyabean cv. Williams, Enrei and T202 at 40 d after sowing. Cv. 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.