Analysis Of Insecticidal Crystal Proteins And Isomaltooligosaccharides By Capillary Electrophoresis

The research results are summarized as following:Purification of crystal proteins (>95%) by petroleum ether-extracted and aqueous two-phase extraction was described. Crude insecticidal crystal proteins (ICPs, 133 kDa) was first dissolved in Na2CO3(pH 10.5), then precipitated in HC1 (1 mol/L) and finally purified by DEAE ion-exchange chromatography. A capillary zone electrophoresis method and a capillary gel electrophoresis for determining ICPs in Bacillus thuringiensis were developed. With 50 mmol/L borax, 3.5 mmol/L SDS, 20% ACN and 0.1% p-mercaptoethanol at pH 9.5 as running buffer, ICPs can be determined within 10 min at the applied voltage of 10 kV and the capillary temperature of 25 . The dimension of capillary was 40 cm 75 um I.D without coating. The linear correlation coefficient between the concentration of ICPs and the peak area was equal to 0.9997 in the concentration range of 0.05-0.40 mg/ml. The experiment conditions of capillary gel electrophoresis: neutrally-coated capillary (50 urn I.D, 40 cm of total length and 30 cm of efficient length), 0.1 mol/L Tris-CHES(pH 8.6), 0.1% SDS, 2.5% PEG as running buffer; the applied voltage of 300 V/cm; the capillary temperature at 25 and the detection wave-length was 214 nm.A capillary electrophoresis method for the determination of main components in isomaltooligosaccharide formulation was described. The influences of various separartion conditions including buffer concentration, pH value, voltage and detection wave-length were investigated. By using an uncoated silica capillary (75 um I.D, 60 cm of total length, and 50 cm of efficient length) and 75 mmol/L borax (pH 10.5) as carrier electrolyte, the derivatized isomaltooligosaccharides with a-naphymethamine, including isomaltose, panose, isomaltotriose, were sufficiently separated at the qualification of an applied voltage of 15 kV and a UV detection wave-length of 214 nm. The results indicate that therelative standard deviations for migration times and peaks area were all less than 0.5%and 2.0% respectively. Quantification by the peak area method allowed reproducibledetermination of the analytes at the concentration range of 0.005-1.000 mg/ml. This method is characterized by its sensitivity, reproducibility and low cost, should be useful for the determination of main components of isomaltooligosaccharide formulation.